Sinko G


Full name : Sinko Goran

First name : Goran

Mail : Institute for Medical Research and Occupational Health\; Ksaverska cesta 2\; PO Box 291\; HR-10000 Zagreb

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Country : Croatia

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Phone : +38514673188

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References (42)

Title : Cholesterol Oxime Olesoxime Assessed as a Potential Ligand of Human Cholinesterases - Kolic_2024_Biomolecules_14_588
Author(s) : Kolic D , Sinko G , Jean L , Chioua M , Dias J , Marco-Contelles J , Kovarik, Z , Kovarik Z
Ref : Biomolecules , 14 :588 , 2024
Abstract : Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10-30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.
ESTHER : Kolic_2024_Biomolecules_14_588
PubMedSearch : Kolic_2024_Biomolecules_14_588
PubMedID: 38785995

Title : Modeling of a near-attack conformation of oxime in phosphorylated acetylcholinesterase via a reactivation product, a phosphorylated oxime - Sinko_2023_Chem.Biol.Interact_14ChEPon_110656
Author(s) : Sinko G
Ref : Chemico-Biological Interactions , :110656 , 2023
Abstract : At the present, only four antidotes are in use in therapy for poisoning by organophosphorus compounds: 2-PAM, HI-6, obidoxime and trimedoxime. Numerous compounds have been designed and synthetized to be more effective reactivators than those currently in use. Many of those new compounds fail at the enzyme level because interactions formed within the AChE active site are not favourable ones that lead to a successful reactivation. The approach in which the modeling of a phosphorylated oxime (POX), a product of successful reactivation in the AChE active site, may be a way to better understand the role of active site residues during the process of formation of the Michaelis type of complex between an enzyme and oxime. After reactivation, a change in phosphorus stereochemistry occurs leading to a different spatial arrangement of attached substituents, now including an oxime. To study interactions between the AChE oxyanion hole and a phosphorylated oxime, an S203G mutant was used to avoid the steric hindrance caused by the catalytic serine. In this way, the POX could be positioned close to the oxyanion hole. In the final step, the oxime without a phosphoester moiety was transferred into the phosphorylated AChE and molecular dynamics was used to test the stability of the near-attack conformation of the oxime near the phosphorylated serine.
ESTHER : Sinko_2023_Chem.Biol.Interact_14ChEPon_110656
PubMedSearch : Sinko_2023_Chem.Biol.Interact_14ChEPon_110656
PubMedID: 37579936

Title : Selected herbicides screened for toxicity and analysed as inhibitors of both cholinesterases - Pehar_2023_Chem.Biol.Interact_14ChEPon_379_110506
Author(s) : Pehar V , Kolic D , Zandona A , Sinko G , Katalinic M , Stepanic V , Kovarik Z
Ref : Chemico-Biological Interactions , 379 :110506 , 2023
Abstract : Sets of 346 herbicides in use and 163 no longer in use were collected from open access online sources and compared in silico with cholinesterases inhibitors (ChI) and drugs in terms of physicochemical profile and estimated toxic effects on human health. The screening revealed at least one potential adverse consequence for each herbicide class assigned according to their mode of action on weeds. The classes with most toxic warnings were K1, K3/N, F1 and E. The selection of 11 commercial herbicides for in vitro biological tests on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the enzymes involved in neurotoxicity and detoxification of various xenobiotics, respectively, was based mainly on the structural similarity with inhibitors of cholinesterases. Organophosphate anilofos and oxyacetanilide flufenacet were the most potent inhibitors of AChE (25 microM) and BChE (6.4 microM), respectively. Glyphosate, oxadiazon, tembotrione and terbuthylazine were poor inhibitors with an estimated IC(50) above 100 microM, while for glyphosate the IC(50) was above 1 mM. Generally, all of the selected herbicides inhibited with a slight preference towards BChE. Cytotoxicity assays showed that anilofos, bensulide, butamifos, piperophos and oxadiazon were cytotoxic for hepatocytes (HepG2) and neuroblastoma cell line (SH-SY5Y). Time-independent cytotoxicity accompanied with induction of reactive oxygen species indicated rapid cell death in few hours. Our results based on in silico and in vitro analyses give insight into the potential toxic outcome of herbicides in use and can be applied in the design of new molecules with a less impact on humans and the environment.
ESTHER : Pehar_2023_Chem.Biol.Interact_14ChEPon_379_110506
PubMedSearch : Pehar_2023_Chem.Biol.Interact_14ChEPon_379_110506
PubMedID: 37141932

Title : Use of connectivity index and simple topological parameters for estimating the inhibition potency of acetylcholinesterase - Milicevic_2022_Saudi.Pharm.J_30_369
Author(s) : Milicevic A , Sinko G
Ref : Saudi Pharm J , 30 :369 , 2022
Abstract : Acetylcholinesterase (AChE) has proven to be an effective drug target in the treatment of neurodegenerative diseases such as Alzheimer's, Parkinson's and dementia. We developed a novel QSAR regression model for estimating potency to inhibit AChE, pK (i), on a set of 75 structurally different compounds including oximes, N-hydroxyiminoacetamides, 4-aminoquinolines and flavonoids. Although the model included only three simple descriptors, the valence molecular connectivity index of the zero-order, (0) (v) , the number of 10-membered rings (nR10) and the number of hydroxyl groups (nOH), it yielded excellent statistics (r = 0.937, S.E. = 0.51). The stability of the model was evaluated when an initial set of 75 compounds was broadened to 165 compounds in total, with the increase of the range of pK (i) (exp) from 6.0 to 10.2, yielding r = 0.882 and S.E. = 0.89. The predictive power of the model was evaluated by calculating pK (i) values for 55 randomly chosen compounds (S.E.(test) = 0.90) from the calibration model created on other 110 compounds (S.E. = 0.89), all taken from the pool of 165 compounds.
ESTHER : Milicevic_2022_Saudi.Pharm.J_30_369
PubMedSearch : Milicevic_2022_Saudi.Pharm.J_30_369
PubMedID: 35527825

