Radic Z


Full name : Radic Zoran

First name : Zoran

Mail : Skaggs School of Pharmacy and Pharmaceutical Sciences\; University of California at San Diego\; 9500 Gilman Drive\; La Jolla\; CA 92093-0650

Zip Code : CA 92093

City : San Diego

Country : USA

Email : zradic@ucsd.edu

Phone : +18588224834

Fax :

Website : \/\/pharmacy.ucsd.edu\/faculty\/bios\/radic.shtml

Directory :

References (147)

Title : Connectivity between surface and interior in catalytic subunits of acetylcholinesterases inferred from their X-ray structures - Radic_2023_J.Neurochem_17thISCM_
Author(s) : Radic Z
Ref : Journal of Neurochemistry , : , 2023
Abstract : Catalytic activity and function of acetylcholinesterase (AChE; EC have been recognized and studied for over a century, its quaternary and primary structures for about half a century and its tertiary structure has been known for about 33 years. Clear understanding of relationships between the structure and the function is still pending for this enzyme. Hundreds of crystallographic, static snapshots of AChEs from different sources reveal largely one general backbone conformation with narrow entry into the active center gorge, tightly fit to accept one acetylcholine (ACh) molecule, in contrast to its high catalytic turnover. This short review of available X-ray structures of AChEs from electric ray Torpedo californica, mouse and human, finds some limited, yet consistent deviations in conformations of selected secondary structure elements of AChE relevant for its function. The observed conformational diversity of the acyl pocket loop of AChE, unlike the large -loop, appears consistent with structurally dynamic INS data and solution-based SAXS experiments to explain its dominant role in controlling the size of the active center gorge opening, as well as connectivity between the immediate surroundings of the buried active Ser, and catalytically relevant sites on the AChE surface.
ESTHER : Radic_2023_J.Neurochem_17thISCM_
PubMedSearch : Radic_2023_J.Neurochem_17thISCM_
PubMedID: 36892323

Title : Structural and dynamic effects of paraoxon binding to human acetylcholinesterase by X-ray crystallography and inelastic neutron scattering - Gerlits_2022_Structure_30_1538
Author(s) : Gerlits O , Fajer M , Cheng X , Blumenthal DK , Radic Z , Kovalevsky A
Ref : Structure , 30 :1538 , 2022
Abstract : Organophosphorus (OP) compounds, including nerve agents and some pesticides, covalently bind to the catalytic serine of human acetylcholinesterase (hAChE), thereby inhibiting acetylcholine hydrolysis necessary for efficient neurotransmission. Oxime antidotes can reactivate the OP-conjugated hAChE, but reactivation efficiency can be low for pesticides, such as paraoxon (POX). Understanding structural and dynamic determinants of OP inhibition and reactivation can provide insights to design improved reactivators. Here, X-ray structures of hAChE with unaged POX, with POX and oximes MMB4 and RS170B, and with MMB4 are reported. A significant conformational distortion of the acyl loop was observed upon POX binding, being partially restored to the native conformation by oximes. Neutron vibrational spectroscopy combined with molecular dynamics simulations showed that picosecond vibrational dynamics of the acyl loop soften in the -20-50 cm(-1) frequency range. The acyl loop structural perturbations may be correlated with its picosecond vibrational dynamics to yield more comprehensive template for structure-based reactivator design.
ESTHER : Gerlits_2022_Structure_30_1538
PubMedSearch : Gerlits_2022_Structure_30_1538
PubMedID: 36265484
Gene_locus related to this paper: human-ACHE

Title : Room temperature crystallography of human acetylcholinesterase bound to a substrate analogue 4K-TMA: Towards a neutron structure - Gerlits_2021_Curr.Res.Struct.Biol_3_206
Author(s) : Gerlits O , Blakeley MP , Keen DA , Radic Z , Kovalevsky A
Ref : Current Research in Structural Biology , 3 :206 , 2021
Abstract : Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently attach to the catalytic Ser203 residue. Reactivation of inhibited hAChE can be achieved with nucleophilic antidotes, such as oximes. Understanding structural and electrostatic (i.e. protonation states) determinants of the catalytic and reactivation processes is crucial to improve design of oxime reactivators. Here we report X-ray structures of hAChE conjugated with a reversible covalent inhibitor 4K-TMA (4K-TMA:hAChE) at 2.8 A resolution and of 4K-TMA:hAChE conjugate with oxime reactivator methoxime, MMB4 (4K-TMA:hAChE:MMB4) at 2.6 A resolution, both at physiologically relevant room temperature, as well as cryo-crystallographic structure of 4K-TMA:hAChE at 2.4 A resolution. 4K-TMA acts as a substrate analogue reacting with the hydroxyl of Ser203 and generating a reversible tetrahedral hemiketal intermediate that closely resembles the first tetrahedral intermediate state during hAChE-catalyzed acetylcholine hydrolysis. Structural comparisons of room temperature with cryo-crystallographic structures of 4K-TMA:hAChE and published mAChE complexes with 4K-TMA, as well as the effect of MMB4 binding to the peripheral anionic site (PAS) of the 4K-TMA:hAChE complex, revealed only discrete, minor differences. The active center geometry of AChE, already highly evolved for the efficient catalysis, was thus indicative of only minor conformational adjustments to accommodate the tetrahedral intermediate in the hydrolysis of the neurotransmitter acetylcholine (ACh). To map protonation states in the hAChE active site gorge we collected 3.5 A neutron diffraction data paving the way for obtaining higher resolution datasets that will be needed to determine locations of individual hydrogen atoms.
ESTHER : Gerlits_2021_Curr.Res.Struct.Biol_3_206
PubMedSearch : Gerlits_2021_Curr.Res.Struct.Biol_3_206
PubMedID: 34541552
Gene_locus related to this paper: human-ACHE

Title : Ligand design for human acetylcholinesterase and nicotinic acetylcholine receptors, extending beyond the conventional and canonical - Taylor_2021_J.Neurochem__
Author(s) : Taylor P , Shong YJ , Samskey N , Ho KY , Radic Z , Fenical W , Sharpless KB , Kovarik Z , Andrea Camacho-Hernandez G
Ref : Journal of Neurochemistry , : , 2021
Abstract : We detail here distinctive departures from lead classical cholinesterase reactivators, the pyridinium aldoximes, to achieve rapid CNS penetration and reactivation of AChE in the CNS (brain and spinal cord). Such reactivation is consistent with these non-canonical reactivators enhancing survival parameters in both mice and macaques following exposure to organophosphates. Thus, the ideal cholinesterase reactivator should show minimal toxicity, limited inhibitory activity in the absence of an organophosphate, and rapid CNS penetration, in addition to its nucleophilic potential at the target, the conjugated AChE active center. These are structural properties directed to reactivity profiles at the conjugated AChE active center, reinforced by the pharmacokinetic and tissue disposition properties of the reactivator leads. In the case of nicotinic acetylcholine receptor (nAChR) agonists and antagonists, with the many existing receptor subtypes in mammals, we prioritize subtype selectivity in their design. In contrast to nicotine and its analogues that react with panoply of AChR subtypes, the substituted di-2-picolyl amine pyrimidines possess distinctive ionization characteristics reflecting in selectivity for the orthosteric site at the alpha7 subtypes of receptor. Here entry to the CNS should be prioritized for the therapeutic objectives of the nicotinic agent influencing aberrant CNS activity in development or in the sequence of CNS ageing (longevity) in mammals, along with general peripheral activities controlling inflammation.
ESTHER : Taylor_2021_J.Neurochem__
PubMedSearch : Taylor_2021_J.Neurochem__
PubMedID: 33638151

Title : Shifts in backbone conformation of acetylcholinesterases upon binding of covalent inhibitors, reversible ligands and substrates - Radic_2021_Crystals_11_1557
Author(s) : Radic Z
Ref : Crystals , 11 :1557 , 2021
Abstract : The influence of ligand binding to human, mouse and Torpedo californica acetylcholinesterase (EC; AChE) backbone structures is analyzed in a pairwise fashion by comparison with X-ray structures of unliganded AChEs. Both complexes with reversible ligands (substrates and inhibitors) as well as covalently interacting ligands leading to the formation of covalent AChE conjugates of tetrahedral and of trigonal-planar geometries are considered. The acyl pocket loop (AP loop) in the AChE backbone is recognized as the conformationally most adaptive, but not necessarily sterically exclusive, structural element. Conformational changes of the centrally located AP loop coincide with shifts in C-terminal alpha-helical positions, revealing interacting components for a potential allosteric interaction within the AChE backbone. The stabilizing power of the aromatic choline binding site, with the potential to attract and pull fitting entities covalently tethered to the active Ser, is recognized. Consequently, the pull can promote catalytic reactions or relieve steric pressure within the impacted space of the AChE active center gorge. These dynamic properties of the AChE backbone inferred from the analysis of static X-ray structures contribute towards a better understanding of the molecular template important in the structure-based design of therapeutically active molecules, including AChE inhibitors as well as reactivators of conjugated, inactive AChE
ESTHER : Radic_2021_Crystals_11_1557
PubMedSearch : Radic_2021_Crystals_11_1557

Title : Backbone Conformation Shifts in X-ray Structures of Human Acetylcholinesterase upon Covalent Organophosphate Inhibition - Luedtke_2021_Crystals_11_1270
Author(s) : Luedtke S , Bojo C , Li Y , Luna E , Bianca Pomar , Radic Z
Ref : Crystals , 11 :1270 , 2021
Abstract : Conformations of Calpha backbones in X-ray structures of most organophosphate (OP)-inhibited human acetylcholinesterases (hAChEs) have been previously shown to be similar to that of the native hAChE. One of the exceptions is the structure of the diethylphosphoryl-hAChE conjugate, where stabilization of a large ethoxy group into the acyl pocket (AP) of hAChE-triggered notable loop distortions and consequential dissociation of the hAChE homodimer. Recently, six X-ray structures of hAChE conjugated with large OP nerve agents of the A-type, Novichoks, have been deposited to PDB. In this study we analyzed backbone conformation shifts in those structures, as well as in OP-hAChE conjugates formed by Paraoxon, Soman, Tabun, and VX. A Java-based pairwise alpha carbon comparison tool (PACCT 3) was used for analysis. Surprisingly, despite the snug fit of large substituents on phosphorus, inside Novichok-conjugated hAChEs only minor conformational changes were detected in their backbones. Small magnitudes of observed changes were due to a 1.2-2.4 A shift of the entire conjugated OP away from the AP. It thus appears that the small AP of AChEs can accommodate, without distortion, substituents of the size of ethoxy or butyryl groups, provided that conjugated OP is "pulled" away from the AP. This observation has practical consequences in the structure-based design of nucleophilic reactivation antidotes as well as in the definition of the AChE specificity that relies on the size of its AP
ESTHER : Luedtke_2021_Crystals_11_1270
PubMedSearch : Luedtke_2021_Crystals_11_1270

Title : Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects - Blumenthal_2021_J.Biol.Chem__
Author(s) : Blumenthal DK , Cheng X , Fajer M , Ho KY , Rohrer J , Gerlits O , Taylor P , Juneja P , Kovalevsky A , Radic Z
Ref : Journal of Biological Chemistry , :101007 , 2021
Abstract : Acetylcholinesterase (EC; AChE), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission, be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human AChE (hAChE) in solution occurs through a C-terminal 4-helix bundle (4HB) at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R(P) enantiomer of sarin promotes a ten-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6 or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of a S(P)-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS (TR-SAXS) occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wild-type hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket towards the 4HB dimerization interface 25 A away.
ESTHER : Blumenthal_2021_J.Biol.Chem__
PubMedSearch : Blumenthal_2021_J.Biol.Chem__
PubMedID: 34324828
Gene_locus related to this paper: human-ACHE

Title : Enhancing target tissue levels and diminishing plasma clearance of ionizing zwitterionic antidotes in organophosphate exposures - Shyong_2021_J.Pharmacol.Exp.Ther__
Author(s) : Shyong YJ , Sepulveda Y , Garcia A , Samskey NM , Radic Z , Sit R , Sharpless KB , Momper JD , Taylor P
Ref : Journal of Pharmacology & Experimental Therapeutics , : , 2021
Abstract : Inhibition of acetylcholinesterase (AChE) by certain organophosphates (OP) can be life-threatening and requires reactivating antidote accessibility to the peripheral and central nervous systems to reverse symptoms and enhance survival parameters. In considering dosing requirements for oxime antidotes in OP exposures that inactivate AChE, clearance of proton ionizable, zwitterionic antidotes is rapid and proceeds with largely the parent antidotal compound being cleared by renal transporters. Such transporters may also control disposition between target tissues and plasma as well as overall elimination from the body. An ideal, small molecule antidote should access and be retained in primary target tissues: CNS (brain), skeletal muscle, and peripheral autonomic sites, for sufficient periods to reactivate AChE and prevent acute toxicity. We show here that we can markedly prolong the antidotal activity of zwitterionic antidotes by inhibiting P-glycoprotein (P-gp) transporters in the brain capillary and renal systems. We employ the P-gp inhibitor, tariquidar (TQD), as a reference compound and show tissue and plasma levels of RS194B, a hydroxyl-imino acetamido alkylamine reactivator, are elevated and plasma clearances are reduced. To examine mechanism, identify the transporter and establish the actions of a transport inhibitor, we compare the pharmacokinetic parameters in a P-glycoprotein knock-out mouse strain and see dramatic enhancements of short-term plasma and tissue levels. Hence, repurposed transport inhibitors, that are candidate or FDA approved drugs, should enhance target tissue concentrations of the zwitterionic antidote through inhibition of both renal elimination and brain capillary extrusion. Significance Statement We examine renal and brain capillary transporter inhibition for lowering dose and frequency of dosing of a blood-brain barrier (BBB) permanent, reactivating antidote, RS194B, an ionizable zwitterion. Through a small molecule, tariqudar (TQD), and gene knock-out mice, CNS antidote concentrations are enhanced and total body clearances are diminished. RS194B with repurposed transport inhibitors should enhance reactivation of central and peripheral OP-inhibited AChE. Activity at both disposition sites are desired features for replacing 2-PAM as an antidote for acute OP exposure.
ESTHER : Shyong_2021_J.Pharmacol.Exp.Ther__
PubMedSearch : Shyong_2021_J.Pharmacol.Exp.Ther__
PubMedID: 34145064

Title : Rational design, synthesis and evaluation of uncharged, smart bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase - Gorecki_2020_J.Biol.Chem_295_4079
Author(s) : Gorecki L , Gerlits O , Kong X , Cheng X , Blumenthal DK , Taylor P , Ballatore C , Kovalevsky A , Radic Z
Ref : Journal of Biological Chemistry , 295 :4079 , 2020
Abstract : Corrected : Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are non-productive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE (hAChE), here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-hAChE conjugates. Oxime group re-orientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.
ESTHER : Gorecki_2020_J.Biol.Chem_295_4079
PubMedSearch : Gorecki_2020_J.Biol.Chem_295_4079
PubMedID: 32019865
Gene_locus related to this paper: human-ACHE

Title : Evaluation of high-affinity phenyltetrahydroisoquinoline aldoximes, linked through anti-triazoles, as reactivators of phosphylated cholinesterases - Macek Hrvat_2019_Toxicol.Lett_321_83
Author(s) : Macek Hrvat N , Kalisiak J , Sinko G , Radic Z , Sharpless KB , Taylor P , Kovarik Z
Ref : Toxicol Lett , 321 :83 , 2019
Abstract : Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a co-regulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1,3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure.
ESTHER : Macek Hrvat_2019_Toxicol.Lett_321_83
PubMedSearch : Macek Hrvat_2019_Toxicol.Lett_321_83
PubMedID: 31863869

Title : Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library-Reactivators of Acetylcholinesterase - Kovarik_2019_Chemistry_25_4100
Author(s) : Kovarik Z , Kalisiak J , Hrvat NM , Katalinic M , Zorbaz T , Zunec S , Green C , Radic Z , Fokin VV , Sharpless KB , Taylor P
Ref : Chemistry , 25 :4100 , 2019
Abstract : Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post-exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun-inhibited human AChE, and in vivo antidotal efficacies in tabun-exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.
ESTHER : Kovarik_2019_Chemistry_25_4100
PubMedSearch : Kovarik_2019_Chemistry_25_4100
PubMedID: 30458057

Title : A new crystal form of human acetylcholinesterase for exploratory room-temperature crystallography studies - Gerlits_2019_Chem.Biol.Interact_13ChEPon_
Author(s) : Gerlits O , Ho KY , Cheng X , Blumenthal D , Taylor P , Kovalevsky A , Radic Z
Ref : Chemico-Biological Interactions , : , 2019
Abstract : Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 A resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under closer-to-physiological temperature conditions.
ESTHER : Gerlits_2019_Chem.Biol.Interact_13ChEPon_
PubMedSearch : Gerlits_2019_Chem.Biol.Interact_13ChEPon_
PubMedID: 31176713
Gene_locus related to this paper: human-ACHE

