Blumenthal Donald KDepartment of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112 USAPhone : Fax : Send E-Mail to Blumenthal Donald K
Title: Structural and dynamic effects of paraoxon binding to human acetylcholinesterase by X-ray crystallography and inelastic neutron scattering Gerlits O, Fajer M, Cheng X, Blumenthal DK, Radic Z, Kovalevsky A Ref: Structure, :, 2022 : PubMed
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.
Acetylcholinesterase (EC 3.1.1.7; 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.
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.
        
Title: Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase Gerlits O, Kong X, Cheng X, Wymore T, Blumenthal DK, Taylor P, Radic Z, Kovalevsky A Ref: Journal of Biological Chemistry, 294:10607, 2019 : PubMed
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.
        
Title: Limitations in current acetylcholinesterase structure-based design of oxime antidotes for organophosphate poisoning Kovalevsky A, Blumenthal DK, Cheng X, Taylor P, Radic Z Ref: Annals of the New York Academy of Sciences, 1378:41, 2016 : PubMed
Acetylcholinesterase (AChE; EC 3.1.1.7), 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.
Synthetic arteriovenous grafts (AVGs) used for hemodialysis frequently fail due to the development of neointimal hyperplasia (NH) at the vein-graft anastomosis. Inflammation and smooth-muscle cell (SMC) and myofibroblast proliferation and migration likely play an important role in the pathogenesis of NH. Epoxyeicosatrienoic acids (EETs), the products of the catabolism of arachidonic acid by cytochrome P450 enzymes, possess anti-inflammatory, antiproliferative, antimigratory and vasodilatory properties that should reduce NH. The degradation of vasculoprotective EETs is catalyzed by the enzyme, soluble epoxide hydrolase (sEH). sEH upregulation may thus contribute to NH development by the enhanced removal of vasculoprotective EETs. In this study, sEH, cytochrome P450 and EETs were examined after AVG placement in a porcine model to explore their potential roles in AVG stenosis. Increased sEH protein expression, decreased P450 epoxygenase activity and dysregulation of 5 oxylipin mediators were observed in the graft-venous anastomotic tissues when compared to control veins. Pharmacological inhibitors of sEH decreased the growth factor-induced migration of SMCs and fibroblasts, although they had no significant effect on the proliferation of these cells. These results provide insights on epoxide biology in vascular disorders and a rationale for the development of novel pharmacotherapeutic strategies to prevent AVG failure due to NH and stenosis.