Title : Evaluation of the Key Structural Features of Various Butyrylcholinesterase Inhibitors Using Simple Molecular Descriptors - Milicevic_2022_Molecules_27_
Author(s) : Milicevic A , Sinko G
Ref : Molecules , 27 : , 2022
Abstract : In this study, we developed several QSAR models based on simple descriptors (such as topological and constitutional) to estimate butyrylcholinesterase (BChE) inhibition potency, pK(i) (or pIC(50)), of a set of 297 (289 after exclusion of outliers) structurally different compounds. The models were similar to the best model that we obtained previously for acetylcholinesterase AChE and were based on the valence molecular connectivity indices of second and third order ((2)(v) and (3)(v)), the number of aliphatic hydroxyl groups (nOH), AlogP Ghose-Crippen octanol-water partition coeff. (logP), and O-060-atom-centred fragments (Al-O-Ar, Ar-O-Ar, R..O..R and R-O-C=X). The best models with two and three descriptors yielded r = 0.787 and S.E. = 0.89, and r = 0.827 and S.E. = 0.81, respectively. We also correlated nine scoring functions, calculated for 20 ligands whose complexes with BChE we found in the Protein Data Bank as crystal structures to pK(i) (or pIC(50)). The best correlations yielded PLP1 and PLP2 (Piecewise Linear Pairwise potential functions) with r = 0.619 and 0.689, respectively. Correlation with certain simple topological and constitutional descriptors yielded better results, e.g., (3)(v) (r = 0.730), on the same set of compounds (N = 20).
ESTHER : Milicevic_2022_Molecules_27_
PubMedSearch : Milicevic_2022_Molecules_27_
PubMedID: 36296489

Title : Assessment of four organophosphorus pesticides as inhibitors of human acetylcholinesterase and butyrylcholinesterase - Cadez_2021_Sci.Rep_11_21486
Author(s) : Cadez T , Kolic D , Sinko G , Kovarik Z
Ref : Sci Rep , 11 :21486 , 2021
Abstract : Toxicity of organophosphorus compounds (OPs) remains a major public health concern due to their widespread use as pesticides and the existence of nerve agents. Their common mechanism of action involves inhibition of enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which are crucial for neurotransmission. Both chronic and acute poisoning by OPs can leave long-lasting health effects even when the patients are treated with standard medical therapy. Therefore, an increasing urgency exists to find more effective oxime reactivators for compounds which are resistant to reactivation, especially phosphoramidates. Here, we investigated in silico and in vitro interactions and kinetics of inhibition for human cholinesterases with four organophosphate pesticides-ethoprophos, fenamiphos, methamidophos and phosalone. Overall, ethoprophos and fenamiphos displayed higher potency as inhibitors for tested cholinesterases. Our results show that methamidophos-inhibited hAChE was more susceptible to reactivation than hAChE inhibited by fenamiphos by selected oximes. Molecular modelling enabled an evaluation of interactions important for specificity and selectivity of both inhibition and reactivation of cholinesterases. Two newly developed reactivators-bispyridinium triazole oxime 14A and zwitterionic oxime RS194B possess remarkable potential for further development of antidotes directed against pesticides and related phosphoramidate exposures, such as nerve agents tabun or Novichoks.
ESTHER : Cadez_2021_Sci.Rep_11_21486
PubMedSearch : Cadez_2021_Sci.Rep_11_21486
PubMedID: 34728713

Title : Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates - Bosak_2020_Molecules_25_
Author(s) : Bosak A , Bavec A , Konte T , Sinko G , Kovarik Z , Golicnik M
Ref : Molecules , 25 : , 2020
Abstract : Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate S-phenyl thioacetate (PTA). Inhibition dissociation constants (K(i)), evaluated by both discontinuous and continuous inhibition measurements (progress curves), were similar and in the mM range. The rePON1 displayed almost the same values of K(i) constants for Ro 02-0683 and physostigmine while, for carbofuran and bambuterol, the values were approximately ten times lower and two times higher, respectively. The affinity of rePON1 towards the tested carbamates was about 3-40 times lower than that of PTA. Molecular modelling of rePON1-carbamate complexes suggested non-covalent interactions with residues of the rePON1 active site that could lead to competitive inhibition of its arylesterase activity. In conclusion, carbamates can reduce the level of PON1 activity, which should be kept in mind, especially in medical conditions characterized by reduced PON1 levels.
ESTHER : Bosak_2020_Molecules_25_
PubMedSearch : Bosak_2020_Molecules_25_
PubMedID: 31947900

Title : Enantioseparation, in vitro testing, and structural characterization of triple-binding reactivators of organophosphate-inhibited cholinesterases - Marakovic_2020_Biochem.J_477_2771
Author(s) : Marakovic N , Knezevic A , Roncevic I , Brazzolotto X , Kovarik Z , Sinko G
Ref : Biochemical Journal , 477 :2771 , 2020
Abstract : The enantiomers of racemic 2-hydroxyimino-N-(azidophenylpropyl)acetamide-derived triple-binding oxime reactivators were separated, and tested for inhibition and reactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibited with tabun (GA), cyclosarin (GF), sarin (GB), and VX. Both enzymes showed the greatest affinity toward the methylimidazole derivative (III) of 2-hydroxyimino-N-(azidophenylpropyl)acetamide (I). The crystal structure was determined for the complex of oxime III within human BChE, confirming that all three binding groups interacted with active site residues. In the case of BChE inhibited by GF, oximes I (kr=207M-1min-1) and III (kr=213M-1min-1) showed better reactivation efficiency than the reference oxime 2-PAM. Finally, the key mechanistic steps in the reactivation of GF-inhibited BChE with oxime III were modeled using the PM7R6 method, stressing the importance of proton transfer from Nsigma of His438 to Ogamma of Ser203 for achieving successful reactivation.
ESTHER : Marakovic_2020_Biochem.J_477_2771
PubMedSearch : Marakovic_2020_Biochem.J_477_2771
PubMedID: 32639532
Gene_locus related to this paper: human-BCHE