Title : Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase - Gerlits_2019_J.Biol.Chem_294_10607
Author(s) : Gerlits O , Kong X , Cheng X , Wymore T , Blumenthal DK , Taylor P , Radic Z , Kovalevsky A
Ref : Journal of Biological Chemistry , 294 :10607 , 2019
Abstract : Exposure to organophosphorus compounds (OPs) may be fatal if untreated, and a clear and present danger posed by nerve agent OPs has become palpable in recent years. OPs inactivate acetylcholinesterase (AChE) by covalently modifying its catalytic serine. Inhibited AChE cannot hydrolyze the neurotransmitter acetylcholine leading to its build-up at the cholinergic synapses and creating an acute cholinergic crisis. Current antidotes, including oxime reactivators that attack the OP-AChE conjugate to free the active enzyme, are inefficient. Better reactivators are sought, but their design is hampered by a conformationally rigid portrait of AChE extracted exclusively from 100K X-ray crystallography and scarcity of structural knowledge on human AChE (hAChE). Here, we present room temperature X-ray structures of native and VX-phosphonylated hAChE with an imidazole-based oxime reactivator, RS-170B. We discovered that inhibition with VX triggers substantial conformational changes in bound RS-170B from a "nonproductive" pose (the reactive aldoxime group points away from the VX-bound serine) in the reactivator-only complex to a "semi-productive" orientation in the VX-modified complex. This observation, supported by concurrent molecular simulations, suggested that the narrow active-site gorge of hAChE may be significantly more dynamic than previously thought, allowing RS-170B to reorient inside the gorge. Furthermore, we found that small molecules can bind in the choline-binding site hindering approach to the phosphorous of VX-bound serine. Our results provide structural and mechanistic perspectives on the reactivation of OP-inhibited hAChE and demonstrate that structural studies at physiologically relevant temperatures can deliver previously overlooked insights applicable for designing next-generation antidotes.
ESTHER : Gerlits_2019_J.Biol.Chem_294_10607
PubMedSearch : Gerlits_2019_J.Biol.Chem_294_10607
PubMedID: 31138650
Gene_locus related to this paper: human-ACHE

Title : Assessment of ionizable, zwitterionic oximes as reactivating antidotal agents for organophosphate exposure - Taylor_2019_Chem.Biol.Interact_13ChEPon_308_194
Author(s) : Taylor P , Yan-Jye S , Momper J , Hou W , Camacho-Hernandez GA , Radic Z , Rosenberg YJ , Kovarik Z , Sit R , Sharpless KB
Ref : Chemico-Biological Interactions , 308 :194 , 2019
Abstract : Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.
ESTHER : Taylor_2019_Chem.Biol.Interact_13ChEPon_308_194
PubMedSearch : Taylor_2019_Chem.Biol.Interact_13ChEPon_308_194
PubMedID: 31100277

Title : Counteracting tabun inhibition by reactivation by pyridinium aldoximes that interact with active center gorge mutants of acetylcholinesterase - Kovarik_2019_Toxicol.Appl.Pharmacol_372_40
Author(s) : Kovarik Z , Macek Hrvat N , Kalisiak J , Katalinic M , Sit RK , Zorbaz T , Radic Z , Fokin VV , Sharpless KB , Taylor P
Ref : Toxicol Appl Pharmacol , 372 :40 , 2019
Abstract : Tabun represents the phosphoramidate class of organophosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. Currently used therapy in counteracting excessive cholinergic stimulation consists of a muscarinic antagonist (atropine) and an oxime reactivator of inhibited AChE, but the classical oximes are particularly ineffective in counteracting tabun exposure. In a recent publication (Kovarik et al., 2019), we showed that several oximes prepared by the Huisgen 1,3 dipolar cycloaddition and related precursors efficiently reactivate the tabun-AChE conjugate. Herein, we pursue the antidotal question further and examine a series of lead precursor molecules, along with triazole compounds, as reactivators of two AChE mutant enzymes. Such studies should reveal structural subtleties that reside within the architecture of the active center gorge of AChE and uncover intimate mechanisms of reactivation of alkylphosphate conjugates of AChE. The designated mutations appear to minimize steric constraints of the reactivating oximes within the impacted active center gorge. Indeed, after initial screening of the triazole oxime library and its precursors for the reactivation efficacy on Y337A and Y337A/F338A human AChE mutants, we found potentially active oxime-mutant enzyme pairs capable of degrading tabun in cycles of inhibition and reactivation. Surprisingly, the most sensitive ex vivo reactivation of mutant AChEs occurred with the alkylpyridinium aldoximes. Hence, although the use of mutant enzyme bio-scavengers in humans may be limited in practicality, bioscavenging and efficient neutralization of tabun itself or phosphoramidate mixtures of organophosphates might be achieved efficiently in vitro or ex vivo with these mutant AChE combinations.
ESTHER : Kovarik_2019_Toxicol.Appl.Pharmacol_372_40
PubMedSearch : Kovarik_2019_Toxicol.Appl.Pharmacol_372_40
PubMedID: 30978400
Gene_locus related to this paper: human-ACHE

Title : Pharmacology, Pharmacokinetics, and Tissue Disposition of Zwitterionic Hydroxyiminoacetamido Alkylamines as Reactivating Antidotes for Organophosphate Exposure - Sit_2018_J.Pharmacol.Exp.Ther_367_363
Author(s) : Sit RK , Kovarik Z , Macek Hrvat N , Zunec S , Green C , Fokin VV , Sharpless KB , Radic Z , Taylor P
Ref : Journal of Pharmacology & Experimental Therapeutics , 367 :363 , 2018
Abstract : In the development of antidotal therapy for treatment of organophosphate exposure from pesticides used in agriculture and nerve agents insidiously employed in terrorism, the alkylpyridinium aldoximes have received primary attention since their early development by I. B. Wilson in the 1950s. Yet these agents, by virtue of their quaternary structure, are limited in rates of crossing the blood-brain barrier, and they require administration parenterally to achieve full distribution in the body. Oximes lacking cationic charges or presenting a tertiary amine have been considered as alternatives. Herein, we examine the pharmacokinetic properties of a lead ionizable, zwitterionic hydroxyiminoacetamido alkylamine in mice to develop a framework for studying these agents in vivo and generate sufficient data for their consideration as appropriate antidotes for humans. Consequently, in vitro and in vivo efficacies of immediate structural congeners were explored as leads or backups for animal studies. We compared oral and parenteral dosing, and we developed an intramuscular loading and oral maintenance dosing scheme in mice. Steady-state plasma and brain levels of the antidote were achieved with sequential administrations out to 10 hours, with brain levels exceeding plasma levels shortly after administration. Moreover, the zwitterionic oxime showed substantial protection after gavage, whereas the classic methylpyridinium aldoxime (2-pyridinealdoxime methiodide) was without evident protection. Although further studies in other animal species are necessary, ionizing zwitterionic aldoximes present viable alternatives to existing antidotes for prophylaxis and treatment of large numbers of individuals in terrorist-led events with nerve agent organophosphates, such as sarin, and in organophosphate pesticide exposure.
ESTHER : Sit_2018_J.Pharmacol.Exp.Ther_367_363
PubMedSearch : Sit_2018_J.Pharmacol.Exp.Ther_367_363
PubMedID: 30190337

Title : Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity - Hagstrom_2018_Arch.Toxicol_92_1161
Author(s) : Hagstrom D , Zhang S , Ho A , Tsai ES , Radic Z , Jahromi A , Kaj KJ , He Y , Taylor P , Collins ES
Ref : Archives of Toxicology , 92 :1161 , 2018
Abstract : The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 microM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.
ESTHER : Hagstrom_2018_Arch.Toxicol_92_1161
PubMedSearch : Hagstrom_2018_Arch.Toxicol_92_1161
PubMedID: 29167930
Gene_locus related to this paper: dugja-CHE1 , dugja-CHE2

Title : The JAVA Based Computational Tool for Pairwise Comparison of Protein Backbone Folds in Liganded and Apo 3D Structures of the alpha\/beta Hydrolase Fold Proteins - Zheng_2018_FaASEB.J_32_527
Author(s) : Zheng Z , Rohrer J , Radic Z
Ref : FASEB Journal , 32 :527 , 2018
Abstract : https://doi.org/10.1096/fasebj.2018.32.1_supplement.527.12 Cholinesterases, carboxylesterases and lipases are some of prominent catalytically active proteins with specifically similar, globular tertiary structure fold termed alpha/beta hydrolase fold. Ligand binding, both reversible and covalent, frequently results in small, yet systematic and potentially functionally important changes in the conformation of their alpha carbon backbones that can be easily overseen by commonly used overlays based on overall minimization of backbone atom RMSD analysis. In particular, backbone conformations of cholinesterases - acetylcholinesterase and butyrylcholinesterase - show only small conformational changes. This is revealed in more than 200 PDB deposited cholinesterase X-ray structures in complexes with structurally diverse ligands that affect their function. We developed a novel, reference point based principle for overlay-independent pairwise comparison of liganded and non-liganded alpha carbon backbone conformations from respective PDB (Protein Data Bank) deposited 3D structure entries and encoded it in JAVA based computer algorithm for quick analysis. Comparisons are based on differences in distances between each alpha carbon and chosen reference point in each pair of compared structures, as well as on differences in the angle between center of mass, reference point and each of alpha carbons in the comparison. Combination of the two comparison criteria reveals subset of backbone alpha carbons in two structures that maintains best their relative positions in the 3D space and that can be used as tethering points for overlay of compared structures. Created overlay has capacity to reveal small but systematic local changes in the backbone conformation frequently masked by the overall RMSD minimization approach. Our systematic analysis of a number of PDB deposited alpha/beta hydrolase fold structures revealed small, yet functionally important conformational changes in their backbone consistent with experimental observations and instrumental in revealing molecular mechanisms in respective catalytic reactions. These findings are important for complete molecular target template characterization in the structure based development of novel antidotes of exposure to organophosphates which covalently inhibit this family of enzymes.
ESTHER : Zheng_2018_FaASEB.J_32_527
PubMedSearch : Zheng_2018_FaASEB.J_32_527

Title : Butyrylcholinesterase identification in a phenylvalerate esterase-enriched fraction sensitive to low mipafox concentrations in chicken brain - Mangas_2017_Arch.Toxicol_91_909
Author(s) : Mangas I , Radic Z , Taylor P , Ghassemian M , Candela H , Vilanova E , Estevez J
Ref : Archives of Toxicology , 91 :909 , 2017
Abstract : Multiple epidemiological and experimental studies have demonstrated that exposure to organophosphorus compounds (OPs) is associated with a variety of neurological disorders. Some of these exposure symptoms cannot be precisely correlated with known molecular targets and mechanisms of toxicity. Most of the known molecular targets of OPs fall in the protein family of serine esterases. We have shown that three esterase components in the soluble fraction of chicken brain (an animal model frequently used in OP neurotoxicity assays) can be kinetically distinguished using paraoxon, mipafox and phenylmethyl sulfonyl fluoride as inhibitors, and phenyl valerate as a substrate; we termed them Ealpha, Ebeta and Egamma. The Ealpha-component, which is highly sensitive to paraoxon and mipafox and resistant to PMSF, has shown sensitivity to the substrate acetylthiocholine, and to ethopropazine and iso-OMPA (specific inhibitors of butyrylcholinesterase; BChE) but not to BW 284C51 (a specific inhibitor of acetylcholinesterase; AChE). In this work, we employed a large-scale proteomic analysis B with a LC/MS/MS TripleTOF system; 259 proteins were identified in a chromatographic fractionated sample enriched in Ealpha activity of the chicken brain soluble fraction. Bioinformatics analysis revealed that BChE is the only candidate protein identified to be responsible for almost all the Ealpha activity. This study demonstrates the potential information to be gained from combining kinetic dissection with large-scale proteomics and bioinformatics analyses for identification of proteins that are targets of OP toxicity and may be involved in detoxification of phosphoryl and carbonyl esters.
ESTHER : Mangas_2017_Arch.Toxicol_91_909
PubMedSearch : Mangas_2017_Arch.Toxicol_91_909
PubMedID: 26838044

Title : Post-exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor - Rosenberg_2017_Chem.Biol.Interact_274_50
Author(s) : Rosenberg YJ , Mao L , Jiang X , Lees J , Zhang L , Radic Z , Taylor P
Ref : Chemico-Biological Interactions , 274 :50 , 2017
Abstract : Organophosphate (OP) nerve agents and pesticides trigger a common mechanism of neurotoxicity resulting from critical targeting and inhibition of acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Therapeutic countermeasures have thus focused on either administering an oxime post-exposure, that can rapidly reactivate OP-inhibited AChE, or by preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets. While several pyridinium aldoxime antidotes are currently approved, their utility is impaired due to their inability to cross the blood-brain barrier (BBB) efficiently. The present study utilized a macaque (Ma) model to demonstrate the efficacy of a novel zwitterionic and centrally acting oxime RS194B to reactivate sarin- and paraoxon-inhibited macaque AChE and butyrylcholinesterase (BChE) in vitro and to further assess the capacity of RS194B to effect a reversal of clinical symptoms following sarin inhalation in vivo. In vitro, oxime reactivation of MaAChE and MaBChE was shown to be comparable to their human orthologs, while the macaque studies indicated that IM administration of 62.5 mg/kg of RS194B and 0.28 mg/kg atropine after continuous exposure to 49.6 mug/kg sarin vapor, rapidly reactivated the inhibited AChE and BChE in blood and reversed both early and advanced clinical symptoms of sarin-induced toxicity following pulmonary exposure within 1 h. The rapid cessation of autonomic and central symptoms, including convulsions, observed in macaques bodes well for the use of RS194B as an intra- or post-exposure human treatment and validates the macaque model in generating efficacy and toxicology data required for approval under the FDA Animal rule.
ESTHER : Rosenberg_2017_Chem.Biol.Interact_274_50
PubMedSearch : Rosenberg_2017_Chem.Biol.Interact_274_50
PubMedID: 28693885

Title : HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants - Macek Hrvat_2016_Chem.Biol.Interact_259_148
Author(s) : Macek Hrvat N , Zunec S , Taylor P , Radic Z , Kovarik Z
Ref : Chemico-Biological Interactions , 259 :148 , 2016
Abstract : The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms.
ESTHER : Macek Hrvat_2016_Chem.Biol.Interact_259_148
PubMedSearch : Macek Hrvat_2016_Chem.Biol.Interact_259_148
PubMedID: 27083141
Gene_locus related to this paper: human-ACHE

Title : Limitations in current acetylcholinesterase structure-based design of oxime antidotes for organophosphate poisoning - Kovalevsky_2016_Ann.N.Y.Acad.Sci_1378_41
Author(s) : Kovalevsky A , Blumenthal DK , Cheng X , Taylor P , Radic Z
Ref : Annals of the New York Academy of Sciences , 1378 :41 , 2016
Abstract : Acetylcholinesterase (AChE; EC, an essential enzyme of cholinergic neurotransmission in vertebrates, is a primary target in acute nerve agent and organophosphate (OP) pesticide intoxication. Catalytically inactive OP-AChE conjugates formed between the active-center serine and phosphorus of OPs can, in principle, be reactivated by nucleophilic oxime antidotes. Antidote efficacy is limited by the structural diversity of OP-AChE conjugates resulting from differences in the structure of the conjugated OP, the different active-center volumes they occupy when conjugated to the active-center serine of AChE, and the distinct chemical characteristics of both OPs and oximes documented in numerous X-ray structures of OP-conjugated AChEs. Efforts to improve oxime reactivation efficacy by AChE structure-based enhancement of oxime structure have yielded only limited success. We outline here the potential limitations of available AChE X-ray structures that preclude an accurate prediction of oxime structures, which are necessary for association in the OP-AChE gorge and nucleophilic attack of the OP-conjugated phosphorus.
ESTHER : Kovalevsky_2016_Ann.N.Y.Acad.Sci_1378_41
PubMedSearch : Kovalevsky_2016_Ann.N.Y.Acad.Sci_1378_41
PubMedID: 27371941

Title : Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation - Hagstrom_2017_Arch.Toxicol_91_2837
Author(s) : Hagstrom D , Hirokawa H , Zhang L , Radic Z , Taylor P , Collins ES
Ref : Archives of Toxicology , 91 :2837 , 2016
Abstract : The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.
ESTHER : Hagstrom_2017_Arch.Toxicol_91_2837
PubMedSearch : Hagstrom_2017_Arch.Toxicol_91_2837
PubMedID: 27990564
Gene_locus related to this paper: dugja-CHE1 , dugja-CHE2

Title : Mechanistic studies of new oximes reactivators of human butyrylcholinesterase inhibited by cyclosarin and sarin - de Lima_2016_J.Biomol.Struct.Dyn__1
Author(s) : de Lima WE , Francisco A , da Cunha EFF , Radic Z , Taylor P , Franca TCC , Ramalho TC
Ref : J Biomol Struct Dyn , :1 , 2016
Abstract : Butyryl cholinesterase (BChE) has been seen as a key enzyme in the search for new strategies in the treatment of poisoning by organophosphates (OPs), since human BChE (HssBChE), complexed with the appropriate oxime, can be a suitable scavenger and deactivator for OPs in the blood stream. However, the efficacy of HssBChE is limited by its strict stoichiometric scavenging, slow reactivation and propensity for aging. The improvement of the reactivation rate by new and more efficient oximes could contribute to mitigate this problem and increase the HssBChE efficiency as scavenger. Several oximes have been synthesized and tested with this goal, some with promising results, but the mechanistic aspects of the reactivation reaction are not fully understood yet. In order to better investigate this mechanism, docking and mixed quantum and molecular mechanics (QM/MM) combined with principal components analysis (PCA) were performed here to evaluate the capacity of reactivation and determine the preferred route for the reactivation reaction of two new oximes on HssBChE inhibited by the neurotoxic agents cyclosarin and sarin. Plots of potential energies were calculated and all the transition states (TS) of the reactional mechanism were determined. Our results showed a good correlation with experimental data and pointed to the most efficient oxime with both OPs. The protocol used could be a suitable tool for a preliminary evaluation of the HssBChE reactivation rates by new oximes.
ESTHER : de Lima_2016_J.Biomol.Struct.Dyn__1
PubMedSearch : de Lima_2016_J.Biomol.Struct.Dyn__1
PubMedID: 27125569

Title : Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches - Mangas_2016_Arch.Toxicol_90_603
Author(s) : Mangas I , Taylor P , Vilanova E , Estevez J , Franca TCC , Komives E , Radic Z
Ref : Archives of Toxicology , 90 :603 , 2016
Abstract : The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.
ESTHER : Mangas_2016_Arch.Toxicol_90_603
PubMedSearch : Mangas_2016_Arch.Toxicol_90_603
PubMedID: 25743373