Title : Targeting organophosphorus compounds poisoning by novel quinuclidine-3 oximes: development of butyrylcholinesterase-based bioscavengers - Zandona_2020_Arch.Toxicol_94_3157
Author(s) : Zandona A , Katalinic M , Sinko G , Radman Kastelic A , Primozic I , Kovarik Z
Ref : Archives of Toxicology , 94 :3157 , 2020
Abstract : A library of 14 mono-oxime quinuclidinium-based compounds with alkyl or benzyl substituent were synthesized and characterized in vitro as potential antidotes for organophosphorus compounds (OP) poisoning treatment. We evaluated their potency for reversible inhibition and reactivation of OP inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and evaluated interactions by molecular docking studies. The reactivation was notable for both AChE and BChE inhibited by VX, cyclosarin, sarin and paraoxon, if quinuclidinium compounds contained the benzyl group attached to the quinuclidinium moiety. Out of all 14, oxime Q8 [4-bromobenzyl-3-(hydroxyimino)quinuclidinium bromide] was singled out as having the highest determined overall reactivation rate of approximately 20,000 M(-1) min(-1) for cyclosarin-inhibited BChE. Furthermore, this oxime in combination with BChE exhibited a capability to act as a bioscavenger of cyclosarin, degrading within 2 h up to 100-fold excess of cyclosarin concentration over the enzyme. Molecular modeling revealed that the position of the cyclohexyl moiety conjugated with the active site serine of BChE directs the favorable positioning of the quinuclidinium ring and the bromophenyl moiety of Q8, which makes phosphonylated-serine easily accessible for the nucleophilic displacement by the oxime group of Q8. This result presents a novel scaffold for the development of new BChE-based bioscavengers. Furthermore, a cytotoxic effect was not observed for Q8, which also makes it promising for further in vivo reactivation studies.
ESTHER : Zandona_2020_Arch.Toxicol_94_3157
PubMedSearch : Zandona_2020_Arch.Toxicol_94_3157
PubMedID: 32583098

Title : Structural aspects of 4-aminoquinolines as reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase - Bosak_2019_Chem.Biol.Interact_13ChEPon_308_101
Author(s) : Bosak A , Opsenica DM , Sinko G , Zlatar M , Kovarik Z
Ref : Chemico-Biological Interactions , 308 :101 , 2019
Abstract : Eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) were synthesised and tested as inhibitors of human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Both enzymes were inhibited by all of the compounds with inhibition constants (Ki) ranging from 0.50 to 50muM exhibiting slight selectivity toward AChE over BChE. The most potent inhibitors of AChE were compounds with an n-octylamino chain or adamantyl group. The shortening of the chain length resulted in a decrease in AChE inhibition by 5-20 times. Docking studies revealed that the quinoline group within the AChE active site was positioned in the choline binding site, while the C(4)-amino group substituents, depending on their lipophilicity, could establish hydrogen bonds or pi-interactions with residues of the peripheral anionic site. The most potent inhibitors of BChE were compounds with the most voluminous substituent on C(4)-amino group (adamantyl) or those with a stronger electron withdrawing substituent on C(7) (trifluormethyl group). Based on AChE inhibition, compounds with an n-octylamino chain or adamantyl substituent were shown to possess the capacity for further development as potential drugs for treatment of neurodegenerative diseases.
ESTHER : Bosak_2019_Chem.Biol.Interact_13ChEPon_308_101
PubMedSearch : Bosak_2019_Chem.Biol.Interact_13ChEPon_308_101
PubMedID: 31100281

Title : Assessment of scoring functions and in silico parameters for AChE-ligand interactions as a tool for predicting inhibition potency - Sinko_2019_Chem.Biol.Interact_13ChEPon_308_216
Author(s) : Sinko G
Ref : Chemico-Biological Interactions , 308 :216 , 2019
Abstract : In this study, 68 crystal structures of complexes between acetylcholinesterase (AChE, EC and its ligands, deposited in the PDB, were analyzed by scoring the functions: LigScore1, LigScore2, PLP1, PLP2, Jain, PMF and PMF04. The scores derived from scoring functions were correlated with an inhibition constant for each ligand (Ki or IC50) in a broad range 10(-3) - 10(-12)M. The linear correlation model resulted in the highest coefficient of determination (r(2)) for the PLP2 function, 0.591. The LigScore1 function resulted in the lowest r(2) value of 0.226. The PubChem database was the source of in silico computed ligand properties which were then correlated with an inhibition constant for each ligand. For the purposes of this study, two additional non-PubChem parameters were evaluated: total and relative number of sp(2) hybridized atoms in the ligand. A high coefficient of determination (r(2)>0.5) was calculated for the following parameters: the number of heavy atoms, molecular mass, and number of atoms with sp(2) hybridization. The PLP2 scoring function is a good candidate for drug discovery related to AChE, although a better scoring function could be developed with a higher number of crystal structures of AChE complexes and more reliable kinetic data.
ESTHER : Sinko_2019_Chem.Biol.Interact_13ChEPon_308_216
PubMedSearch : Sinko_2019_Chem.Biol.Interact_13ChEPon_308_216
PubMedID: 31150627

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 : Synthesis and In Vitro Screening of Novel Heterocyclic beta-d-Gluco- and beta-d-Galactoconjugates as Butyrylcholinesterase Inhibitors - Baumann_2019_Molecules_24_
Author(s) : Baumann K , Kordic L , Mocibob M , Sinko G , Tomic S
Ref : Molecules , 24 : , 2019
Abstract : The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer's disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic beta-d-gluco- and beta-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted beta-d-gluco- and beta-d-galacto-derivatives in a 10-50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood-brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.
ESTHER : Baumann_2019_Molecules_24_
PubMedSearch : Baumann_2019_Molecules_24_
PubMedID: 31382668

Title : The estimation of oxime efficiency is affected by the experimental design of phosphylated acetylcholinesterase reactivation - Macek Hrvat_2018_Toxicol.Lett_293_222
Author(s) : Macek Hrvat N , Zorbaz T , Sinko G , Kovarik Z
Ref : Toxicol Lett , 293 :222 , 2018
Abstract : Reactivation of acetylcholinesterase (AChE), an essential enzyme in neurotransmission, is a key point in the treatment of acute poisoning by nerve agents and pesticides, which structurally belong to organophosphorus compounds (OP). Due to the high diversity of substituents on the phosphorous atom, there is a variety of OP-AChE conjugates deriving from AChE inhibition, and therefore not only is there no universal reactivator efficient enough for the most toxic OPs, but for some nerve agents there is still a lack of any reactivator at all. The endeavor of many chemists to find more efficient reactivators resulted in thousands of newly-designed and synthesized oximes-potential reactivators of AChE. For an evaluation of the oximes reactivation efficiency, many research groups employ a simple spectrophotometric Ellman method. Since parameters that describe reactivator efficiency are often incomparable among laboratories, we tried to emphasize the critical steps in the determination of reactivation parameters as well as in the experimental design of a reactivation assay. We highlighted the important points in evaluation of reactivation kinetic parameters with an aim to achieve better agreement and comparability between the results obtained by different laboratories and overall, a more efficient evaluation of in vitro reactivation potency.
ESTHER : Macek Hrvat_2018_Toxicol.Lett_293_222
PubMedSearch : Macek Hrvat_2018_Toxicol.Lett_293_222
PubMedID: 29180286