Title : Quaternary and tertiary aldoxime antidotes for organophosphate exposure in a zebrafish model system - Schmidt_2015_Toxicol.Appl.Pharmacol_284_197
Author(s) : Schmidt HR , Radic Z , Taylor P , Fradinger EA
Ref : Toxicol Appl Pharmacol , 284 :197 , 2015
Abstract : The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The ki values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, kr, in both zebrafish and human AChE. However, differences between the Kox and k2 constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, Ki and alphaKi, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure.
ESTHER : Schmidt_2015_Toxicol.Appl.Pharmacol_284_197
PubMedSearch : Schmidt_2015_Toxicol.Appl.Pharmacol_284_197
PubMedID: 25701203

Title : Catalytic Soman Scavenging by the Y337A\/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes - Kovarik_2015_Chem.Res.Toxicol_28_1036
Author(s) : Kovarik Z , Macek Hrvat N , Katalinic M , Sit RK , Paradyse A , Zunec S , Musilek K , Fokin VV , Taylor P , Radic Z
Ref : Chemical Research in Toxicology , 28 :1036 , 2015
Abstract : Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of the soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin, and paraoxon, inhibition. We here demonstrate that ex vivo, in whole human blood, 1 muM soman was detoxified within 30 min when supplemented with 0.5 muM Y337A/F338A AChE and 100 muM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of 42 pyridinium aldoximes, and 5 imidazole 2-aldoxime N-propylpyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2-3-fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack.
ESTHER : Kovarik_2015_Chem.Res.Toxicol_28_1036
PubMedSearch : Kovarik_2015_Chem.Res.Toxicol_28_1036
PubMedID: 25835984

Title : Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal alpha7 from Muscle alpha1(2)betagammadelta nAChRs - Bourne_2015_Structure_23_1106
Author(s) : Bourne Y , Sulzenbacher G , Radic Z , Araoz R , Reynaud M , Benoit E , Zakarian A , Servent D , Molgo J , Taylor P , Marchot P
Ref : Structure , 23 :1106 , 2015
Abstract : Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal alpha7, alpha4beta2, alpha3beta2, and muscle-type alpha12betagammadelta nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the alpha7 and alpha12betagammadelta subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 A resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.
ESTHER : Bourne_2015_Structure_23_1106
PubMedSearch : Bourne_2015_Structure_23_1106
PubMedID: 26004441

Title : Imidazole aldoximes effective in assisting butyrylcholinesterase catalysis of organophosphate detoxification - Sit_2014_J.Med.Chem_57_1378
Author(s) : Sit RK , Fokin VV , Amitai G , Sharpless KB , Taylor P , Radic Z
Ref : Journal of Medicinal Chemistry , 57 :1378 , 2014
Abstract : Intoxication by organophosphate (OP) nerve agents and pesticides should be addressed by efficient, quickly deployable countermeasures such as antidotes reactivating acetylcholinesterase or scavenging the parent OP. We present here synthesis and initial in vitro characterization of 14 imidazole aldoximes and their structural refinement into three efficient reactivators of human butyrylcholinesterase (hBChE) inhibited covalently by nerve agent OPs, sarin, cyclosarin, VX, and the OP pesticide metabolite, paraoxon. Rapid reactivation of OP-hBChE conjugates by uncharged and nonprotonated tertiary imidazole aldoximes allows the design of a new OP countermeasure by conversion of hBChE from a stoichiometric to catalytic OP bioscavenger with the prospect of oral bioavailability and central nervous system penetration. The enhanced in vitro reactivation efficacy determined for tertiary imidazole aldoximes compared to that of their quaternary N-methyl imidazolium analogues is attributed to ion pairing of the cationic imidazolium with Asp 70, altering a reactive alignment of the aldoxime with the phosphorus in the OP-hBChE conjugate.
ESTHER : Sit_2014_J.Med.Chem_57_1378
PubMedSearch : Sit_2014_J.Med.Chem_57_1378
PubMedID: 24571195

Title : Centrally acting oximes in reactivation of tabun-phosphoramidated AChE - Kovarik_2013_Chem.Biol.Interact_203_77
Author(s) : Kovarik Z , Macek Hrvat N , Sit RK , Radic Z , Fokin VV , Sharpless KB , Taylor P
Ref : Chemico-Biological Interactions , 203 :77 , 2013
Abstract : Organophosphates (OP) inhibit acetylcholinesterase (AChE, EC, both in peripheral tissues and central nervous system (CNS), causing adverse and sometimes fatal effects due to the accumulation of neurotransmitter acetylcholine (ACh). The currently used therapy, focusing on the reactivation of inhibited AChE, is limited to peripheral tissues because commonly used quaternary pyridinium oxime reactivators do not cross the blood brain barrier (BBB) at therapeutically relevant levels. A directed library of thirty uncharged oximes that contain tertiary amine or imidazole protonable functional groups that should cross the BBB as unionized species was tested as tabun-hAChE conjugate reactivators along with three reference oximes: DAM (diacetylmonoxime), MINA (monoisonitrosoacetone), and 2-PAM. The oxime RS150D [N-((1-(3-(2-((hydroxyimino)methyl)-1H-imidazol-1-yl)propyl)-1H-1,2,3-triazol-4-y l)methyl)benzamide] was highlighted as the most promising reactivator of the tabun-hAChE conjugate. We also observed that oximes RS194B [N-(2-(azepan-1-yl)ethyl)-2-(hydroxyimino)acetamide] and RS41A [2-(hydroxyimino)-N-(2-(pyrrolidin-1-yl)ethyl)acetamide], which emerged as lead uncharged reactivators of phosphylated hAChE with other OPs (sarin, cyclosarin and VX), exhibited only moderate reactivation potency for tabun inhibited hAChE. This implies that geometry of oxime access to the phosphorus atom conjugated to the active serine is an important criterion for efficient reactivation, along with the chemical nature of the conjugated moiety: phosphorate, phosphonate, or phosphoramidate. Moreover, modification of the active center through mutagenesis enhances the rates of reactivation. The phosphoramidated-hAChE choline-binding site mutant Y337A showed three-times enhanced reactivation capacity with non-triazole imidazole containing aldoximes (RS113B, RS113A and RS115A) and acetamide derivative (RS194B) than with 2PAM.
ESTHER : Kovarik_2013_Chem.Biol.Interact_203_77
PubMedSearch : Kovarik_2013_Chem.Biol.Interact_203_77
PubMedID: 22960624

Title : Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes - Radic_2013_Biochem.J_450_231
Author(s) : Radic Z , Dale T , Kovarik Z , Zunec S , Garcia E , Zhang LM , Amitai G , Green C , Radic B , Duggan BM , Ajami D , Rebek J , Taylor P
Ref : Biochemical Journal , 450 :231 , 2013
Abstract : In the present paper we show a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide OPs (organophosphates) catalysed by purified hBChE (human butyrylcholinesterase) in combination with novel non-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient reactivator of OP-hBChE conjugates formed by the nerve agents VX and cyclosarin, and the pesticide paraoxon. It was also functional in reactivation of sarin- and tabun-inhibited hBChE. A 3-5-fold enhancement of in vitro reactivation of VX-, cyclosarin- and paraoxon-inhibited hBChE was observed when compared with the commonly used N-methylpyridinium aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide). Kinetic analysis showed that the enhancement resulted from improved molecular recognition of corresponding OP-hBChE conjugates by TAB2OH. The unique features of TAB2OH stem from an exocyclic quaternary nitrogen and a hydroxy group, both ortho to an oxime group on a benzene ring. pH-dependences reveal participation of the hydroxy group (pKa=7.6) forming an additional ionizing nucleophile to potentiate the oxime (pKa=10) at physiological pH. The TAB2OH protective indices in therapy of sarin- and paraoxon-exposed mice were enhanced by 30-60% when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establish that oxime-assisted catalysis is feasible for OP bioscavenging.
ESTHER : Radic_2013_Biochem.J_450_231
PubMedSearch : Radic_2013_Biochem.J_450_231
PubMedID: 23216060

Title : Automated docking with protein flexibility in the design of femtomolar click chemistry inhibitors of acetylcholinesterase - Morris_2013_J.Chem.Inf.Model_53_898
Author(s) : Morris GM , Green LG , Radic Z , Taylor P , Sharpless KB , Olson AJ , Grynszpan F
Ref : J Chem Inf Model , 53 :898 , 2013
Abstract : The use of computer-aided structure-based drug design prior to synthesis has proven to be generally valuable in suggesting improved binding analogues of existing ligands. (1) Here we describe the application of the program AutoDock (2) to the design of a focused library that was used in the "click chemistry in-situ" generation of the most potent noncovalent inhibitor of the native enzyme acetylcholinesterase (AChE) yet developed (Kd = approximately 100 fM). (3) AutoDock version 3.0.5 has been widely distributed and successfully used to predict bound conformations of flexible ligands. Here, we also used a version of AutoDock which permits additional conformational flexibility in selected amino acid side chains of the target protein.
ESTHER : Morris_2013_J.Chem.Inf.Model_53_898
PubMedSearch : Morris_2013_J.Chem.Inf.Model_53_898
PubMedID: 23451944

Title : Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates - Radic_2013_Chem.Biol.Interact_203_67
Author(s) : Radic Z , Sit RK , Garcia E , Zhang LM , Zunec S , Kovarik Z , Amitai G , Fokin VV , Sharpless KB , Taylor P
Ref : Chemico-Biological Interactions , 203 :67 , 2013
Abstract : A library of more than 200 novel uncharged oxime reactivators was used to select and refine lead reactivators of human acetylcholinesterase (hAChE) covalently conjugated with sarin, cyclosarin, VX, paraoxon and tabun. N-substituted 2-hydroxyiminoacetamido alkylamines were identified as best reactivators and reactivation kinetics of the lead oximes, RS41A and RS194B, were analyzed in detail. Compared to reference pyridinium reactivators, 2PAM and MMB4, molecular recognition of RS41A reflected in its Kox constant was compromised by an order of magnitude on average for different OP-hAChE conjugates, without significant differences in the first order maximal phosphorylation rate constant k2. Systematic structural modifications of the RS41A lead resulted in several-fold improvement with reactivator, RS194B. Kinetic analysis indicated Kox reduction for RS194B as the main kinetic constant leading to efficient reactivation. Subtle structural modifications of RS194B were used to identify essential determinants for efficient reactivation. Computational molecular modeling of RS41A and RS194B interactions with VX inhibited hAChE, bound reversibly in Michaelis type complex and covalently in the pentacoordinate reaction intermediate suggests that the faster reactivation reaction is a consequence of a tighter RS194B interactions with hAChE peripheral site (PAS) residues, in particular with D74, resulting in lower interaction energies for formation of both the binding and reactivation states. Desirable in vitro reactivation properties of RS194B, when coupled with its in vivo pharmacokinetics and disposition in the body, reveal the potential of this oxime design as promising centrally and peripherally active antidotes for OP toxicity.
ESTHER : Radic_2013_Chem.Biol.Interact_203_67
PubMedSearch : Radic_2013_Chem.Biol.Interact_203_67
PubMedID: 22975155

Title : Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases - Radic_2012_J.Biol.Chem_287_11798
Author(s) : Radic Z , Sit RK , Kovarik Z , Zunec S , Garcia E , Zhang L , Amitai G , Green C , Radic B , Fokin VV , Sharpless KB , Taylor P
Ref : Journal of Biological Chemistry , 287 :11798 , 2012
Abstract : We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.
ESTHER : Radic_2012_J.Biol.Chem_287_11798
PubMedSearch : Radic_2012_J.Biol.Chem_287_11798
PubMedID: 22343626

Title : New structural scaffolds for centrally acting oxime reactivators of phosphylated cholinesterases - Sit_2011_J.Biol.Chem_286_19422
Author(s) : Sit RK , Radic Z , Gerardi V , Zhang L , Garcia E , Katalinic M , Amitai G , Kovarik Z , Fokin VV , Sharpless KB , Taylor P
Ref : Journal of Biological Chemistry , 286 :19422 , 2011
Abstract : We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K(ox)) and maximum reactivation rate (k(2)), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.
ESTHER : Sit_2011_J.Biol.Chem_286_19422
PubMedSearch : Sit_2011_J.Biol.Chem_286_19422
PubMedID: 21464125

Title : Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging - Cochran_2011_J.Biol.Chem_286_29718
Author(s) : Cochran R , Kalisiak J , Kucukkilinc TT , Radic Z , Garcia E , Zhang L , Ho KY , Amitai G , Kovarik Z , Fokin VV , Sharpless KB , Taylor P
Ref : Journal of Biological Chemistry , 286 :29718 , 2011
Abstract : The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bio-scavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for aging-prone alkyl phosphate inhibitors.
ESTHER : Cochran_2011_J.Biol.Chem_286_29718
PubMedSearch : Cochran_2011_J.Biol.Chem_286_29718
PubMedID: 21730071

Title : Naturally occurring variations in the human cholinesterase genes: heritability and association with cardiovascular and metabolic traits - Valle_2011_J.Pharmacol.Exp.Ther_338_125
Author(s) : Valle AM , Radic Z , Rana BK , Mahboubi V , Wessel J , Shih PA , Rao F , O'Connor DT , Taylor P
Ref : Journal of Pharmacology & Experimental Therapeutics , 338 :125 , 2011
Abstract : Cholinergic neurotransmission in the central and autonomic nervous systems regulates immediate variations in and longer-term maintenance of cardiovascular function with acetylcholinesterase (AChE) activity that is critical to temporal responsiveness. Butyrylcholinesterase (BChE), largely confined to the liver and plasma, subserves metabolic functions. AChE and BChE are found in hematopoietic cells and plasma, enabling one to correlate enzyme levels in whole blood with hereditary traits in twins. Using both twin and unrelated subjects, we found certain single nucleotide polymorphisms (SNPs) in the ACHE gene correlated with catalytic properties and general cardiovascular functions. SNP discovery from ACHE resequencing identified 19 SNPs: 7 coding SNPs (cSNPs), of which 4 are nonsynonymous, and 12 SNPs in untranslated regions, of which 3 are in a conserved sequence of an upstream intron. Both AChE and BChE activity traits in blood were heritable: AChE at 48.8 +/- 6.1% and BChE at 81.4 +/- 2.8%. Allelic and haplotype variations in the ACHE and BCHE genes were associated with changes in blood AChE and BChE activities. AChE activity was associated with BP status and SBP, whereas BChE activity was associated with features of the metabolic syndrome (especially body weight and BMI). Gene products from cDNAs with nonsynonymous cSNPs were expressed and purified. Protein expression of ACHE nonsynonymous variant D134H (SNP6) is impaired: this variant shows compromised stability and altered rates of organophosphate inhibition and oxime-assisted reactivation. A substantial fraction of the D134H instability could be reversed in the D134H/R136Q mutant. Hence, common genetic variations at ACHE and BCHE loci were associated with changes in corresponding enzymatic activities in blood.
ESTHER : Valle_2011_J.Pharmacol.Exp.Ther_338_125
PubMedSearch : Valle_2011_J.Pharmacol.Exp.Ther_338_125
PubMedID: 21493754

Title : Interaction kinetics of oximes with native, phosphylated and aged human acetylcholinesterase - Radic_2010_Chem.Biol.Interact_187_163
Author(s) : Radic Z , Kalisiak J , Fokin VV , Sharpless KB , Taylor P
Ref : Chemico-Biological Interactions , 187 :163 , 2010
Abstract : Oximes are commonly used nucleophilic reactivators of alkyl phosphorylated and alkyl methylphosphonylated acetylcholinesterase (AChE) and butyrylcholinesterase. Covalent inhibition of these enzymes by organophosphate (OP) pesticides results typically in phosphorylated enzymes, while covalent inhibition by nerve agent OPs results in methyl phosphonylated cholinesterases. In this study we determined kinetic constants for interaction of three triazole containing oximes with native human AChE, enzyme diethylphosphorylated by paraoxon, enzyme phosphonylated by VX and cyclosarin as well as enzyme aged upon phosphonylation by soman. Stopped-flow kinetics of oxime interaction was monitored using quenching of intrinsic tryptophan fluorescence of AChE as an indicator of oxime binding. Triazole oximes were efficiently synthesized using copper catalyzed cycloaddition between azide and alkyne building blocks ("Click chemistry"). Equilibrium dissociation constants determined for both native enzymes were in low micromolar range for all three oximes, while dissociation constants for phosphylated (phosphorylated and phosphonylated) enzymes were typically one to two orders of magnitude larger. Dissociation constants for interaction with aged enzymes were similar or smaller than those determined for native enzymes. Similar results were obtained with reference oximes, 2PAM and HI6. Association rate constants for formation of oxime complexes were similar for both native, phosphylated and aged enzymes. In summary our data suggest that modification of active site gorge in AChEs by phosphylation of the active serine compromises oxime binding. Dealkylation of phosphonylated enzyme, however opens space in the gorge allowing oximes to bind tighter.
ESTHER : Radic_2010_Chem.Biol.Interact_187_163
PubMedSearch : Radic_2010_Chem.Biol.Interact_187_163
PubMedID: 20412789

Title : Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation - Kucukkilinc_2010_Chem.Biol.Interact_187_238
Author(s) : Kucukkilinc TT , Cochran R , Kalisiak J , Garcia E , Valle A , Amitai G , Radic Z , Taylor P
Ref : Chemico-Biological Interactions , 187 :238 , 2010
Abstract : Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and to a lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of varying structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. In this study wild type human AChE and human AChE with residue mutations D134H, D134H_E202Q and D134H_F338A were characterized and investigated for inhibition by OPs and consequent oxime reactivation of phosphylated enzymes. The rationale for selecting these substitution positions was based on D134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs. Inhibition of D134H by paraoxon and analogues of cyclosarin was 2-8 times slower than inhibition of wild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Both inhibition and reactivation of D134H_E202Q and D134H_F338A double mutants were up to two orders of magnitude slower than the wt indicating that introduction of the active center substitutions abolished fully the effect of the peripherally located D134H. These results indicate that selected residues outside the active center influence inhibition, reactivation and catalysis rates through longer range interactions.
ESTHER : Kucukkilinc_2010_Chem.Biol.Interact_187_238
PubMedSearch : Kucukkilinc_2010_Chem.Biol.Interact_187_238
PubMedID: 20382137