Title : Oxime-assisted reactivation of tabun-inhibited acetylcholinesterase analysed by active site mutations - Katalinic_2018_Toxicology_406-407_104
Author(s) : Katalinic M , Sinko G , Macek Hrvat N , Zorbaz T , Bosak A , Kovarik Z
Ref : Toxicology , 406-407 :104 , 2018
Abstract : The antidotal property of oximes is attributed to their ability to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OP) such as pesticides and nerve warfare agents. Understanding their interactions within the active site of phosphylated AChE is of great significance for the search for more efficient reactivators, especially in the case of the most resistant OP to reactivation, tabun. Therefore, herein we studied the interactions and reactivation of tabun-inhibited AChE by site-directed mutagenesis and a series of bispyridinium oximes. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilisation of the oxime's pyridinium ring(s) within the active site gorge needed to obtain the proper orientation of the oxime group toward the phosphorylated active site serine. However, in the case of W286A, the mutation in the peripheral binding site by preventing a pi-pi interaction with one of the oxime's pyridinium rings allowed a more favourable position of the oxime for a nucleophilic attack on the phosphorylated catalytic serine. The mutation resulted in a 2-5 fold increase in the reactivation rates when compared to the AChE wild type. Therefore, it seems that aromatic amino acids at the peripheral binding site presented a limitation in bispyridinium oxime reactivation efficiency of tabun-phosphorylated AChE. Moreover, this is further corroborated by the reactivation by mono-pyridinium oxime 2-PAM, in which mutations at the peripheral site did not influence either the affinity or reactivation of tabun-inhibited AChE.
ESTHER : Katalinic_2018_Toxicology_406-407_104
PubMedSearch : Katalinic_2018_Toxicology_406-407_104
PubMedID: 29772260

Title : The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry - Marakovic_2017_Acta.Chim.Slov_64_15
Author(s) : Marakovic N , Sinko G
Ref : Acta Chim Slov , 64 :15 , 2017
Abstract : Modern drug discovery is mainly based on the de novo synthesis of a large number of compounds with a diversity of chemical functionalities. Though the introduction of combinatorial chemistry enabled the preparation of large libraries of compounds from so-called building blocks, the problem of successfully identifying leads remains. The introduction of a dynamic combinatorial chemistry method served as a step forward due to the involvement of biological macromolecular targets (receptors) in the synthesis of high affinity products. The major breakthrough was a synthetic method in which building blocks are irreversibly combined due to the presence of a receptor. Here we present various receptor-based combinatorial chemistry approaches. Huisgen's cycloaddition (1,3-dipolar cycloaddition of azides and alkynes) forms stabile 1,2,3-triazoles with very high receptor affinity that can reach femtomolar levels, as the case with acetylcholinesterase inhibitors shows. Huisgen's cycloaddition can be applied to various receptors including acetylcholinesterase, acetylcholine binding protein, carbonic anhydrase-II, serine/threonine-protein kinase and minor groove of DNA.
ESTHER : Marakovic_2017_Acta.Chim.Slov_64_15
PubMedSearch : Marakovic_2017_Acta.Chim.Slov_64_15
PubMedID: 28380234

Title : Resorcinol-, catechol- and saligenin-based bronchodilating beta2-agonists as inhibitors of human cholinesterase activity - Bosak_2017_J.Enzyme.Inhib.Med.Chem_32_789
Author(s) : Bosak A , Knezevic A , Gazic Smilovic I , Sinko G , Kovarik Z
Ref : J Enzyme Inhib Med Chem , 32 :789 , 2017
Abstract : We investigated the influence of bronchodilating beta2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with Ki constants ranging from 9.4 muM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional pi-pi interaction of salmeterol's benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.
ESTHER : Bosak_2017_J.Enzyme.Inhib.Med.Chem_32_789
PubMedSearch : Bosak_2017_J.Enzyme.Inhib.Med.Chem_32_789
PubMedID: 28573890

Title : Pyridoxal oxime derivative potency to reactivate cholinesterases inhibited by organophosphorus compounds - Busic_2016_Toxicol.Lett_262_114
Author(s) : Busic V , Katalinic M , Sinko G , Kovarik Z , Gaso-Sokac D
Ref : Toxicol Lett , 262 :114 , 2016
Abstract : Organophosphorus (OP) nerve agents (sarin, tabun VX and soman) inhibit the enzyme acetylcholinesterase (AChE, EC by binding to its active site while preventing neurotransmission in the cholinergic synapses. The protection and treatment of this kind of poisoning are still a challenge as we are yet to discover an antidote that would be effective in all cases of poisoning. To aid the search for more efficient antidotes, we evaluated the ability of nine pyridoxal oxime derivatives, prepared by a novel synthetic pathway, to reactivate recombinant human AChE and the related purified human plasma butyrylcholinesterase (BChE, EC inhibited by VX, tabun and paraoxon. Oximes are derivatives of vitamin B6 bearing a phenacyl moiety attached to the quaternary nitrogen atom and having various substituents on the phenyl ring. As the results have shown, the tested oximes were in general more efficient in the reactivation of OP-inhibited BChE than AChE. The highest observed rate was in the case of VX-inhibited BChE reactivation, where kobs was 0.0087min-1 and the reactivation maximum of 90% was achieved within 5h. The cholinesterases displayed a binding affinity for these derivatives in a mumolar range no matter the substituent on their rings which was in accordance with the molecular modelling results showing a similar binding pattern for all oximes within the active site of both AChE and BChE. Such a positioning reveals also that hydroxy and a metoxy substituents at the vicinity of the oxime moiety present a possible steric hindrance explaining the reactivation results.
ESTHER : Busic_2016_Toxicol.Lett_262_114
PubMedSearch : Busic_2016_Toxicol.Lett_262_114
PubMedID: 27693733