Title : Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism - Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
Author(s) : Bourne Y , Radic Z , Araoz R , Talley TT , Benoit E , Servent D , Taylor P , Molgo J , Marchot P
Ref : Proc Natl Acad Sci U S A , 107 :6076 , 2010
Abstract : Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.
ESTHER : Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
PubMedSearch : Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
PubMedID: 20224036

Title : Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state - Bourne_2010_J.Am.Chem.Soc_132_18292
Author(s) : Bourne Y , Radic Z , Taylor P , Marchot P
Ref : Journal of the American Chemical Society , 132 :18292 , 2010
Abstract : The active center of acetylcholinesterase (AChE), a target site for competitive inhibitors, resides centrosymmetric to the subunit at the base of a deep, narrow gorge lined by aromatic residues. At the gorge entry, a peripheral site encompasses overlapping binding loci for noncompetitive inhibitors, which alter substrate access to the gorge. The click-chemistry inhibitor TZ2PA6 links the active center ligand, tacrine, to the peripheral site ligand, propidium, through a biorthogonal reaction of an acetylene and an azide that forms either a syn1 or an anti1 triazole. Compared with wild-type mouse AChE, a Tyr337Ala mutant displays full catalytic activity, albeit with 2-3 orders of magnitude higher affinities for the TZ2PA6 syn1 and anti1 regioisomers, reflected in low femtomolar K(d) values, diffusion-limited association, and dissociation half-times greater than 1 month and 1 week, respectively. Three structures of each of the co-crystallized syn1 and anti1 complexes of the Tyr337Ala mutant were solved at three distinct times of crystal maturation, consistent with or exceeding the half-lives of the complexes in solution, while crystalline complexes obtained from soaked Tyr337Ala crystals led to picturing "freshly formed" complexes. The structures, at 2.55-2.75 A resolution, reveal a range of unprecedented conformations of the bound regioisomers, not observed in the wild-type AChE complexes, associated with concerted positional rearrangements of side chains in the enzyme gorge. Moreover, time-dependent conformational remodeling of the crystalline complexes appears to correlate with the dissociation half-times of the solution complexes. Hence, for the tight-binding TZ2PA6 inhibitors, the initial complexes kinetically driven in solution slowly form more stable complexes governed by thermodynamic equilibrium and observable in mature crystals.
ESTHER : Bourne_2010_J.Am.Chem.Soc_132_18292
PubMedSearch : Bourne_2010_J.Am.Chem.Soc_132_18292
PubMedID: 21090615
Gene_locus related to this paper: mouse-ACHE

Title : Acetylcholinesterase: mechanisms of covalent inhibition of H447I mutant determined by computational analyses - Cheng_2008_Chem.Biol.Interact_175_196
Author(s) : Cheng YH , Cheng XL , Radic Z , McCammon JA
Ref : Chemico-Biological Interactions , 175 :196 , 2008
Abstract : The reaction mechanisms of two inhibitor TFK(+) and TFK(0) binding to H447I mutant mouse acetylcholinesterase (mAChE) have been investigated by using a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach and classical molecular dynamics (MD) simulations. TFK(+) binding to the H447I mutant may proceed with a different reaction mechanism from the wild-type. A water molecule takes over the role of His447 and participates in the bond breaking and forming as a "charge relayer". Unlike in the wild-type mAChE case, Glu334, a conserved residue from the catalytic triad, acts as a catalytic base in the reaction. The calculated energy barrier for this reaction is about 8kcal/mol. These predictions await experimental verification. In the case of the neutral ligand TFK(0), however, multiple MD simulations on the TFK(0)/H447I complex reveal that none of the water molecules can be retained in the active site as a "catalytic" water. Taken together our computational studies confirm that TFK(0) is almost inactive in the H447I mutant, and also provide detailed mechanistic insights into the experimental observations.
ESTHER : Cheng_2008_Chem.Biol.Interact_175_196
PubMedSearch : Cheng_2008_Chem.Biol.Interact_175_196
PubMedID: 18657802

Title : Atomic interactions of neonicotinoid agonists with AChBP: molecular recognition of the distinctive electronegative pharmacophore - Talley_2008_Proc.Natl.Acad.Sci.U.S.A_105_7606
Author(s) : Talley TT , Harel M , Hibbs RE , Radic Z , Tomizawa M , Casida JE , Taylor P
Ref : Proc Natl Acad Sci U S A , 105 :7606 , 2008
Abstract : Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 A in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.
ESTHER : Talley_2008_Proc.Natl.Acad.Sci.U.S.A_105_7606
PubMedSearch : Talley_2008_Proc.Natl.Acad.Sci.U.S.A_105_7606
PubMedID: 18477694

Title : The cholinesterases: analysis by pharmacogenomics in man - Valle_2008_Chem.Biol.Interact_175_343
Author(s) : Valle AM , Radic Z , Rana BK , Whitfield JB , O'Connor DT , Martin NG , Taylor P
Ref : Chemico-Biological Interactions , 175 :343 , 2008
Abstract : We have undertaken a study on variations in cholinesterase (ChE) genes in relation to cardiovascular (CV) function and the metabolic syndrome. Peripheral and central nervous system control of cardiovascular (CV) function mediated through cholinergic pathways is critical in homeostatic maintenance of blood pressure and responsiveness to stress. For acetylcholinesterase (AChE; EC our focus is to identify single nucleotide polymorphisms (SNPs) in the gene that are linked to cardiovascular function. For butyrylcholinesterase (BChE; EC we examined whether BChE activity correlated with parameters of the metabolic syndrome and cardiovascular function. ChE can be found in whole blood enabling a characterization of biochemical phenotype in addition to correlating genotype with phenotypic physiologic responses. Analysis of enzymatic activity was determined spectrophotometrically in blood samples from twin and other subject registries. Correlation analysis revealed significant relationships between enzyme activity and certain CV endpoints. Linkage analysis with data from a dizygotic (DZ) twin set showed a suggestive linkage at the BChE locus, and statistical analysis revealed a high correlation between BChE activity and variables associated with cardiovascular risk and the metabolic syndrome. Pattern of within-pair twin correlations by zygosity and the ACE model-fitting findings suggest the major source of this variation (65%) is attributable to an additive genetic component. To date 19 SNPs have been identified by the re-sequencing of AChE including four nonsynonymous coding SNPs (cSNPs).
ESTHER : Valle_2008_Chem.Biol.Interact_175_343
PubMedSearch : Valle_2008_Chem.Biol.Interact_175_343
PubMedID: 18541229

Title : Probing gorge dimensions of cholinesterases by freeze-frame click chemistry - Radic_2008_Chem.Biol.Interact_175_161
Author(s) : Radic Z , Manetsch R , Fournier D , Sharpless KB , Taylor P
Ref : Chemico-Biological Interactions , 175 :161 , 2008
Abstract : Freeze-frame click chemistry is a proven approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks and macromolecular template targets. We recently described in situ design of femtomolar reversible inhibitors of fish and mammalian acetylcholinesterases (EC; AChEs) using several different libraries of acetylene and azide building blocks. Active center gorge geometries of those AChEs are rather similar and identical triazole inhibitors were detected in situ when incubating the same building block libraries in different AChEs. Drosophila melanogaster AChE crystal structure and other insect AChE homology models differ more in their overall 3D structure than other members of the cholinesterase family. The portion of the gorge proximal to the catalytic triad and choline binding site has a approximately 50% reduction in volume, and the gorge entrance at the peripheral anionic site (PAS) is more constricted than in the fish and mammalian AChEs. In this communication we describe rationale for using purified recombinant Drosophila AChE as a template for in situ reaction of tacrine and propidium based libraries of acetylene and azide building blocks. The structures of resulting triazole inhibitors synthesized in situ are expected to differ appreciably from the fish and mammalian AChEs. While the latter AChEs exclusively promote synthesis of syn-substituted triazoles, the best Drosophila AChE triazole inhibitors were always anti-substituted. The anti-regioisomer triazoles were by about one order of magnitude better inhibitors of Drosophila than mammalian and fish AChEs. Moreover, the preferred site of acetylene+azide reaction in insect AChE and the resulting triazole ring formation shifts from near the base of the gorge to closer to its rim due to substantial differences of the gorge geometry in Drosophila AChE. Thus, in addition to synthesizing high affinity, lead inhibitors in situ, freeze-frame, click chemistry has capacity to generate species-specific AChE ligands that conform to the determinants in the gorge.
ESTHER : Radic_2008_Chem.Biol.Interact_175_161
PubMedSearch : Radic_2008_Chem.Biol.Interact_175_161
PubMedID: 18555981

Title : Acetylcholinesterase: mechanisms of covalent inhibition of wild-type and H447I mutant determined by computational analyses - Cheng_2007_J.Am.Chem.Soc_129_6562
Author(s) : Cheng Y , Cheng X , Radic Z , McCammon JA
Ref : Journal of the American Chemical Society , 129 :6562 , 2007
Abstract : The reaction mechanisms of two inhibitors TFK+ and TFK0 binding to both the wild-type and H447I mutant mouse acetylcholinesterase (mAChE) have been investigated by using a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach and classical molecular dynamics (MD) simulations. In the wild-type mAChE, the binding reactions of TFK+ and TFK0 are both spontaneous processes, which proceed through the nucleophilic addition of the Ser203-Ogamma to the carbonyl-C of TFK+ or TFK0, accompanied with a simultaneous proton transfer from Ser203 to His447. No barrier is found along the reaction paths, consistent with the experimental reaction rates approaching the diffusion-controlled limit. By contrast, TFK+ binding to the H447I mutant may proceed with a different reaction mechanism. A water molecule takes over the role of His447 and participates in the bond breaking and forming as a "charge relayer". Unlike in the wild-type mAChE case, Glu334, a conserved residue from the catalytic triad, acts as a catalytic base in the reaction. The calculated energy barrier for this reaction is about 8 kcal/mol. These predictions await experimental verification. In the case of the neutral ligand TFK0, however, multiple MD simulations on the TFK0/H447I complex reveal that none of the water molecules can be retained in the active site as a "catalytic" water. Furthermore, our alchemical free energy calculation also suggests that the binding of TFK0 to H447I is much weaker than that of TFK+. Taken together, our computational studies confirm that TFK0 is almost inactive in the H447I mutant and also provide detailed mechanistic insights into the experimental observations.
ESTHER : Cheng_2007_J.Am.Chem.Soc_129_6562
PubMedSearch : Cheng_2007_J.Am.Chem.Soc_129_6562
PubMedID: 17461584

Title : Mechanisms of organophosphate toxicity and detoxication with emphasis on studies in Croatia - Reiner_2007_Arh.Hig.Rada.Toksikol_58_329
Author(s) : Reiner E , Radic Z , Simeon-Rudolf V
Ref : Arh Hig Rada Toksikol , 58 :329 , 2007
Abstract : This review comprises studies on the mechanisms of toxicity and detoxication of organophosphorus (OP) compounds done in Croatia in different research areas. One area is the synthesis of antidotes against OP poisoning and their in vivo testing in experimental animals. In vitro studies included in this review focus on the mechanisms of reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), protection of cholinesterases from inhibition by OPs, and reactivation of phosphylated cholinesterases. The third area comprises distribution profiles of BChE and paraoxonase (PON) phenotypes in selected population groups and the detection of OPs and metabolites in humans. Finally, methods are described for the detection of OP compounds in human blood and other media by means of cholinesterase inhibition.
ESTHER : Reiner_2007_Arh.Hig.Rada.Toksikol_58_329
PubMedSearch : Reiner_2007_Arh.Hig.Rada.Toksikol_58_329
PubMedID: 17913688

Title : Application of recombinant DNA methods for production of cholinesterases as organophosphate antidotes and detectors - Taylor_2007_Arh.Hig.Rada.Toksikol_58_339
Author(s) : Taylor P , Reiner E , Kovarik Z , Radic Z
Ref : Arh Hig Rada Toksikol , 58 :339 , 2007
Abstract : To develop new avenues for synthesizing novel antidotes for organophosphate poisoning and for detection of the organophosphates, we have turned to recombinant DNA methods to synthesize cholinesterases with unusual properties. For antidotal therapy we describe mutations of the native mouse and human enzymes that allow for enhanced rates of oxime reactivation. Such enzymes, when localized in the circulation, would enable the circulating cholinesterase to become a catalytic rather than simply a stoichiometric scavenger. Hence, "oxime-assisted catalysis" provides a means for scavenging the organophosphates in the circulation thereby minimizing their tissue penetration and toxicity. Accordingly, the oxime antidote or prophylactic agent has a dual action within the circulation and at the tissue level. Second, through a novel chemistry, termed freeze-frame, click chemistry, we have used organophosphate conjugates of acetylcholinesterase as templates for the synthesis of novel nucleophilic reactivating agents. Finally, acetylcholinesterase can be modified through cysteine substitution mutagenesis and attachment of fluorophores at the substitution positions. When linked at certain locations in the molecule, the attached fluorophore is sensitive to organophosphate conjugation with acetylcholinesterase, and thus the very target of insecticide or nerve agent action becomes a detection molecule for organophosphate exposure.
ESTHER : Taylor_2007_Arh.Hig.Rada.Toksikol_58_339
PubMedSearch : Taylor_2007_Arh.Hig.Rada.Toksikol_58_339
PubMedID: 17913689

Title : Structure-guided drug design: conferring selectivity among neuronal nicotinic receptor and acetylcholine-binding protein subtypes - Taylor_2007_Biochem.Pharmacol_74(8)_1164
Author(s) : Taylor P , Talley TT , Radic Z , Hansen SB , Hibbs RE , Shi J
Ref : Biochemical Pharmacology , 74 :1164 , 2007
Abstract : Neuronal nicotinic receptors, encoded by nine genes of the alpha and three of the beta type of subunits, and whose gene products assemble in distinct permutations as pentameric molecules, constitute a fertile area for structure-guided drug design. Design strategies are augmented by a wide variety of peptide, alkaloid and terpenoid toxins from various marine and terrestrial species that interact with nicotinic receptors. Also, acetylcholine-binding proteins from mollusks, as structural surrogates of the receptor that mimic its extracellular domain, provide atomic resolution templates for analysis of structure and response. Herein, we describe a structure-guided approach to nicotinic ligand design that employs crystallography of this protein as the basic template, but also takes into consideration the dynamic properties of the receptor molecules in their biological media. We present the crystallographic structures of several complexes of various agonists and antagonists that associate with the agonist site and can competitively block the action of acetylcholine. In so far as the extracellular domain is involved, we identify additional non-competitive sites at those subunit interfaces where agonists do not preferentially bind. Ligand association at these interface sites may modulate receptor function. Ligand binding is also shown by solution-based spectroscopic and spectrometric methods to affect the dynamics of discrete domains of the receptor molecule. The surrogate receptor molecules can then be employed to design ligands selective for receptor subtype through the novel methods of freeze-frame, click chemistry that uses the very structure of the target molecule as a template for synthesis of the inhibitor.
ESTHER : Taylor_2007_Biochem.Pharmacol_74(8)_1164
PubMedSearch : Taylor_2007_Biochem.Pharmacol_74(8)_1164
PubMedID: 17826748

Title : Acetylcholinesterase: converting a vulnerable target to a template for antidotes and detection of inhibitor exposure - Taylor_2007_Toxicology_233_70
Author(s) : Taylor P , Kovarik Z , Reiner E , Radic Z
Ref : Toxicology , 233 :70 , 2007
Abstract : Applications of recombinant DNA technology, chemical synthesis on biological templates and fluorescence detection of organophosphorylation provide unexplored avenues for development of antidotes and approaches for remote detection of organophosphate nerve agents and pesticides. We discuss here how acetylcholinesterase (AChE), through appropriate mutations, becomes more susceptible to oxime reactivation. Since the reaction between organophosphate and the mutated enzyme remains rapid, regeneration of active enzyme by oxime becomes the rate-limiting step in the process to complete a catalytic cycle for generation of active enzyme. Accordingly, "Oxime-assisted Catalysis" by AChE provides a potential means for catalyzing the hydrolysis of organophosphates in plasma prior to their reaching the cellular target site. In turn, AChE, when conjugated with organophosphate, is employed as a template for 'click-chemistry, freeze-frame' synthesis of new nucleophilic reactivating agents that could potentially prove useful in AChE reactivation at the target site as well as in catalytic scavenging of organophosphates in plasma. Finally, substituted AChE molecules can be conjugated to fluorophores giving rise to shifts in emission spectra for detection of dispersed organophosphates. Since external reagents do not have to be added to detect the fluorescence change, the modified enzyme would serve as a remote sensor.
ESTHER : Taylor_2007_Toxicology_233_70
PubMedSearch : Taylor_2007_Toxicology_233_70
PubMedID: 17196318

Title : Mutation of acetylcholinesterase to enhance oxime-assisted catalytic turnover of methylphosphonates - Kovarik_2007_Toxicology_233_79
Author(s) : Kovarik Z , Radic Z , Berman HA , Taylor P
Ref : Toxicology , 233 :79 , 2007
Abstract : Selected mutagenesis of acetylcholinesterase (AChE; EC may enable one to develop more effective scavenging agents in which AChE itself, in combination with an oxime, will complete a catalytic cycle of hydrolysis of the organophosphate by rapid conjugation followed by enhanced nucleophile-mediated hydrolysis of the phosphonyl enzyme conjugate. Through enlargement of the active site gorge of mouse AChE by mutations Y337A, F295L and F297I, we studied continuous enzymatic degradation of S(P)-cycloheptyl methylphosphonyl thiocholine (S(P)-CHMPTCh) in the presence of HI-6. Continuous hydrolysis of S(P)-CHMPTCh was measured spectrophotometrically from thiocholine released during hydrolysis with DTNB as the thiol reagent. The rates of hydrolysis expressed as moles of formed thiocholine per mole of enzyme per minute were 3.3, 0.69, 0.34 and 0.15min(-1) for F295L/Y337A, Y337A, F297I/Y337A and AChE wild-type, respectively. These rates did not depend on the initial S(P)-CHMPTCh concentration range employed. However, by increasing HI-6 concentrations, the rates approached a limiting value, indicating that oxime reactivation is the rate-limiting step in S(P)-CHMPTCh hydrolysis. Our results confirm that a mixture of a mutant enzyme and an oxime might serve as an in vivo catalytic scavenger of organophosphates.
ESTHER : Kovarik_2007_Toxicology_233_79
PubMedSearch : Kovarik_2007_Toxicology_233_79
PubMedID: 17046138

Title : Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP - Kovarik_2006_Biochem.Biophys.Res.Commun_342_973
Author(s) : Kovarik Z , Ciban N , Radic Z , Simeon-Rudolf V , Taylor P
Ref : Biochemical & Biophysical Research Communications , 342 :973 , 2006
Abstract : We used mouse recombinant wild-type acetylcholinesterase (AChE; EC, butyrylcholinesterase (BChE; EC, and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.
ESTHER : Kovarik_2006_Biochem.Biophys.Res.Commun_342_973
PubMedSearch : Kovarik_2006_Biochem.Biophys.Res.Commun_342_973
PubMedID: 16598855

Title : A mutation linked with autism reveals a common mechanism of endoplasmic reticulum retention for the alpha,beta-hydrolase fold protein family - De Jaco_2006_J.Biol.Chem_281_9667
Author(s) : De Jaco A , Comoletti D , Kovarik Z , Gaietta G , Radic Z , Lockridge O , Ellisman MH , Taylor P
Ref : Journal of Biological Chemistry , 281 :9667 , 2006
Abstract : A mutation linked to autistic spectrum disorders encodes an Arg to Cys replacement in the C-terminal portion of the extracellular domain of neuroligin-3. The solvent-exposed Cys causes virtually complete retention of the protein in the endoplasmic reticulum when the protein is expressed in transfected cells. An identical Cys substitution was reported for butyrylcholinesterase through genotyping patients with post-succinylcholine apnea. Neuroligin, butyrylcholinesterase, and acetylcholinesterase are members of the alpha,beta-hydrolase fold family of proteins sharing sequence similarity and common tertiary structures. Although these proteins have distinct oligomeric assemblies and cellular dispositions, homologous Arg residues in neuroligin-3 (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcholinesterase (Arg-395) are conserved in all studied mammalian species. To examine whether an homologous Arg to Cys mutation affects related proteins similarly despite their differing capacities to oligomerize, we inserted homologous mutations in the acetylcholinesterase and butyrylcholinesterase cDNAs. Using confocal fluorescence microscopy and analysis of oligosaccharide processing, we find that the homologous Arg to Cys mutation also results in endoplasmic reticulum retention of the two cholinesterases. Small quantities of mutated acetylcholinesterase exported from the cell retain activity but show a greater K(m), a much smaller k(cat), and altered substrate inhibition. The nascent proteins associate with chaperones during processing, but the mutation presumably restricts processing through the endoplasmic reticulum and Golgi apparatus, because of local protein misfolding and inability to oligomerize. The mutation may alter the capacity of these proteins to dissociate from their chaperone prior to oligomerization and processing for export.
ESTHER : De Jaco_2006_J.Biol.Chem_281_9667
PubMedSearch : De Jaco_2006_J.Biol.Chem_281_9667
PubMedID: 16434405

Title : Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding - Bourne_2006_J.Biol.Chem_281_29256
Author(s) : Bourne Y , Radic Z , Sulzenbacher G , Kim E , Taylor P , Marchot P
Ref : Journal of Biological Chemistry , 281 :29256 , 2006
Abstract : Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N,N,N-trimethylammonio)-trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-A resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface.
ESTHER : Bourne_2006_J.Biol.Chem_281_29256
PubMedSearch : Bourne_2006_J.Biol.Chem_281_29256
PubMedID: 16837465
Gene_locus related to this paper: mouse-ACHE

Title : Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase - Bui_2006_Biophys.J_90_3280
Author(s) : Bui JM , Radic Z , Taylor P , McCammon JA
Ref : Biophysical Journal , 90 :3280 , 2006
Abstract : The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.
ESTHER : Bui_2006_Biophys.J_90_3280
PubMedSearch : Bui_2006_Biophys.J_90_3280
PubMedID: 16473897

Title : Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein - Hibbs_2006_J.Biol.Chem_281_39708
Author(s) : Hibbs RE , Radic Z , Taylor P , Johnson DA
Ref : Journal of Biological Chemistry , 281 :39708 , 2006
Abstract : Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.
ESTHER : Hibbs_2006_J.Biol.Chem_281_39708
PubMedSearch : Hibbs_2006_J.Biol.Chem_281_39708
PubMedID: 17068341

Title : In situ selection of lead compounds by click chemistry: target-guided optimization of acetylcholinesterase inhibitors - Krasinski_2005_J.Am.Chem.Soc_127_6686
Author(s) : Krasinski A , Radic Z , Manetsch R , Raushel J , Taylor P , Sharpless KB , Kolb HC
Ref : Journal of the American Chemical Society , 127 :6686 , 2005
Abstract : The target-guided, in situ click chemistry approach to lead discovery has been successfully employed for discovering acetylcholinesterase (AChE) inhibitors by incubating a selected enzyme/tacrine azide combination with a variety of acetylene reagents that were not previously known to interact with the enzyme's peripheral binding site. The triazole products, formed by the enzyme, were identified by HPLC-mass spectrometry analysis of the crude reaction mixtures. The target-guided lead discovery search was also successful when performed with reagent mixtures containing up to 10 components. From 23 acetylene reagents, the enzyme selected two phenyltetrahydroisoquinoline (PIQ) building blocks that combined with the tacrine azide within the active center gorge to form multivalent inhibitors that simultaneously associate with the active and peripheral binding sites. These new inhibitors are up to 3 times as potent as our previous phenylphenanthridinium-derived compounds, and with dissociation constants as low as 33 femtomolar, they are the most potent noncovalent AChE inhibitors known. In addition, the new compounds lack a permanent positive charge and aniline groups and possess fewer fused aromatic rings. Remarkably, despite the high binding affinity, the enzyme displayed a surprisingly low preference for one PIQ enantiomer over the other.
ESTHER : Krasinski_2005_J.Am.Chem.Soc_127_6686
PubMedSearch : Krasinski_2005_J.Am.Chem.Soc_127_6686
PubMedID: 15869290

Title : Tetrameric mouse acetylcholinesterase: continuum diffusion rate calculations by solving the steady-state Smoluchowski equation using finite element methods - Zhang_2005_Biophys.J_88_1659
Author(s) : Zhang D , Suen J , Zhang Y , Song Y , Radic Z , Taylor P , Holst MJ , Bajaj C , Baker NA , McCammon JA
Ref : Biophysical Journal , 88 :1659 , 2005
Abstract : The tetramer is the most important form for acetylcholinesterase in physiological conditions, i.e., in the neuromuscular junction and the nervous system. It is important to study the diffusion of acetylcholine to the active sites of the tetrameric enzyme to understand the overall signal transduction process in these cellular components. Crystallographic studies revealed two different forms of tetramers, suggesting a flexible tetramer model for acetylcholinesterase. Using a recently developed finite element solver for the steady-state Smoluchowski equation, we have calculated the reaction rate for three mouse acetylcholinesterase tetramers using these two crystal structures and an intermediate structure as templates. Our results show that the reaction rates differ for different individual active sites in the compact tetramer crystal structure, and the rates are similar for different individual active sites in the other crystal structure and the intermediate structure. In the limit of zero salt, the reaction rates per active site for the tetramers are the same as that for the monomer, whereas at higher ionic strength, the rates per active site for the tetramers are approximately 67%-75% of the rate for the monomer. By analyzing the effect of electrostatic forces on ACh diffusion, we find that electrostatic forces play an even more important role for the tetramers than for the monomer. This study also shows that the finite element solver is well suited for solving the diffusion problem within complicated geometries.
ESTHER : Zhang_2005_Biophys.J_88_1659
PubMedSearch : Zhang_2005_Biophys.J_88_1659
PubMedID: 15626705

Title : Molecular basis of interactions of cholinesterases with tight binding inhibitors - Radic_2005_Chem.Biol.Interact_157-158_133
Author(s) : Radic Z , Manetsch R , Krasinski A , Raushel J , Yamauchi J , Garcia C , Kolb HC , Sharpless KB , Taylor P
Ref : Chemico-Biological Interactions , 157-158 :133 , 2005
Abstract : Among the large variety of reversible inhibitors that bind to cholinesterases (ChE), only a few exhibit exquisitely strong binding reflected in low femtomolar to picomolar equilibrium dissociation constants. These tight binding inhibitors owe their high affinity to distinctive modes of interaction with the enzyme: naturally occurring snake toxins, the fasciculins, share a large 1000 angstroms2 complementary surface for its complex with acetylcholinesterases (AChE; EC; transition state analogs trifluoroacetophenones form a covalent bond with the active serine; disubstituted 1,2,3-triazole inhibitors formed in situ are selected by AChE for optimal interaction surface over the length of the active center gorge. All these inhibitors bind with higher affinity to AChEs than to the closely related butyrylcholinesterases (BuChE; EC Selectivity of individual inhibitors towards BuChE increases with increasing their molecular size. Interaction kinetics for all three classes of compounds reveal very slow rates of dissociation of the AChE-inhibitor complexes or conjugates combined with very fast association rates. The influence of conformational flexibility of the active center gorge on the affinity of inhibitor binding was demonstrated by comparing binding properties of a series of disubstituted 1,2,3-triazoles having systematically varied structures. Analysis of the linear free energy relationships of binding to both mouse and Electrophorus AChE reveals independent contributions of individual structural elements of inhibitors to their binding with the triazole ring emerging as an independently contributing pharmacophore. These tight binding inhibitor interactions reveal useful information not only on the conformational flexibility of ChEs, but also on the diversity of modes of interaction that achieve inhibition.
ESTHER : Radic_2005_Chem.Biol.Interact_157-158_133
PubMedSearch : Radic_2005_Chem.Biol.Interact_157-158_133
PubMedID: 16289416

Title : Species-related differences in the oxime-induced reactivation of organophosphate-inhibited acetylcholinesterases -
Author(s) : Luo C , Dawson M , Chambers C , Chilukuri N , Radic Z , Taylor P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :393 , 2005
PubMedID: 16429528

Title : Structural insights into conformational flexibility at the peripheral site and within the active center gorge of AChE - Bourne_2005_Chem.Biol.Interact_157-158_159
Author(s) : Bourne Y , Radic Z , Kolb HC , Sharpless KB , Taylor P , Marchot P
Ref : Chemico-Biological Interactions , 157-158 :159 , 2005
Abstract : The peripheral anionic site on acetylcholinesterase (AChE), located at the active site gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors. Yet the molecular mechanisms coupling this site to the active center at the base of the gorge to modulate catalysis remain unclear. Crystal structures of mAChE bound with decidium, propidium and gallamine unveiled new determinants contributing to ligand interactions at the peripheral site. Subsequent studies using the syn and anti regioisomers of the click-chemistry inhibitor, TZ2PA6, that link propidium and tacrine moieties via distinctively substituted triazoles, revealed the inherent flexibility and a unique conformation of the peripheral site, along with substantial binding contributions from the triazoles with the Tyr337 region within the gorge. The recently solved structures of the mAChE mutant, Tyr337Ala, complexed with the TZ2PA6 isomers now reveals distinctive and time-dependent conformations of the complexes that are consistent with the triazole contribution to the energetics of inhibitor binding manifested in the respective dissociation rates of the complexes.
ESTHER : Bourne_2005_Chem.Biol.Interact_157-158_159
PubMedSearch : Bourne_2005_Chem.Biol.Interact_157-158_159
PubMedID: 16259971

Title : Acetylcholinesterase mutants: oxime-assisted catalytic scavengers of organophosphonates -
Author(s) : Kovarik Z , Radic Z , Simeon-Rudolf V , Reiner E , Taylor P
Ref : Chemico-Biological Interactions , 157-158 :388 , 2005
PubMedID: 16429526

Title : Ligand-induced conformational changes in residues flanking the active site gorge of acetylcholinesterase -
Author(s) : Pezzementi L , Shi J , Johnson DA , Radic Z , Boyd A , Taylor P
Ref : Chemico-Biological Interactions , 157-158 :413 , 2005
PubMedID: 16429560

Title : Contemporary paradigms for cholinergic ligand design guided by biological structure - Taylor_2004_Bioorg.Med.Chem.Lett_14_1875
Author(s) : Taylor P , Hansen SB , Talley TT , Hibbs RE , Radic Z
Ref : Bioorganic & Medicinal Chemistry Lett , 14 :1875 , 2004
Abstract : The identification of the various nicotinic receptor subtypes, when coupled with the recent development of three-dimensional structures of surrogate extracellular receptor domains, offers new opportunities to design nicotinic ligands. Conformation and fluctuations in receptor structure are critical to ligand selectivity, and we present here how a flexible receptor template can be used in the development of selective ligands affecting cholinergic neurotransmission.
ESTHER : Taylor_2004_Bioorg.Med.Chem.Lett_14_1875
PubMedSearch : Taylor_2004_Bioorg.Med.Chem.Lett_14_1875
PubMedID: 15050619

Title : Poster (50) Insights into the interactions at the acetylcholinesterase peripheral anionic site. -
Author(s) : Bourne Y , Taylor P , Radic Z , Berman HA , Marchot P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004

Title : Poster (51) Novel compounds with distinctive interactions at the acetylcholinesterase peripheral anionic site -
Author(s) : Bourne Y , Taylor P , Radic Z , Lewis WG , Grynszpan F , Finn MG , Sharpless KB , Marchot P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :348 , 2004

Title : Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates - Kovarik_2004_Biochemistry_43_3222
Author(s) : Kovarik Z , Radic Z , Berman HA , Simeon-Rudolf V , Reiner E , Taylor P
Ref : Biochemistry , 43 :3222 , 2004
Abstract : Selective mutants of mouse acetylcholinesterase (AChE; EC phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.
ESTHER : Kovarik_2004_Biochemistry_43_3222
PubMedSearch : Kovarik_2004_Biochemistry_43_3222
PubMedID: 15023072

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase. -
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Cholinergic Mechanisms, CRC Press :627 , 2004

Title : Nanosecond dynamics of acetylcholinesterase near the active center gorge - Boyd_2004_J.Biol.Chem_279_26612
Author(s) : Boyd AE , Dunlop CS , Wong L , Radic Z , Taylor P , Johnson DA
Ref : Journal of Biological Chemistry , 279 :26612 , 2004
Abstract : To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible omega loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys(69)-Cys(96) omega loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.
ESTHER : Boyd_2004_J.Biol.Chem_279_26612
PubMedSearch : Boyd_2004_J.Biol.Chem_279_26612
PubMedID: 15078872

Title : Kinetics of interaction of ethopropazine enantiomers with butyrylcholinesterase and acetylcholinesterase. -
Author(s) : Reiner E , Sinko G , Radic Z , Taylor P , Simeon-Rudolf V
Ref : Cholinergic Mechanisms, CRC Press :705 , 2004

Title : Poster (89) Phosphorylation of mouse cholinesterases and mutants by ddvp and reactivation of conjugates by Hi-6 and 2-PAM -
Author(s) : Kovarik Z , Wong L , Radic Z , Taylor P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :367 , 2004

Title : Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation - Bourne_2004_Proc.Natl.Acad.Sci.U.S.A_101_1449
Author(s) : Bourne Y , Kolb HC , Radic Z , Sharpless KB , Taylor P , Marchot P
Ref : Proc Natl Acad Sci U S A , 101 :1449 , 2004
Abstract : The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1- and anti1-mouse AChE complexes at 2.45- to 2.65-A resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.
ESTHER : Bourne_2004_Proc.Natl.Acad.Sci.U.S.A_101_1449
PubMedSearch : Bourne_2004_Proc.Natl.Acad.Sci.U.S.A_101_1449
PubMedID: 14757816
Gene_locus related to this paper: mouse-ACHE

Title : Intrinsic tryptophan fluorescence of cholinesterases. -
Author(s) : Radic Z , Kim E , Taylor P
Ref : Cholinergic Mechanisms, CRC Press :171 , 2004

Title : Phosphonylation of acetylcholinesterase and the propensity for reactivation analysed by chirality and mutagenesis. -
Author(s) : Kovarik Z , Radic Z , Berman HA , Taylor P
Ref : Cholinergic Mechanisms, CRC Press :611 , 2004

Title : Poster (13) Conformational dynamics accompanying substrate and inhibitor binding to acetylcholinesterase. -
Author(s) : Taylor P , Shi J , Johnson DA , Boyd AE , Radic Z
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :327 , 2004

Title : Spectroscopic approaches to the study of acetylcholinesterase structure and function -
Author(s) : Taylor P , Shi J , Radic Z , Boyd AE , Johnson DA
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :177 , 2004

Title : Kinetics of ethopropazine binding to butyrylcholinesterase in the absence and presence of acetylthiocholine -
Author(s) : Reiner E , Sinko G , Bosak A , Simeon-Rudolf V , Radic Z , Taylor P , Stojan J , Golicnik M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :187 , 2004

Title : Monitoring cholinesterase - ligand interactions -
Author(s) : Radic Z , Kim E , Taylor P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :201 , 2004

Title : Poster (105) Analysis of acetylcholinesterase structure and dynamics by steady state and time-resolved fluorescence -
Author(s) : Shi J , Radic Z , Johnson DA , Taylor P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :375 , 2004

Title : Ligand-induced conformational changes in the omega loop of acetylcholinesterase revealed by fluorescence spectroscopy. -
Author(s) : Shi J , Radic Z , Boyd AE , Johnson DA , Taylor P
Ref : Cholinergic Mechanisms, CRC Press :695 , 2004