Title : Design and synthesis of N-substituted-2-hydroxyiminoacetamides and interactions with cholinesterases - Marakovic_2016_Chem.Biol.Interact_259_122
Author(s) : Marakovic N , Knezevic A , Vinkovic V , Kovarik Z , Sinko G
Ref : Chemico-Biological Interactions , 259 :122 , 2016
Abstract : Within this study, we designed and synthesized four new oxime compounds of the N-substituted 2-hydroxyiminoacetamide structure and evaluated their interactions with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Our aim was to explore the possibility of extending the dual-binding mode of interaction between the enzyme and the inhibitor to a so-called triple-binding mode of interaction through the introduction of an additional binding moiety. N-substituted 2-hydroxyiminoacetamide 1 was prepared via BOP catalyzed amidation of hydroxyiminoacetic acid with 3-azido-1-phenylpropylamine. An azide group enabled us to prepare more elaborate structures 2-4 by the copper-catalyzed azide-alkyne cycloaddition. The new compounds 1-4 differed in their presumed AChE peripheral site binding moiety, which ranged from an azide group to functionalized heterocycles. Molecular docking studies revealed that all three binding moieties are involved in the non-covalent interactions with ChEs for all of the four compounds, albeit not always in the complete accordance with the proposed hypothesis. All of the four compounds reversibly inhibited the ChEs with their inhibition potency increasing in the same order for both enzymes (1 < 2 < 4 < 3). A higher preference for binding to BChE (KI from 0.30 mumol/L to 130 mumol/L) over AChE (KI from 50 mumol/L to 1200 mumol/L) was observed for all of the compounds. Compounds were screened for reactivation of cyclosarin-, sarin- and VX-inhibited AChE and BChE.
ESTHER : Marakovic_2016_Chem.Biol.Interact_259_122
PubMedSearch : Marakovic_2016_Chem.Biol.Interact_259_122
PubMedID: 27238725

Title : Response of biochemical biomarkers in the aquatic crustacean Daphnia magna exposed to silver nanoparticles - Ulm_2015_Environ.Sci.Pollut.Res.Int_22_19990
Author(s) : Ulm L , Krivohlavek A , Jurasin D , Ljubojevic M , Sinko G , Crnkovic T , Zuntar I , Sikic S , Vinkovic Vrcek I
Ref : Environ Sci Pollut Res Int , 22 :19990 , 2015
Abstract : The proliferation of silver nanoparticle (AgNP) production and use owing to their antimicrobial properties justifies the need to examine the resulting environmental impacts. The discharge of biocidal nanoparticles to water bodies may pose a threat to aquatic species. This study evaluated the effects of citrate-coated AgNPs on the standardized test organism Daphnia magna Straus clone MBP996 by means of biochemical biomarker response. AgNP toxicity was compared against the toxic effect of Ag(+). The toxicity endpoints were calculated based upon measured Ag concentrations in exposure media. For AgNPs, the NOAEC and LOAEC values at 48 h were 5 and 7 mug Ag/L, respectively, while these values were 0.5 and 1 mug Ag/L, respectively, for Ag(+). The EC50 at 48 h was computed to be 12.4 +/- 0.6 and 2.6 +/- 0.1 mug Ag/L for AgNPs and Ag(+), respectively, with 95 % confidence intervals of 12.1-12.8 and 2.3-2.8 mug Ag/L, respectively. These results indicate significant less toxicity of AgNP compared to free Ag(+) ions. Five biomarkers were evaluated in Daphnia magna neonates after acute exposure to Ag(+) or AgNPs, including glutathione (GSH) level, reactive oxygen species (ROS) content, and catalase (CAT), acetylcholinesterase (AChE), and superoxide dismutase (SOD) activity. AgNPs induced toxicity and oxidative stress responses in D. magna neonates at tenfold higher concentrations than Ag. Biochemical methods revealed a clear increase in AChE activity, decreased ROS level, increased GSH level and CAT activity, but no significant changes in SOD activity. As Ag(+) may dissolve from AgNPs, these two types of Ag could act synergistically and produce a greater toxic response. The observed remarkably high toxicity of AgNPs (in the parts-per-billion range) to crustaceans indicates that these organisms are a vulnerable link in the aquatic food chain with regard to contamination by nanosilver. Graphical Abstract .
ESTHER : Ulm_2015_Environ.Sci.Pollut.Res.Int_22_19990
PubMedSearch : Ulm_2015_Environ.Sci.Pollut.Res.Int_22_19990
PubMedID: 26296504

Title : Alteration of cholinesterase activity as possible mechanism of silver nanoparticle toxicity - Sinko_2014_Environ.Sci.Pollut.Res.Int_21_1391
Author(s) : Sinko G , Vinkovic Vrcek I , Goessler W , Leitinger G , Dijanosic A , Miljanic S
Ref : Environ Sci Pollut Res Int , 21 :1391 , 2014
Abstract : Due to their broad-spectrum antimicrobial activity, silver nanoparticles (AgNPs) have been used in a large number of commercial and medical products. Such proliferated AgNP production poses toxicological and environmental issues which need to be addressed. The present study aimed to investigate the effects of AgNPs on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), important enzymes in areas of neurobiology, toxicology and pharmacology. Three different AgNPs, prepared by the chemical reduction using trisodium citrate, hydroxylamine hydrochloride (Cl-AgNPs), and borohydride following stabilization with poly(vinyl alcohol), were purified and characterised with respect to their sizes, shapes and optical properties. Their inhibition potential on AChE and BChE was evaluated in vitro using an enzyme assay with o-nitrophenyl acetate or o-nitrophenyl butyrate as substrates, respectively. All three studied AgNPs were reversible inhibitors of ChEs. Among tested nanoparticles, Cl-AgNP was found to be the most potent inhibitor of both AChE and BChE. Although the detailed mechanism by which the AgNPs inhibit esterase activities remains unknown, structural perturbation of the enzyme may be the common mode of ChE inhibition by AgNPs.
ESTHER : Sinko_2014_Environ.Sci.Pollut.Res.Int_21_1391
PubMedSearch : Sinko_2014_Environ.Sci.Pollut.Res.Int_21_1391
PubMedID: 23904256