Title : Poster (19) Kinetics of ethopropazine binding to butyrylcholinesterase in the absence and presence of acetylthiocholine. -
Author(s) : Reiner E , Sinko G , Stuglin A , Simeon-Rudolf V , Radic Z , Taylor P , Stojan J , Golicnik M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :330 , 2004

Title : Phosphorylation of mouse cholinesterases and mutants by DDVP and reactivation of conjugates by HI-6 and 2-PAM -
Author(s) : Kovarik Z , Wong L , Radic Z , Taylor P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :219 , 2004

Title : In situ click chemistry: enzyme inhibitors made to their own specifications - Manetsch_2004_J.Am.Chem.Soc_126_12809
Author(s) : Manetsch R , Krasinski A , Radic Z , Raushel J , Taylor P , Sharpless KB , Kolb HC
Ref : Journal of the American Chemical Society , 126 :12809 , 2004
Abstract : The in situ click chemistry approach to lead discovery employs the biological target itself for assembling inhibitors from complementary building block reagents via irreversible connection chemistry. The present publication discusses the optimization of this target-guided strategy using acetylcholinesterase (AChE) as a test system. The application of liquid chromatography with mass spectroscopic detection in the selected ion mode for product identification greatly enhanced the sensitivity and reliability of this method. It enabled the testing of multicomponent mixtures, which may dramatically increase the in situ screening throughput. In addition to the previously reported in situ product syn-TZ2PA6, we discovered three new inhibitors, syn-TZ2PA5, syn-TA2PZ6, and syn-TA2PZ5, derived from tacrine and phenylphenanthridinium azides and acetylenes, in the reactions with Electrophorus electricus and mouse AChE. All in situ-generated compounds were extremely potent AChE inhibitors, because of the presence of multiple sites of interaction, which include the newly formed triazole nexus as a significant pharmacophore.
ESTHER : Manetsch_2004_J.Am.Chem.Soc_126_12809
PubMedSearch : Manetsch_2004_J.Am.Chem.Soc_126_12809
PubMedID: 15469276

Title : Poster (22) Monitoring cholinesterase-ligand interactions -
Author(s) : Radic Z , Taylor P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :332 , 2004

Title : Analysis of acetylcholinesterase structure and dynamics by steady state and time-resolved fluorescence -
Author(s) : Shi J , Radic Z , Johnson DA , Taylor P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :225 , 2004

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase - Luo_2003_Biochem.Pharmacol_66_387
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Biochemical Pharmacology , 66 :387 , 2003
Abstract : The inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) causes acute toxicity or death of the intoxicated individual. One group of these compounds, the OP nerve agents, pose an increasing threat in the world due to their possible use in the battlefield or terrorist acts. Antidotes containing oxime compounds to reactivate the inhibited enzyme are highly valued for treatment against OP poisoning. One of these reactivators, HI-6, was shown to be significantly more effective in treating soman toxicity than other oximes, such as 2-PAM, TMB4, and obidoxime. However, HI-6 was less effective in reactivating AChE inhibited by the OP pesticide, paraoxon. In this study, the mechanism for HI-6-induced reactivation of OP-AChE conjugates was investigated using mouse mutant AChEs inhibited with different OPs including organophosphate paraoxon, and several methylphosphonates. Results indicate that the HI-6 molecule may assume two different orientations in the reactivation of AChE inhibited by organophosphate and Sp methylphosphonates. These conclusions were further corroborated by reactivation studies using an analog of HI-6 in which the bispyridinium moieties are linked by a methylene bridge rather than an ether oxygen.
ESTHER : Luo_2003_Biochem.Pharmacol_66_387
PubMedSearch : Luo_2003_Biochem.Pharmacol_66_387
PubMedID: 12907237

Title : Acetylcholinesterase active centre and gorge conformations analyzed by combinatorial mutations and enantiomeric phosphonates - Kovarik_2003_Biochem.J_373_33
Author(s) : Kovarik Z , Radic Z , Berman HA , Simeon-Rudolf V , Reiner E , Taylor P
Ref : Biochemical Journal , 31 :33 , 2003
Abstract : A series of eight double and triple mutants of mouse acetylcholinesterase (AChE; EC, with substitutions corresponding to residues found largely within the butyrylcholinesterase (BChE; EC active-centre gorge, was analysed to compare steady-state kinetic constants for substrate turnover and inhibition parameters for enantiomeric methylphosphonate esters. The mutations combined substitutions in the acyl pocket (Phe(295)-->Leu and Phe(297)-->Ile) with the choline-binding site (Tyr(337)-->Ala and Phe(338)-->Ala) and with a side chain (Glu(202)--> Gln) N-terminal to the active-site serine, Ser(203). The mutations affected catalysis by increasing K (m) and decreasing k (cat), but these constants were typically affected by an order of magnitude or less, a relatively small change compared with the catalytic potential of AChE. To analyse the constraints on stereoselective phosphonylation, the mutant enzymes were reacted with a congeneric series of S (P)- and R (P)-methylphosphonates of known absolute stereochemistry. Where possible, the overall reaction rates were deconstructed into the primary constants for formation of the reversible complex and intrinsic phosphonylation. The multiple mutations greatly reduced the reaction rates of the more reactive S (P)-methylphosphonates, whereas the rates of reaction with the R (P)-methylphosphonates were markedly enhanced. With the phosphonates of larger steric bulk, the enhancement of rates for the R (P) enantiomers, coupled with the reduction of the S (P) enantiomers, was sufficient to invert markedly the enantiomeric preference. The sequence of mutations to enlarge the size of the AChE active-centre gorge, resembling in part the more spacious gorge of BChE, did not show an ordered conversion into BChE reactivity as anticipated for a rigid template. Rather, the individual aromatic residues may mutually interact to confer a distinctive stereospecificity pattern towards organophosphates.
ESTHER : Kovarik_2003_Biochem.J_373_33
PubMedSearch : Kovarik_2003_Biochem.J_373_33
PubMedID: 12665427

Title : Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases - Saxena_2003_Eur.J.Biochem_270_4447
Author(s) : Saxena A , Fedorko JM , Vinayaka CR , Medhekar R , Radic Z , Taylor P , Lockridge O , Doctor BP
Ref : European Journal of Biochemistry , 270 :4447 , 2003
Abstract : E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.
ESTHER : Saxena_2003_Eur.J.Biochem_270_4447
PubMedSearch : Saxena_2003_Eur.J.Biochem_270_4447
PubMedID: 14622273

Title : Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site - Bourne_2003_Embo.J_22_1
Author(s) : Bourne Y , Taylor P , Radic Z , Marchot P
Ref : EMBO Journal , 22 :1 , 2003
Abstract : The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.
ESTHER : Bourne_2003_Embo.J_22_1
PubMedSearch : Bourne_2003_Embo.J_22_1
PubMedID: 12505979
Gene_locus related to this paper: mouse-ACHE

Title : Inhibitors of different structure induce distinguishing conformations in the omega loop, Cys 69- Cys 96, of mouse acetylcholinesterase - Shi_2002_J.Biol.Chem_277_43301
Author(s) : Shi J , Radic Z , Taylor P
Ref : Journal of Biological Chemistry , 277 :43301 , 2002
Abstract : We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Omega loop), Cys(69)-Cys(96), of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the Omega loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the Omega loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors.
ESTHER : Shi_2002_J.Biol.Chem_277_43301
PubMedSearch : Shi_2002_J.Biol.Chem_277_43301
PubMedID: 12196517

Title : Cover picture: angew. Chem. Int. Ed. 6\/2002 - Lewis_2002_Angew.Chem.Int.Ed.Engl_41_875
Author(s) : Lewis WG , Green LG , Grynszpan F , Radic Z , Carlier PR , Taylor P , Finn MG , Sharpless KB
Ref : Angew Chem Int Ed Engl , 41 :875 , 2002
Abstract : The cover picture shows the electric eel, Electrophorus electricus, a source for commercially available acetylcholinesterase. In an experiment described by K. B. Sharpless and M. G. Finn and co-workers on pp. 1053+/-1057, a femtomolar inhibitor was assembled by the enzyme from a collection of building blocks containing azide and alkyne functional groups, shown floating in solution. The templated 1,3-dipolar cycloaddition reaction, producing the inhibitor, is represented by the flare of light at the center of the image.
ESTHER : Lewis_2002_Angew.Chem.Int.Ed.Engl_41_875
PubMedSearch : Lewis_2002_Angew.Chem.Int.Ed.Engl_41_875

Title : Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks -
Author(s) : Lewis WG , Green LG , Grynszpan F , Radic Z , Carlier PR , Taylor P , Finn MG , Sharpless KB
Ref : Angew Chem Int Ed Engl , 41 :1053 , 2002
PubMedID: 12491310

Title : Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein - Hansen_2002_J.Biol.Chem_277_41299
Author(s) : Hansen SB , Radic Z , Talley TT , Molles BE , Deerinck TJ , Tsigelny I , Taylor P
Ref : Journal of Biological Chemistry , 277 :41299 , 2002
Abstract : The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.
ESTHER : Hansen_2002_J.Biol.Chem_277_41299
PubMedSearch : Hansen_2002_J.Biol.Chem_277_41299
PubMedID: 12235129

Title : Reversibly bound and covalently attached ligands induce conformational changes in the Omega loop, Cys 69-Cys 96, of mouse acetylcholinesterase - Shi_2001_J.Biol.Chem_276_42196
Author(s) : Shi J , Boyd AE , Radic Z , Taylor P
Ref : Journal of Biological Chemistry , 276 :42196 , 2001
Abstract : We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu(76), Glu(81), Glu(84), Tyr(124), Ala(262), and His(287)) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (Omega loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the Omega loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the Omega loop accompany ligand binding.
ESTHER : Shi_2001_J.Biol.Chem_276_42196
PubMedSearch : Shi_2001_J.Biol.Chem_276_42196
PubMedID: 11517229

Title : Peripheral site ligands accelerate inhibition of acetylcholinesterase by neutral organophosphates - Radic_2001_J.Appl.Toxicol_21 Suppl 1_S13
Author(s) : Radic Z , Taylor P
Ref : J Appl Toxicol , 21 Suppl 1 :S13 , 2001
Abstract : The rates of inhibition of mouse acetylcholinesterase (AChE; EC by paraoxon, haloxon, DDVP and enantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholines were measured in the presence and absence of AChE peripheral site inhibitors: gallamine, d-tubocurarine, propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolar concentrations enhanced the rates of inhibition by neutral organophosphates, whereas inhibition rates by cationic organophosphates were decreased. When peripheral site ligand concentrations extended to millimolar concentrations the extent of the enhancement decreased, creating a well-shaped activation profile. Analysis of inhibition by DDVP revealed that peripheral site inhibitors increase the second-order reaction rates by increasing maximal rates of phosphorylation. These observations suggest that peripheral site ligands are capable of allosterically affecting the conformation of residues in the choline binding site of AChE, thus optimizing the position of the leaving group of uncharged organophosphates during the inhibition reaction.
ESTHER : Radic_2001_J.Appl.Toxicol_21 Suppl 1_S13
PubMedSearch : Radic_2001_J.Appl.Toxicol_21 Suppl 1_S13
PubMedID: 11920914

Title : Synthesis of fluorescent probes directed to the active site gorge of acetylcholinesterase - Saltmarsh_2000_Bioorg.Med.Chem.Lett_10_1523
Author(s) : Saltmarsh JR , Boyd AE , Rodriguez OP , Radic Z , Taylor P , Thompson CM
Ref : Bioorganic & Medicinal Chemistry Lett , 10 :1523 , 2000
Abstract : Six organophosphorus compounds linked to fluorophore groups were prepared in an effort to selectively modify the active site of acetylcholinesterase and deliver probes to the gorge region. Two compounds that vary by the length of a methylene (CH2) group, pyrene-SO2NH(CH2)nNHC(O)CH2CH2P(O)(OEt)(F) (where n = 2 or 3) were found to be potent, irreversible inhibitors of recombinant mouse AChE (Ki approximately 10(5) M(-1) min(-1)). Size exclusion chromatography afforded a fluorescently-labeled cholinesterase conjugate.
ESTHER : Saltmarsh_2000_Bioorg.Med.Chem.Lett_10_1523
PubMedSearch : Saltmarsh_2000_Bioorg.Med.Chem.Lett_10_1523
PubMedID: 10915041

Title : Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase - Milatovic_2000_Free.Radic.Biol.Med_28_597
Author(s) : Milatovic D , Radic Z , Zivin M , Dettbarn W
Ref : Free Radic Biol Med , 28 :597 , 2000
Abstract : N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.
ESTHER : Milatovic_2000_Free.Radic.Biol.Med_28_597
PubMedSearch : Milatovic_2000_Free.Radic.Biol.Med_28_597
PubMedID: 10719241

Title : Mechanism of oxime reactivation of acetylcholinesterase analyzed by chirality and mutagenesis - Wong_2000_Biochemistry_39_5750
Author(s) : Wong L , Radic Z , Bruggemann RJ , Hosea NA , Berman HA , Taylor P
Ref : Biochemistry , 39 :5750 , 2000
Abstract : Organophosphates inactivate acetylcholinesterase by reacting covalently with the active center serine. We have examined the reactivation of a series of resolved enantiomeric methylphosphonate conjugates of acetylcholinesterase by two oximes, 2-pralidoxime (2-PAM) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium) (HI-6). The S(p) enantiomers of the methylphosphonate esters are far more reactive in forming the conjugate with the enzyme, and we find that rates of oxime reactivation also show an S(p) versus R(p) preference, suggesting that a similar orientation of the phosphonyl oxygen toward the oxyanion hole is required for both efficient inactivation and reactivation. A comparison of reactivation rates of (S(p))- and (R(p))-cycloheptyl, 3,3-dimethylbutyl, and isopropyl methylphosphonyl conjugates shows that steric hindrance by the alkoxy group precludes facile access of the oxime to the tetrahedral phosphorus. To facilitate access, we substituted smaller side chains in the acyl pocket of the active center and find that the Phe295Leu substitution enhances the HI-6-elicited reactivation rates of the S(p) conjugates up to 14-fold, whereas the Phe297Ile substitution preferentially enhances 2-PAM reactivation by as much as 125-fold. The fractional enhancement of reactivation achieved by these mutations of the acyl pocket is greatest for the conjugated phosphonates of the largest steric bulk. By contrast, little enhancement of the reactivation rate is seen with these mutants for the R(p) conjugates, where limitations on oxime access to the phosphonate and suboptimal positioning of the phosphonyl oxygen in the oxyanion hole may both slow reactivation. These findings suggest that impaction of the conjugated organophosphate within the constraints of the active center gorge is a major factor in influencing oxime access and reactivation rates. Moreover, the individual oximes differ in attacking orientation, leading to the presumed pentavalent transition state. Hence, their efficacies as reactivating agents depend on the steric bulk of the intervening groups surrounding the tetrahedral phosphorus.
ESTHER : Wong_2000_Biochemistry_39_5750
PubMedSearch : Wong_2000_Biochemistry_39_5750
PubMedID: 10801325

Title : Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4'-bipyridine and by a coumarin derivative - Simeon-Rudolf_1999_Chem.Biol.Interact_119-120_119
Author(s) : Simeon-Rudolf V , Kovarik Z , Radic Z , Reiner E
Ref : Chemico-Biological Interactions , 119-120 :119 , 1999
Abstract : Inhibition of recombinant mouse wild type AChE (EC and BChE (EC, and AChE peripheral site-directed mutants and human serum BChE variants by 4,4'-bipyridine (4,4'-BP) and the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) was studied. The enzyme activity was measured with acetylthiocholine as substrate. Enzyme-inhibitor dissociation constants for the catalytic and peripheral sites were evaluated from the apparent dissociation constants as a function of the substrate concentration. Inhibition by 4,4'-BP of AChE, BChE and the AChE mutant Y72N/Y124Q/W286A, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than for the catalytic site. CHMC displayed binding only to the catalytic site of Y72N/Y124Q/W286A mutant and only to the peripheral site of w.t. AChE and the human usual BChE. Modelling of the 4,4'-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic studies.
ESTHER : Simeon-Rudolf_1999_Chem.Biol.Interact_119-120_119
PubMedSearch : Simeon-Rudolf_1999_Chem.Biol.Interact_119-120_119
PubMedID: 10421445

Title : Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline - Kovarik_1999_Biochim.Biophys.Acta_1433_261
Author(s) : Kovarik Z , Radic Z , Grgas B , Skrinjaric-Spoljar M , Reiner E , Simeon-Rudolf V
Ref : Biochimica & Biophysica Acta , 1433 :261 , 1999
Abstract : In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC and butyrylcholinesterase (BChE; EC with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.
ESTHER : Kovarik_1999_Biochim.Biophys.Acta_1433_261
PubMedSearch : Kovarik_1999_Biochim.Biophys.Acta_1433_261
PubMedID: 10446376

Title : The influence of peripheral site ligands on the reaction of symmetric and chiral organophosphates with wildtype and mutant acetylcholinesterases - Radic_1999_Chem.Biol.Interact_119-120_111
Author(s) : Radic Z , Taylor P
Ref : Chemico-Biological Interactions , 119-120 :111 , 1999
Abstract : The rates of inhibition of mouse acetylcholinesterase (AChE) (EC by paraoxon, haloxon, DDVP, and enantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholines were measured in the presence and absence of AChE peripheral site inhibitors: gallamine, D-tubocurarine, propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolar concentrations enhanced the rates of inhibition by neutral organophosphorus compounds (OPs) while inhibition rates by cationic OPs were slowed down. When peripheral site ligand concentrations extended to millimolar, the extent of the enhancement decreased creating a bell shaped activation profile. Analysis of inhibition by DDVP and haloxon revealed that peripheral site inhibitors increased the second order reaction rates by increasing maximal rates of phosphylation.
ESTHER : Radic_1999_Chem.Biol.Interact_119-120_111
PubMedSearch : Radic_1999_Chem.Biol.Interact_119-120_111
PubMedID: 10421444

Title : Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime - Luo_1999_Chem.Biol.Interact_119-120_129
Author(s) : Luo C , Saxena A , Ashani Y , Leader H , Radic Z , Taylor P , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :129 , 1999
Abstract : We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LuH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LuH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LuH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LuH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LuH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.
ESTHER : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedSearch : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedID: 10421446

Title : Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity - Taylor_1999_Chem.Biol.Interact_119-120_3
Author(s) : Taylor P , Wong L , Radic Z , Tsigelny I , Bruggemann R , Hosea NA , Berman HA
Ref : Chemico-Biological Interactions , 119-120 :3 , 1999
Abstract : We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.
ESTHER : Taylor_1999_Chem.Biol.Interact_119-120_3
PubMedSearch : Taylor_1999_Chem.Biol.Interact_119-120_3
PubMedID: 10421434

Title : Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site - Sentjurc_1999_Biochim.Biophys.Acta_1430_349
Author(s) : Sentjurc M , Pecar S , Stojan J , Marchot P , Radic Z , Grubic Z
Ref : Biochimica & Biophysica Acta , 1430 :349 , 1999
Abstract : Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.
ESTHER : Sentjurc_1999_Biochim.Biophys.Acta_1430_349
PubMedSearch : Sentjurc_1999_Biochim.Biophys.Acta_1430_349
PubMedID: 10082962

Title : Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium - Luo_1999_Biochemistry_38_9937
Author(s) : Luo C , Saxena A , Smith M , Garcia GE , Radic Z , Taylor P , Doctor BP
Ref : Biochemistry , 38 :9937 , 1999
Abstract : Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.
ESTHER : Luo_1999_Biochemistry_38_9937
PubMedSearch : Luo_1999_Biochemistry_38_9937
PubMedID: 10433700

Title : Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge - Tara_1998_Biopolymers_46_465
Author(s) : Tara S , Elcock AH , Kirchhoff PD , Briggs JM , Radic Z , Taylor P , McCammon JA
Ref : Biopolymers , 46 :465 , 1998
Abstract : It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.
ESTHER : Tara_1998_Biopolymers_46_465
PubMedSearch : Tara_1998_Biopolymers_46_465
PubMedID: 9838872

Title : Oxime Reactivation of Acetylcholinesterase Inhibited by Enantiomeric Organophosphates -
Author(s) : Bruggemann RJ , Radic Z , Tsigelny I , Taylor P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :377 , 1998

Title : Inhibition of Acetylcholinesterase (ACHE\; E.C. and Butyrylcholinesterase (BCHE\; E.C. by Terbutaline -
Author(s) : Kovarik Z , Radic Z , Skrinjaric-Spoljar M , Reiner E , Simeon-Rudolf V
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :243 , 1998

Title : Isomalathion Stereoisomers -
Author(s) : Talley TT , Jianmongkol S , Richardson RJ , Radic Z , Thompson CM
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :531 , 1998

Title : The Effect of Peripheral Site Ligands on the Reaction Kinetics of Phosphyl and Carboxyl Esters with Acetylcholinesterase -
Author(s) : Radic Z , Taylor P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :211 , 1998

Title : Reactivation of Enantiomeric Organophosphonyl Conjugates of Acetylcholinesterase Mutants, F295L and F297I by Mono- and Bis-Quarternary Oximes -
Author(s) : Wong L , Radic Z , Hosea NA , Berman HA , Taylor P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :250 , 1998

Title : Expression and activity of mutants of fasciculin, a peptidic acetylcholinesterase inhibitor from mamba venom - Marchot_1997_J.Biol.Chem_272_3502
Author(s) : Marchot P , Prowse CN , Kanter J , Camp S , Ackermann EJ , Radic Z , Bougis PE , Taylor P
Ref : Journal of Biological Chemistry , 272 :3502 , 1997
Abstract : Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, DeltaPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.
ESTHER : Marchot_1997_J.Biol.Chem_272_3502
PubMedSearch : Marchot_1997_J.Biol.Chem_272_3502
PubMedID: 9013597

Title : Electrostatic influence on the kinetics of ligand binding to acetylcholinesterase. Distinctions between active center ligands and fasciculin - Radic_1997_J.Biol.Chem_272_23265
Author(s) : Radic Z , Kirchhoff PD , Quinn DM , McCammon JA , Taylor P
Ref : Journal of Biological Chemistry , 272 :23265 , 1997
Abstract : To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4.
ESTHER : Radic_1997_J.Biol.Chem_272_23265
PubMedSearch : Radic_1997_J.Biol.Chem_272_23265
PubMedID: 9287336

Title : Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning - Saxena_1997_Biochem.Pharmacol_54_269
Author(s) : Saxena A , Maxwell DM , Quinn DM , Radic Z , Taylor P , Doctor BP
Ref : Biochemical Pharmacology , 54 :269 , 1997
Abstract : It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.
ESTHER : Saxena_1997_Biochem.Pharmacol_54_269
PubMedSearch : Saxena_1997_Biochem.Pharmacol_54_269
PubMedID: 9271331

Title : Determining ligand orientation and transphosphonylation mechanisms on acetylcholinesterase by Rp, Sp enantiomer selectivity and site-specific mutagenesis - Taylor_1997_Enantiomer_2_249
Author(s) : Taylor P , Hosea NA , Tsigelny I , Radic Z , Berman HA
Ref : Enantiomer , 2 :249 , 1997
Abstract : Acetylcholinesterase, an enzyme of the serine hydrolase family, catalyzes the rapid hydrolysis of certain carboxyl esters. Other acyl esters efficiently transacylate the enzyme with a subsequent, slow deacylation step. Of these, the phosphoryl and phosphonyl esters are perhaps of greatest mechanistic interest since individual enantiomers of known absolute stereochemistry can be isolated and their interactions with the dissymmetric enzyme active site examined. We describe here studies of a series of enantiomeric Rp- and Sp-alkylphosphonates interacting with mouse acetylcholinesterase. Since the acetylcholinesterase is generated by recombinant DNA methods, mutant enzymes can be made with specific replacements of individual amino acid side chains. Individual amino acid replacements in the acyl pocket, the choline subsite and at the active center gorge entry have been generated, and the reaction kinetics of the mutant enzymes analyzed. These studies have shown that substitution of aliphatic amino acids for phenylalanines 295 and 297 in the acyl pocket diminishes, and in some cases, actually inverts chiral preferences. The combined structure-activity approach, where both ligand and enzyme are modified systematically, has enabled us to show that the restricted dimensions of the acyl pocket in the active center dictate enantiomeric selectivity. Moreover, the reactions of compounds of known absolute stereochemistry show three requirements for efficient transphosphonylation: (a) apposition of the phosphate with the gamma-oxygen on Ser 203 to form a pentavalent, presumed trigonal bipyramidal intermediate, (b) polarization of the phosphonyl oxygen bond by its positioning in the oxyanion hole, and (c) positioning the leaving group towards the gorge exit.
ESTHER : Taylor_1997_Enantiomer_2_249
PubMedSearch : Taylor_1997_Enantiomer_2_249
PubMedID: 9676269

Title : Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates - Hosea_1996_Biochemistry_35_10995
Author(s) : Hosea NA , Radic Z , Tsigelny I , Berman HA , Quinn DM , Taylor P
Ref : Biochemistry , 35 :10995 , 1996
Abstract : Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.
ESTHER : Hosea_1996_Biochemistry_35_10995
PubMedSearch : Hosea_1996_Biochemistry_35_10995
PubMedID: 8718893

Title : Ligand Specificity and Gene Expression in the Cholinergic Synapse -
Author(s) : Taylor P , Radic Z , Kreienkamp HJ , Luo ZD , Pickering NA , Camp S
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :15 , 1995

Title : Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes - Ashani_1995_J.Biol.Chem_270_6370
Author(s) : Ashani Y , Radic Z , Tsigelny I , Vellom DC , Pickering NA , Quinn DM , Doctor BP , Taylor P
Ref : Journal of Biological Chemistry , 270 :6370 , 1995
Abstract : Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.
ESTHER : Ashani_1995_J.Biol.Chem_270_6370
PubMedSearch : Ashani_1995_J.Biol.Chem_270_6370
PubMedID: 7890775

Title : Amino Acid Residues in Acetylcholinesterase which Influence Fasciculin Inhibition -
Author(s) : Radic Z , Quinn DM , Vellom DC , Camp S , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :183 , 1995

Title : Structural Determinants of Fasciculin Specificity for Acetylcholinesterase -
Author(s) : Marchot P , Camp S , Radic Z , Bougis PE , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :197 , 1995

Title : Amino Acid Residues that Control Mono- and Bisquaternary Oxime-Induced Reactivation of O-Ethyl Methylphosphonylated Cholinesterases -
Author(s) : Ashani Y , Radic Z , Tsigelny I , Vellom DC , Pickering NA , Quinn DM , Doctor BP , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :133 , 1995

Title : Allosteric control of acetylcholinesterase catalysis by fasciculin - Radic_1995_J.Biol.Chem_270_20391
Author(s) : Radic Z , Quinn DM , Vellom DC , Camp S , Taylor P
Ref : Journal of Biological Chemistry , 270 :20391 , 1995
Abstract : The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp86 is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp86 substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp86, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp86 which lies at the gorge base.
ESTHER : Radic_1995_J.Biol.Chem_270_20391
PubMedSearch : Radic_1995_J.Biol.Chem_270_20391
PubMedID: 7657613

Title : London Dispersion Interactions in Molecular Recognition by Acetylcholinesterase -
Author(s) : Quinn DM , Nair HK , Seravalli J , Lee K , Arbuckle T , Radic Z , Vellom DC , Pickering NA , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :228 , 1995

Title : The Function of Electrostatics in Acetylcholinesterase Catalysis -
Author(s) : Quinn DM , Seravalli J , Nair HK , Medhekar R , Husseini B , Radic Z , Vellom DC , Pickering NA , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :203 , 1995

Title : Theoretical analysis of the structure of the peptide fasciculin and its docking to acetylcholinesterase - van den Born_1995_Prot.Sci_4_703
Author(s) : van den Born HK , Radic Z , Marchot P , Taylor P , Tsigelny I
Ref : Protein Science , 4 :703 , 1995
Abstract : The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound fasciculin.
ESTHER : van den Born_1995_Prot.Sci_4_703
PubMedSearch : van den Born_1995_Prot.Sci_4_703
PubMedID: 7613468

Title : Structural bases for the specificity of cholinesterase catalysis and inhibition. - Taylor_1995_Toxicol.Lett_83_453
Author(s) : Taylor P , Radic Z , Hosea NA , Camp S , Marchot P , Berman HA
Ref : Toxicology Letters , 83 :453 , 1995
Abstract : The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.
ESTHER : Taylor_1995_Toxicol.Lett_83_453
PubMedSearch : Taylor_1995_Toxicol.Lett_83_453
PubMedID: 8597093

Title : Site of fasciculin interaction with acetylcholinesterase - Radic_1994_J.Biol.Chem_269_11233
Author(s) : Radic Z , Duran R , Vellom DC , Li Y , Cervenansky C , Taylor P
Ref : Journal of Biological Chemistry , 269 :11233 , 1994
Abstract : Fasciculin, a 6750-Da peptide from the venom of Dendroaspis, is known to inhibit reversibly mammalian and fish acetylcholinesterases at picomolar concentrations, but is a relatively weak inhibitor of avian, reptile, and insect acetylcholinesterases and mammalian butyryl-cholinesterases. An examination of fasciculin association with several mutant forms of recombinant DNA-derived acetylcholinesterase from mouse shows that it interacts with a cluster of residues near the rim of the gorge on the enzyme. The aromatic residues, Trp286, Tyr72, and Tyr124, have the most marked influence on fasciculin binding, whereas Asp74, a charged residue in the vicinity of the binding site that affects the binding of low molecular weight inhibitors, has little influence on fasciculin binding. The 3 aromatic residues are unique to the susceptible acetylcholinesterases and, along with Asp74, constitute part of the peripheral anionic site. Fasciculin falls in the family of three-loop toxins that include the receptor blocking alpha-toxins and cardiotoxins. From this basic structural motif, a binding site has evolved on fasciculin to be highly specific for the peripheral site on acetylcholinesterase. Acetylthiocholine affects rates of fasciculin binding at concentrations causing substrate inhibition. In the case of the mutant cholinesterases where rates of fasciculin dissociation are more rapid, steady state kinetic parameters also show acetylthiocholine-fasciculin competition to be consistent with occupation at a peripheral or substrate inhibition site rather than the active center.
ESTHER : Radic_1994_J.Biol.Chem_269_11233
PubMedSearch : Radic_1994_J.Biol.Chem_269_11233
PubMedID: 8157652

Title : Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase - Bucht_1994_Biochim.Biophys.Acta_1209_265
Author(s) : Bucht G , Haggstrom B , Radic Z , Osterman A , Hjalmarsson K
Ref : Biochimica & Biophysica Acta , 1209 :265 , 1994
Abstract : The three-dimensional crystal structure of the glycosyl phosphatidylinositol (GPI)-modified form of Torpedo acetylcholinesterase reveals the participation of Arg-44 and Glu-92 in a salt bridge and a hydrogen bond between Asp-93 and Tyr-96. To investigate the biological significance of these interactions, we have made amino acid replacements in this form of AChE: R44E, R44K, E92Q, E92L, D93N, and D93V. None of the introduced mutations affected the production of the acetylcholinesterase polypeptide significantly. However, the mutations introduced at position 92, as well as the D93V and R44E mutations, resulted in a total loss of surface located, active acetylcholinesterase. Replacement of Asp-93 with Asn resulted in a reduced amount of active enzyme. This mutant enzyme was indistinguishable from the wild-type enzyme regarding catalytic constants, but was more sensitive to thermal inactivation. The results show that the salt bridge and hydrogen bond involving residues Arg-44, Glu-92, and Asp-93 have important structural roles and are needed for correct folding, required for transport to the cell surface of TcAChE. The GPI-modified form of acetylcholinesterase is a disulfide bonded dimer. Cys-537 is shown to be required for the formation of the intersubunit disulfide bond in the dimer. Replacement with Ser resulted in the production of an enzyme, that migrates as a monomer upon non-reducing SDS-PAGE and has a lower stability compared to the dimeric wild-type enzyme.
ESTHER : Bucht_1994_Biochim.Biophys.Acta_1209_265
PubMedSearch : Bucht_1994_Biochim.Biophys.Acta_1209_265
PubMedID: 7811701

Title : Expression and ligand specificity of acetylcholinesterase and the nicotinic receptor: a tale of two cholinergic sites - Taylor_1994_Biochem.Soc.Trans_22_740
Author(s) : Taylor P , Radic Z , Kreienkamp HJ , Maeda RK , Luo ZD , Fuentes ME , Vellom DC , Pickering NA
Ref : Biochemical Society Transactions , 22 :740 , 1994
Abstract : The functional design of the nAChR and AChE rather than their recognition capacities requires divergence in structure of the two binding sites. The receptor requires co-operativity to link ligand occupation to the response, rapid conformational transitions of activation, and slower transitions of desensitization. Hence, its binding sites have evolved at subunit interfaces. By contrast, AChE functions with a large kcat and a comparatively large Km. To do so, it must force acetylcholine through a low-energy transition site that features tetrahedral rather than the ground-state, trigonal conformation around the carbonyl carbon. This requires a high affinity (KD approximately 10(-17) M) for the enzyme complex of the transient transition state. Interestingly, the three-finger peptide toxins (alpha-bungarotoxin and fasciculin), though closely homologous, use different interaction sites on the receptor (the agonist recognition site) and AChE (a peripheral site). Finally, although the two proteins show co-ordinated expression during muscle differentiation, the receptor relies primarily on transcriptional control while AChE expression is post-transcriptional, being controlled by mRNA stability.
ESTHER : Taylor_1994_Biochem.Soc.Trans_22_740
PubMedSearch : Taylor_1994_Biochem.Soc.Trans_22_740
PubMedID: 7821676

Title : Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases - Saxena_1994_Protein.Sci_3_1770
Author(s) : Saxena A , Qian N , Kovach IM , Kozikowski AP , Pang YP , Vellom DC , Radic Z , Quinn DM , Taylor P , Doctor BP
Ref : Protein Science , 3 :1770 , 1994
Abstract : Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.
ESTHER : Saxena_1994_Protein.Sci_3_1770
PubMedSearch : Saxena_1994_Protein.Sci_3_1770
PubMedID: 7849595

Title : The cholinesterases: from genes to proteins - Taylor_1994_Annu.Rev.Pharmacol.Toxicol_34_281
Author(s) : Taylor P , Radic Z
Ref : Annual Review of Pharmacology & Toxicology , 34 :281 , 1994
Abstract :
ESTHER : Taylor_1994_Annu.Rev.Pharmacol.Toxicol_34_281
PubMedSearch : Taylor_1994_Annu.Rev.Pharmacol.Toxicol_34_281
PubMedID: 8042853

Title : Differentiation of esterases reacting with organophosphorus compounds - Reiner_1993_Chem.Biol.Interact_87_77
Author(s) : Reiner E , Pavkovic E , Radic Z , Simeon-Rudolf V
Ref : Chemico-Biological Interactions , 87 :77 , 1993
Abstract : The hydrolysis of paraoxon (POX), phenylacetate (PA) and beta-naphthylacetate (BNA) was studied in human serum. Based upon correlations between enzyme activities, upon reversible inhibition by EDTA and upon progressive inhibition by iso-OMPA, tabun, eserine and bis-4 nitrophenylphosphate, the following conclusions were drawn about the number and specificity of enzymes involved in the hydrolysis. Two paraxonases hydrolyse paraoxon: one sensitive and the other insensitive to EDTA. The EDTA-sensitive paraoxonase also hydrolysed BNA. The EDTA-insensitive hydrolysis of BNA and PA was attributed to a serine esterase. The EDTA-sensitive hydrolysis of PA is probably due to more than one enzyme, which might be an arylesterase and a carboxylesterase.
ESTHER : Reiner_1993_Chem.Biol.Interact_87_77
PubMedSearch : Reiner_1993_Chem.Biol.Interact_87_77
PubMedID: 8393750