Title : Peripheral site and acyl pocket define selective inhibition of mouse butyrylcholinesterase by two biscarbamates - Bosak_2013_Arch.Biochem.Biophys_529_140
Author(s) : Bosak A , Smilovic IG , Stimac A , Vinkovic V , Sinko G , Kovarik Z
Ref : Archives of Biochemistry & Biophysics , 529 :140 , 2013
Abstract : In this study we related metacarb (N-(2-(3,5-bis(dimethylcarbamoyloxy)phenyl)-2-hydroxyethyl)propan-2-aminium chloride) and isocarb (N-(2-(3,4-bis(dimethylcarbamoyloxy)phenyl)-2-hydroxyethyl)propan-2-aminium chloride) inhibition selectivity, as well as stereoselectivity of mouse acetylcholinesterase (AChE; and butyrylcholinesterase (BChE; to the active site residues by studying the progressive inhibition of AChE, BChE and six AChE mutants with racemic and (R)-enantiomers of metacarb and isocarb. Metacarb and isocarb proved to be very potent BChE inhibitors with inhibition rate constants in the range of 10(3)-10(4)M(-1)s(-1). For metacarb and isocarb, inhibition of BChE w.t. was 260 and 35 times, respectively, faster than inhibition of AChE w.t. For four mutants inhibition was faster than for AChE w.t. but none reached the inhibition rate of BChE. The highest increase in the inhibition rate (about 30 times for metacarb and 13 times for isocarb) was achieved with mutants F295L/Y337A and Y124Q meaning that selective inhibition of mouse BChE is dictated mainly by two amino acids from BChE: leucine 286 from the acyl pocket and glutamine 119 from the peripheral site. Wild type enzymes displayed pronounced stereoselectivity for (R)-enantiomers of metacarb and isocarb. Interestingly, the residues that define selective inhibition of mouse BChE by biscarbamates also affect the stereoselectivity of enzymes.
ESTHER : Bosak_2013_Arch.Biochem.Biophys_529_140
PubMedSearch : Bosak_2013_Arch.Biochem.Biophys_529_140
PubMedID: 23219600

Title : Metaproterenol, isoproterenol, and their bisdimethylcarbamate derivatives as human cholinesterase inhibitors - Bosak_2012_J.Med.Chem_55_6716
Author(s) : Bosak A , Gazic Smilovic I , Sinko G , Vinkovic V , Kovarik Z
Ref : Journal of Medicinal Chemistry , 55 :6716 , 2012
Abstract : Metaproterenol and isoproterenol are bronchodilators that provide a structural basis for many other bronchodilators currently in use. One of these structurally related bronchodilators is terbutaline; it is administered as a prodrug, bambuterol, and is metabolized (bioconverted) into terbutaline by butyrylcholinesterase (BChE). The metabolism rate can be affected by BChE gene polymorphism in the human population and BChE stereoselectivity. The aim of our study was to investigate inhibition of human BChE and acetylcholinesterase (AChE) with metaproterenol, isoproterenol, and newly synthesized racemic bisdimethylcarbamate derivatives of metaproterenol (metacarb) and isoproterenol (isocarb) and their (R)-enantiomers to see if their bioconversion is affected by BChE inhibition in the same way as that for bambuterol. Metacarb and isocarb proved to be selective BChE inhibitors, as they progressively inhibited AChE 960 to 80 times more slowly than BChE(UU). All studied cholinesterases displayed poor affinity for metaproterenol and isoproterenol, yet BChE(UU) had an affinity about five times higher than that of AChE.
ESTHER : Bosak_2012_J.Med.Chem_55_6716
PubMedSearch : Bosak_2012_J.Med.Chem_55_6716
PubMedID: 22817559

Title : Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers - Sinko_2011_Biochimie_93_1797
Author(s) : Sinko G , Kovarik Z , Reiner E , Simeon-Rudolf V , Stojan J
Ref : Biochimie , 93 :1797 , 2011
Abstract : Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002-250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine 'slides down' the enzyme gorge, resulting in interaction of the three rings of ethopropazine through pi-pi interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme-substrate, enzyme-inhibitor and enzyme-substrate-inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.
ESTHER : Sinko_2011_Biochimie_93_1797
PubMedSearch : Sinko_2011_Biochimie_93_1797
PubMedID: 21740955

Title : Interactions of pyridinium oximes with acetylcholinesterase - Sinko_2010_Chem.Biol.Interact_187_172
Author(s) : Sinko G , Brglez J , Kovarik Z
Ref : Chemico-Biological Interactions , 187 :172 , 2010
Abstract : Catalytic activity of acetylcholinesterase (AChE; EC was studied in the presence of oximes HI-6, K114, K127 and K203, and inhibition constants were determined for the reversible enzyme-inhibitor complex (K(I)). Based on the mixed inhibition model, inhibition constants were 0.020 mM for HI-6, 0.0021 mM for K114, 0.175 mM for K127, and 0.036 mM for K203. Molecular modelling of AChE-oxime complexes was used to determine amino acid residues of the active site involved in the interactions. Bis-oxime K114 achieved the best stabilization in the active site due to pi-pi interaction between its three aromatic rings and Tyr124, Tyr341 and Trp86, and hydrogen bonds formed by its oxime groups with Gly121 and Glu285. Mono-oximes HI-6 and K203, which inhibited the enzyme with similar potency, showed similar positions of their pyridinium rings in the active site. The weakest inhibitor, K127, also formed several hydrogen bonds with the active site residues, but due to its long linker it was more likely stabilized at the peripheral site (Tyr124), which could explain lower AChE affinity for this oxime.
ESTHER : Sinko_2010_Chem.Biol.Interact_187_172
PubMedSearch : Sinko_2010_Chem.Biol.Interact_187_172
PubMedID: 20417629

Title : Structural aspects of flavonoids as inhibitors of human butyrylcholinesterase - Katalinic_2010_Eur.J.Med.Chem_45_186
Author(s) : Katalinic M , Rusak G , Domacinovic Barovic J , Sinko G , Jelic D , Antolovic R , Kovarik Z
Ref : Eur Journal of Medicinal Chemistry , 45 :186 , 2010
Abstract : Selected flavonoids: galangin, kaempferol, quercetin, myricetin, fisetin, apigenin, luteolin and rutin, reversibly inhibited human butyrylcholinesterase (BChE, EC Inhibition potency of the flavonoids we attributed to their chemical structure, i.e., the number of OH groups and their side on the phenyl ring. The most potent BChE inhibitor among the tested flavonoids was galangin, which showed 12 times higher preference for binding to BChE (7 micromol/L) than to the related enzyme human acetylcholinesterase (AChE, EC Docking study showed that flavonoids bind to the BChE active site by forming multiple hydrogen bonds and pi-pi interactions. The UV-VIS (200-500 nm) absorption spectra of the flavonoid phosphate buffer solution (pH 7.4), with the exception of rutin, revealed time dependant changes indicating precipitation of flavonoids or in the case of myricetin, a change in the chemical structure resulting in a BChE non-inhibiting specie. Selected flavonoids showed no cytotoxic effect on HepG2 and A549 cell lines at concentrations up to 200 micromol/L. Cytotoxicity was observed only for fisetin, apigenin and luteolin in the THP-1 cell line with IC50 of 30, 60 and 70 micromol/L, respectively.
ESTHER : Katalinic_2010_Eur.J.Med.Chem_45_186
PubMedSearch : Katalinic_2010_Eur.J.Med.Chem_45_186
PubMedID: 19879672

Title : Pseudo-catalytic scavenging: searching for a suitable reactivator of phosphorylated butyrylcholinesterase - Kovarik_2010_Chem.Biol.Interact_187_167
Author(s) : Kovarik Z , Katalinic M , Sinko G , Binder J , Holas O , Jung YS , Musilova L , Jun D , Kuca K
Ref : Chemico-Biological Interactions , 187 :167 , 2010
Abstract : Butyrylcholinesterase is considered to be an endogenous stoichiometric bioscavenger of organophosphorus compounds (OPs), but due to limited concentration of BChE in the organism, stoichiometric reduction of OP is not always sufficient. This can be improved by creating a pseudo-catalytic scavenger adding oximes as reactivators of inhibited exogenous BChE. In order to improve the BChE bioscavenging function in tabun or paraoxon poisoning, we tested in vitro reactivation of phosphorylated human plasma BChE by bispyridinium oximes varying in the length and type of the linker between rings, and in the position of the oxime group on the ring. Among the tested oximes, the most potent reactivators of tabun-inhibited BChE were K117 [1,1'-(2,2'-oxybis(ethane-2,1-diyl))bis(4-hydroxyiminomethyl pyridinium) bromide] and K127 [4-carbamoyl-1-(2-(2-(4-(hydroxyiminomethyl) pyridinium-1-yl)ethoxy)ethyl)pyridinium bromide]. Reactivation by these oximes (1mM) reached about 50% of control activity after only 20 min; however, reactivation stopped at 70%. Reactivation of paraoxon-inhibited BChE by all of the selected oximes was slow. Using molecular mechanics, we performed docking of the oximes to tabun-inhibited BChE in order to discuss possible structural modifications of bispyridinium oximes to improve reactivation of phosphorylated BChE.
ESTHER : Kovarik_2010_Chem.Biol.Interact_187_167
PubMedSearch : Kovarik_2010_Chem.Biol.Interact_187_167
PubMedID: 20206154

Title : Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning - Kovarik_2008_Chem.Biol.Interact_175_173
Author(s) : Kovarik Z , Calic M , Sinko G , Bosak A , Zunec S , Vrdoljak AL , Radic B
Ref : Chemico-Biological Interactions , 175 :173 , 2008
Abstract : One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.
ESTHER : Kovarik_2008_Chem.Biol.Interact_175_173
PubMedSearch : Kovarik_2008_Chem.Biol.Interact_175_173
PubMedID: 18501341

Title : Preparative HPLC separation of bambuterol enantiomers and stereoselective inhibition of human cholinesterases - Gazic_2008_Anal.Bioanal.Chem_385_1513
Author(s) : Gazic I , Bosak A , Sinko G , Vinkovic V , Kovarik Z
Ref : Anal Bioanal Chem , 385 :1513 , 2008
Abstract : We separated and characterized the enantiomers of bambuterol (5-[-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride), which is used in racemic form as a prodrug of terbutaline, a beta(2)-adrenoceptor agonist. The enantioseparation was attempted on several chiral HPLC columns, and the most effective separation was achieved on the amylose-based Chiralpak AD column. Since in vivo conversion of bambuterol into terbutaline involves hydrolysis by butyrylcholinesterase (EC, we studied the reaction of enantiomers with eight human BChE variants. Both enantiomers inhibited all studied BChE variants; however, the rate of inhibition with the (R)-enantiomer was about five times faster than with the (S)-enantiomer. (R)-bambuterol inhibition rate constants for homozygous usual (UU), fluoride-resistant (FF) or atypical (AA) variant ranged from 6.4 to 0.11 min(-1)microM(-1). The inhibition rates for heterozygotes were between the respective constants for the corresponding homozygotes.
ESTHER : Gazic_2008_Anal.Bioanal.Chem_385_1513
PubMedSearch : Gazic_2008_Anal.Bioanal.Chem_385_1513
PubMedID: 16865342

Title : Structure-activity approach in the reactivation of tabun-phosphorylated human acetylcholinesterase with bispyridinium para-aldoximes - Kovarik_2007_Arh.Hig.Rada.Toksikol_58_201
Author(s) : Kovarik Z , Calic M , Sinko G , Bosak A
Ref : Arh Hig Rada Toksikol , 58 :201 , 2007
Abstract : We investigated interactions of bispyridinium para-aldoximes N,N'-(propano)bis(4-hydroxyiminomethyl) pyridinium bromide (TMB-4(Trimedoxime)), N,N'-(ethano)bis(4-hydroxyiminomethyl)pyridinium methanosulphonate (DMB-4), and N,N'-(methano)bis(4-hydroxyiminomethyl)pyridinium chloride (MMB-4) with human erythrocyte acetylcholinesterase phosphorylated by tabun. We analysed aldoxime conformations to determine the flexibility of aldoxime as an important feature for binding to the acetylcholinesterase active site. Tabun-inhibited human erythrocyte acetylcholinesterase was completely reactivated only by the most flexible bispyridinium aldoxime - TMB-4(Trimedoxime) with a propylene chain between two rings. Shorter linkers than propylene (methylene or ethylene) as in MMB-4 and DMB-4 did not allow appropriate orientation in the active site, and MMB-4 and DMB-4 were not efficient reactivators of tabun-phosphorylated acetylcholinesterase. Since aldoximes are also reversible inhibitors of native acetylcholinesterase, we determined dissociation constants and their protective index against acetylcholinesterase inactivation by tabun.
ESTHER : Kovarik_2007_Arh.Hig.Rada.Toksikol_58_201
PubMedSearch : Kovarik_2007_Arh.Hig.Rada.Toksikol_58_201
PubMedID: 17562604