Title : Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity - Vellom_1993_Biochemistry_32_12
Author(s) : Vellom DC , Radic Z , Li Y , Pickering NA , Camp S , Taylor P
Ref : Biochemistry , 32 :12 , 1993
Abstract : Acetyl- and butyrylcholinesterase have 51-54% sequence identity in mammalian species; they exhibit distinct substrate and inhibitor specificities. The crystal structure of acetylcholinesterase enables one to predict folding of related esterases as well as assign residues responsible for differences in substrate specificity. These predictions were tested by expression of esterase chimeras and site-specific mutants using mouse acetylcholinesterase as a template. Chimeras of acetylcholinesterase in which the amino-terminal 174 and the carboxyl-terminal 88 amino acids have been converted to the butyrylcholinesterase sequences still exhibit acetyl-like substrate specificity. Four nonconserved amino acids which are within the central sequence and appear to surround the acyl pocket, F295, R296, F297, and V300, have been mutated alone and in combination to the corresponding residues found in butyrylcholinesterase, L286, S287, I288, and G291. The V300 and R296 mutants slightly enhance butyrylthiocholine hydrolysis while the F295 and F297 mutants, alone and in combination, confer butyrylcholinesterase character by enhancing activity to butyrylthiocholine, and diminishing activity to acetylthiocholine. The F297 mutation eliminates substrate inhibition. F295 and F297 may form a clamp around the acetoxy methyl group. They have distinctive roles in affecting catalysis of the two acylcholines and precisely control acyl ester specificity. Comparison of the susceptibilities of the chimeras and site-specific mutants to cholinesterase-specific inhibitors isoOMPA, ethopropazine, and BW284c51 suggests that inhibitor selectivity for isoOMPA is attributable to residues limiting the size of the acyl pocket, while residues in the amino-terminal domain presumably near the lip of the gorge affect BW284c51 selectivity.
ESTHER : Vellom_1993_Biochemistry_32_12
PubMedSearch : Vellom_1993_Biochemistry_32_12
PubMedID: 8418833

Title : Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors - Radic_1993_Biochemistry_32_12074
Author(s) : Radic Z , Pickering NA , Vellom DC , Camp S , Taylor P
Ref : Biochemistry , 32 :12074 , 1993
Abstract : By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.
ESTHER : Radic_1993_Biochemistry_32_12074
PubMedSearch : Radic_1993_Biochemistry_32_12074
PubMedID: 8218285

Title : Structure and regulation of expression of the acetylcholinesterase gene - Taylor_1993_Chem.Biol.Interact_87_199
Author(s) : Taylor P , Li Y , Camp S , Rachinsky TL , Ekstrom TJ , Getman DK , Fuentes ME , Vellom DC , Radic Z
Ref : Chemico-Biological Interactions , 87 :199 , 1993
Abstract : Acetylcholinesterase, an enzyme essential for the termination of the action of acetylcholine, is encoded by a single gene. Alternative mRNA processing gives rise to the expression of enzyme forms with three distinct carboxyl-termini. These structural differences govern the cellular disposition of the expressed enzyme but do not influence catalytic activity. Alternative polyadenylation signals give rise to distinct 3' non-coding regions which are likely to affect mRNA stability. Alternative splicing also occurs at the 5' end of the gene where two promoter regions can be identified. Hence, regulation of expression of the gene occurs at 3 levels, transcriptional through alternative promoters, translational by affecting mRNA stability and processing of distinct mRNAs and post-translationally by giving rise to distinct peptide chains which are processed differently. Recombinant DNA studies have also been extended to modifying protein structure through site-specific mutagenesis and studying the function of the mutant enzymes.
ESTHER : Taylor_1993_Chem.Biol.Interact_87_199
PubMedSearch : Taylor_1993_Chem.Biol.Interact_87_199
PubMedID: 8343976

Title : The role of glutamate-199 in the aging of cholinesterase - Saxena_1993_Biochem.Biophys.Res.Commun_197_343
Author(s) : Saxena A , Doctor BP , Maxwell DM , Lenz DE , Radic Z , Taylor P
Ref : Biochemical & Biophysical Research Communications , 197 :343 , 1993
Abstract : Aging of organophosphate-conjugated acetylcholinesterase results from the loss of an alkoxy group with concomitant stabilization of the conjugate to spontaneous or nucleophile-induced deacylation. We have examined the kinetics of aging in a pinacolylmethylphosphonofluoridate (soman)-inhibited mutant enzyme in which the glutamate (E199) located at the amino-terminal to the active-site serine (S200) was converted to glutamine (Q). For wild type enzyme, the soman-acetylcholinesterase conjugate aged immediately, giving rise to a form of enzyme resistant to reactivation by oximes. In contrast, the E199Q mutant enzyme was largely resistant to aging and could be reactivated by oximes. Since the pH dependence for aging was not altered appreciably, the primary influence of the loss of charge appears to be on the intrinsic rate of aging. The negative charge on E199 likely imparts an inductive effect on the conjugated organophosphate to facilitate removal of the alkoxy group.
ESTHER : Saxena_1993_Biochem.Biophys.Res.Commun_197_343
PubMedSearch : Saxena_1993_Biochem.Biophys.Res.Commun_197_343
PubMedID: 7902714

Title : Expression of recombinant acetylcholinesterase in a baculovirus system: kinetic properties of glutamate 199 mutants - Radic_1992_Biochemistry_31_9760
Author(s) : Radic Z , Gibney G , Kawamoto S , MacPhee-Quigley K , Bongiorno C , Taylor P
Ref : Biochemistry , 31 :9760 , 1992
Abstract : The glycophospholipid-linked, amphiphilic form of acetylcholinesterase (AChE) from Torpedo californica and the hydrophilic form from mouse were overexpressed in Sf9 insect cells using the baculovirus expression system. Recombinant baculovirus, constructed by inserting AChE cDNA's into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedron promoter, yielded recombinant enzyme varying between 0.5 and 3.8 mg/L. The recombinant enzyme was glycosylated although it migrated slightly more rapidly in SDS gel electrophoresis than enzyme purified from the electric organ of Torpedo. Kinetic properties of the recombinant DNA- and tissue-derived enzymes are identical. The detailed catalytic properties and susceptibility to inhibitors were examined for two enzyme mutations of the glutamate residue N-terminal to the active site serine. The Glu199 to Gln mutation shifted both the Km and Kss to higher substrate concentrations and resulted in a kcat of 28% of the wild type. Mutation of Glu199 to Asp also yielded a reduction in kcat but with no change in Km. Substrate inhibition normally apparent in wild-type AChE was eliminated with the Asp mutation, suggesting that substrate catalysis and substrate inhibition are not directly linked. Both mutations decreased the affinity of reversible inhibitors and reduced the rates of phosphorylation and carbamoylation; these changes were more striking with the Gln199 mutation. Decarbamoylation rates were unaffected by these mutations. Glu199 is the charged residue found deep within the active center gorge close to the site of acetylcholine binding, and our findings indicate it influences, but is not essential for, efficient catalysis.
ESTHER : Radic_1992_Biochemistry_31_9760
PubMedSearch : Radic_1992_Biochemistry_31_9760
PubMedID: 1356436

Title : Dialkylphosphorus metabolites in the urine and activities of esterases in the serum as biochemical indices for human absorption of organophosphorus pesticides - Drevenkar_1991_Arch.Environ.Contam.Toxicol_20_417
Author(s) : Drevenkar V , Radic Z , Vasilic Z , Reiner E
Ref : Archives of Environmental Contamination & Toxicology , 20 :417 , 1991
Abstract : Ninety-seven agricultural workers were monitored for absorption of the organophosphorus pesticides methidathion, vamidothion, and azinphos-methyl, which were sprayed in an orchard during two seasons. Low levels of only one dialkylphosphorus metabolite (dimethyl phosphorothioate) were found in only eight workers in pre-exposure urine samples. More than one dialkylphosphorus metabolite was detected in almost all exposed individuals in after-exposure urine samples. The highest concentrations were measured after exposure to azinphos-methyl; the median concentrations of dimethyl phosphorodithioate and dimethyl phosphorothioate were 0.92 and 0.78 nmol/mg creatinine with a concentration range up to 14.3 and 53.7, respectively. Three diethylphosphorus metabolites were also detected in some samples, but at lower concentrations. Cholinesterase activities were decreased (31-48%) in the serum of 12 workers; four of those workers had no dialkylphosphorus metabolites in the urine. Paraoxonase and arylesterase activities in the serum were unaffected by the absorption of pesticides, and there was no correlation between the activities of these esterases and the metabolite concentrations in the urine. This study confirmed that dialkylphosphorus metabolites in the urine are a more sensitive index of absorption than cholinesterase inhibition in the serum but lack of correlation between cholinesterase inhibition and metabolite concentration indicates that both parameters should be monitored.
ESTHER : Drevenkar_1991_Arch.Environ.Contam.Toxicol_20_417
PubMedSearch : Drevenkar_1991_Arch.Environ.Contam.Toxicol_20_417
PubMedID: 1650168

Title : Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives - Radic_1991_Mol.Pharmacol_39_98
Author(s) : Radic Z , Reiner E , Taylor P
Ref : Molecular Pharmacology , 39 :98 , 1991
Abstract : Propidium has been demonstrated in previous studies to be a selective ligand for the peripheral anionic site on acetylcholinesterase (EC Its association with this site can be advantageously monitored by direct fluorescent titration. We have measured the ability of acetylcholine, acetylthiocholine, haloxon [di-(2-chloroethyl)3-chloro-4-methylcoumarin-7-ylphosphate] , and a coumarin derivative (3-chloro-7-hydroxy-4-methylcoumarin) to dissociate propidium from the peripheral anionic site of Torpedo californica acetylcholinesterase. Measurements were made by back-titration of propidium after complete inhibition of the active center with diisopropylfluorophosphate. Both acetylcholine and acetylthiocholine show substrate inhibition at high substrate concentrations. The concentrations required for occupation of the peripheral site, as ascertained by competition with propidium, correlated well with the concentration dependence for the kinetics of substrate inhibition. These observations are consistent with substrate inhibition being due to binding of acetylcholine or acetylthiocholine at a peripheral anionic site. Displacement of propidium by haloxon and coumarin indicated that these inhibitors also bind to the peripheral anionic site. The dissociation constants ascertained from peripheral site occupation are in agreement with the constants obtained from inhibition kinetics. Evidence is presented that competition with propidium obtained by direct fluorescence titrations, when combined with inhibition kinetics, provides a more reliable means for ascertaining site selectivity of various inhibitors than does a kinetic analysis alone.
ESTHER : Radic_1991_Mol.Pharmacol_39_98
PubMedSearch : Radic_1991_Mol.Pharmacol_39_98
PubMedID: 1987454

Title : Mechanism of Substrate Inhibition of Acetylcholinesterase -
Author(s) : Reiner E , Aldridge WN , Simeon-Rudolf V , Radic Z , Taylor P
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :227 , 1991

Title : Poster: Pyrydinium oximes: reaction with cholinesterase substrates -
Author(s) : Skrinjaric-Spoljar M , Radic Z , Simeon-Rudolf V
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :305 , 1991

Title : Poster: Onchidal: a naturally occurring irreversible inhibitor of acetylcholinesterase with a novel mechanism of action -
Author(s) : Abramson SN , Radic Z , Manker D , Faulkner DJ , Taylor P
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :296 , 1991

Title : Poster: Human serum esterases: differentiation of EDTA-insensitive enzymes -
Author(s) : Radic Z , Pavkovic E , Reiner E
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :288 , 1991

Title : Inhibition of serum cholinesterase by trialkylphosphorothiolates - Franciskovic_1989_Arch.Toxicol_63_489
Author(s) : Franciskovic L , Radic Z , Reiner E
Ref : Archives of Toxicology , 63 :489 , 1989
Abstract : The kinetics of inhibition of horse serum cholinesterase (EC by six trialkylphosphorothiolates was studies (25 degrees C, pH 7.4). The compounds were : OOS-trimethylphosphorothiolate (OOS-Me), OSS-trimethylphosphorodithiolate (OSS-Me), SSS-trimethylphosphorotrithiolate (SSS-Me) and their corresponding ethyl analogues (OOS-Et, OSS-Et, SSS-Et). The second order rate constants of inhibition ranged from 7.2 to 2128 mol-1 1 min-1, of inhibition ranged from 7.2 to 2128 mol-1 1 min-1, and the enzyme/inhibitor dissociation constants from 0.079 to 1.5 mM. The ethyl esters were better inhibitors than their methyl analogues and the OSS-compounds were better inhibitors than the OOS- or SSS-compounds. The same structure-activity relationship is known to hold for the reaction of the compounds with acetylcholinesterase (EC
ESTHER : Franciskovic_1989_Arch.Toxicol_63_489
PubMedSearch : Franciskovic_1989_Arch.Toxicol_63_489
PubMedID: 2619563

Title : Onchidal: a naturally occurring irreversible inhibitor of acetylcholinesterase with a novel mechanism of action - Abramson_1989_Mol.Pharmacol_36_349
Author(s) : Abramson SN , Radic Z , Manker D , Faulkner DJ , Taylor P
Ref : Molecular Pharmacology , 36 :349 , 1989
Abstract : Onchidal has been identified as the major lipid-soluble component of the defensive secretion of the mollusc Onchidella binneyi, and it has been proposed as the compound responsible for the chemical protection of Onchidella [Bioorg. Chem. 7:125-131 (1978)]. In support of this hypothesis, we now report that onchidal can be found in several different species of Onchidella and that it is toxic to fish. Because onchidal is an acetate ester similar to acetylcholine, its ability to interact with nicotinic acetylcholine receptors and acetylcholinesterase was investigated. Although onchidal did not prevent the binding of 125I-alpha-bungarotoxin to nicotinic acetylcholine receptors, it inhibited acetylcholinesterase in a progressive, apparently irreversible, manner. The apparent affinity of onchidal for the initial reversible binding to acetylcholinesterase (Kd) was approximately 300 microM, and the apparent rate constant for the subsequent irreversible inhibition of enzyme activity (kintact) was approximately 0.1 min-1. Onchidal was a substrate for acetylcholinesterase, and approximately 3250 mol of onchidal were hydrolyzed/mol of enzyme irreversibly inhibited. The calculated kcat for onchidal was 325 min-1. Irreversible inhibition resulted from either onchidal itself or a reactive intermediate in the enzyme-catalyzed hydrolysis of onchidal, rather than from the hydrolysis products of onchidal. Irreversible inhibition of enzyme activity was prevented by coincubation with reversible agents that either sterically block (edrophonium and decamethonium) or allosterically modify (propidium) the acetylcholine binding site. Enzyme activity was not regenerated by incubation with oxime reactivators; therefore, the mechanism of irreversible inhibition does not appear to involve acylation of the active site serine. Because onchidal contains a potentially reactive alpha,beta-unsaturated aldehyde, irreversible inhibition of acetylcholinesterase may result from formation of a novel covalent bond between the toxin and the enzyme. Thus, this novel toxin could potentially be exploited in the design of a new class of anticholinesterase insecticides and in the identification of amino acids that contribute to the binding and hydrolysis of acetylcholine.
ESTHER : Abramson_1989_Mol.Pharmacol_36_349
PubMedSearch : Abramson_1989_Mol.Pharmacol_36_349
PubMedID: 2779521

Title : An enzyme test for determining isomalathion impurities in water-dispersible powders of malathion - Reiner_1986_Bull.World.Health.Organ_64_397
Author(s) : Reiner E , Radic Z
Ref : Bulletin of the World Health Organization , 64 :397 , 1986
Abstract : An enzyme test for determining isomalathion (O,S-dimethyl-S-(1,2-dicarbethoxyethyl) phosphorodithioate) impurities in water-dispersible powders of malathion (WDP malathion) is described. The test is based on inhibition of acetylcholinesterase (EC by isomalathion extracted from WDP malathion. The lower limit of detection of the test is 0.01% (w/w) isomalathion. For 18 samples of WDP malathion there was good correlation between the levels of isomalathion found using the enzyme test and those obtained by thin-layer chromatography.
ESTHER : Reiner_1986_Bull.World.Health.Organ_64_397
PubMedSearch : Reiner_1986_Bull.World.Health.Organ_64_397
PubMedID: 3490319

Title : Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents. A theoretical model tested on haloxon and phosphostigmine - Radic_1984_Biochem.Pharmacol_33_671
Author(s) : Radic Z , Reiner E , Simeon-Rudolf V
Ref : Biochemical Pharmacology , 33 :671 , 1984
Abstract : The reaction of acetylcholinesterase (EC; human erythrocytes) with phosphostigmine, haloxon and VX was studied, and the effect of three reversible ligands (TMA, edrophonium, coumarin) and of acetylthiocholine upon the time-dependent and time-independent (reversible) inhibition by the organophosphates was evaluated. The three ligands and acetylthiocholine decreased the second-order rate constant of phosphorylation by a factor proportional to the enzyme-ligand dissociation constant, or to both Km and Kss (Michaelis constant and the substrate-inhibition constant for acetylthiocholine) irrespective of the organophosphate. However, the time-independent inhibitions by phosphostigmine and haloxon were differently affected. Acetylthiocholine affected the time-independent inhibition by phosphostigmine by a factor proportional to Km, and that by haloxon by a factor proportional to Kss. Coumarin had no effect on the time-independent inhibition by phosphostigmine, while TMA and edrophonium displaced phosphostigmine from its complex. Coumarin displaced haloxon from its complex with the enzyme, while TMA and edrophonium had no effect. We conclude that phosphostigmine and haloxon bind reversibly to different sites on the enzyme and the experiments agree with a theoretical model that haloxon binds reversibly to a peripheral site on acetylcholinesterase, and phosphostigmine to the catalytic site.
ESTHER : Radic_1984_Biochem.Pharmacol_33_671
PubMedSearch : Radic_1984_Biochem.Pharmacol_33_671
PubMedID: 6704184