Title : Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes - Sinko_2007_Anal.Biochem_370_223
Author(s) : Sinko G , Calic M , Bosak A , Kovarik Z
Ref : Analytical Biochemistry , 370 :223 , 2007
Abstract : The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.
ESTHER : Sinko_2007_Anal.Biochem_370_223
PubMedSearch : Sinko_2007_Anal.Biochem_370_223
PubMedID: 17716616

Title : para- and ortho-Pyridinium aldoximes in reaction with acetylthiocholine - Sinko_2006_FEBS.Lett_580_3167
Author(s) : Sinko G , Calic M , Kovarik Z
Ref : FEBS Letters , 580 :3167 , 2006
Abstract : In the oximolysis reaction para-aldoximes K027 and TMB-4(Trimedoxime) react faster with ATCh than ortho-aldoximes HI-6 and K033. The reaction rate constants at 25 degrees C were 22 M(-1) min(-1) for HI-6 and K033, 230 M(-1) min(-1) for TMB-4(Trimedoxime) and 306 M(-1) min(-1) for K027. Semi-empirical calculations showed that differences in rates do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance of the oxime group within the molecule. Thermodynamic parameters, DeltaG, DeltaH and DeltaS, were also determined for oximolysis reaction.
ESTHER : Sinko_2006_FEBS.Lett_580_3167
PubMedSearch : Sinko_2006_FEBS.Lett_580_3167
PubMedID: 16684539

Title : Structure-inhibition relationships in the interaction of butyrylcholinesterase with bambuterol, haloxon and their leaving groups -
Author(s) : Sinko G , Bosak A , Kovarik Z , Simeon-Rudolf V
Ref : Chemico-Biological Interactions , 157-158 :421 , 2005
PubMedID: 16429568

Title : Comparison of two reaction schemes for the hydrolysis of acetylthiocholine by butyrylcholinesterase. -
Author(s) : Simeon-Rudolf V , Sinko G , Stuglin A , Stojan J , Golicnik M
Ref : Cholinergic Mechanisms, CRC Press :701 , 2004

Title : Peripheral binding of ethopropazine to horse serum butyrylcholinesterase. -
Author(s) : Reiner E , Sinko G , Stuglin A , Simeon-Rudolf V
Ref : Cholinergic Mechanisms, CRC Press :673 , 2004

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 : 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 : 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 : Molar absorption coefficients for the reduced Ellman reagent: reassessment - Eyer_2003_Anal.Biochem_312_224
Author(s) : Eyer P , Worek F , Kiderlen D , Sinko G , Stuglin A , Simeon-Rudolf V , Reiner E
Ref : Analytical Biochemistry , 312 :224 , 2003
Abstract : The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.
ESTHER : Eyer_2003_Anal.Biochem_312_224
PubMedSearch : Eyer_2003_Anal.Biochem_312_224
PubMedID: 12531209

Title : Exploring the Active Sites of Cholinesterases by Inhibition with Bambuterol and Haloxon - Kovarik_2003_Croatica.Chem.Acta_76_63
Author(s) : Kovarik Z , Bosac A , Sinko G , Latas T
Ref : Croatica Chemica Acta , 76 :63 , 2003
Abstract : he paper describes the inhibition of mouse acetylcholinesterase (AChE; EC and mouse, human, and horse butyrylcholinesterase (BChE; EC by 5-[2-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride (bambuterol) and by O,O-bis-(2-chloroethyl)-O-(3-chloro-4-methylcoumarin-7-yl) phosphate (haloxon). The haloxon inhibition rate constant (ki) for mouse BChE was 3.7 x 107 min1 mol1 dm3, which was 40-fold higher than the rate constant for mouse AChE. Bambuterol inhibition of horse BChE (ki = 2.1 x 10e5 min1 mol1 dm3) was about 25-fold slower than that of human or mouse BChE, whereas the respective haloxon inhibition of horse BChE (ki = 1.2 x 107 min1 mol1 dm3) was about 2-3-fold slower. Sequence alignments and the computational model of the three-dimensional structure of horse BChE suggest that residues inside the active site at positions 69, 277 and 285 are important for the differences in the inhibition of these three BChE species.
ESTHER : Kovarik_2003_Croatica.Chem.Acta_76_63
PubMedSearch : Kovarik_2003_Croatica.Chem.Acta_76_63
Gene_locus related to this paper: horse-BCHE

Title : Kinetic Model of Ethopropazine Interaction with Horse Serum Butyrylcholinesterase and Its Docking into the Active Site - Golicnik_2002_Arch.Biochem.Biophys_398_23
Author(s) : Golicnik M , Sinko G , Simeon-Rudolf V , Grubic Z , Stojan J
Ref : Archives of Biochemistry & Biophysics , 398 :23 , 2002
Abstract : The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase EC was investigated at 25 and 37 C The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis The model which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase Stojan et al 1998 FEBS Lett 440 85?88 is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena apparent activation at low substrate concentrations and substrate inhibition by excess of substrate For the analysis of the data in the presence of ethopropazine at two temperatures we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site but the competition at the acylation site was not excluded The proposed reaction scheme revealed upon analysis competitive effects of ethopropazine at both sites at 25 C three enzyme inhibitor dissociation constants could be evaluated at 37 C only two constants could be evaluated Although the model considers both cooperative phenomena it appears that decreased enzyme sensitivity at higher temperature predominantly for the ligands at the peripheral binding site makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible The same reason might also account for one of the paradoxes in cholinesterases activities at 25 C at low substrate concentrations are higher than at 37 C Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328 W82 D70 and Y332 amino acid residues stabilize binding of the inhibitor 2002 Elsevier Science
ESTHER : Golicnik_2002_Arch.Biochem.Biophys_398_23
PubMedSearch : Golicnik_2002_Arch.Biochem.Biophys_398_23
PubMedID: 11811945

Title : Comparison of protocols for measuring activities of human blood cholinesterases by the Ellman method - Reiner_2000_Arh.Hig.Rada.Toksikol_51_13
Author(s) : Reiner E , Sinko G , Skrinjaric-Spoljar M , Simeon-Rudolf V
Ref : Arh Hig Rada Toksikol , 51 :13 , 2000
Abstract : This paper presents a protocol for routine assays of human blood cholinesterase activities which separates erythrocytes from plasma by centrifugation and measures acetylcholinesterase activity in unwashed erythrocytes and butyrylcholinesterase activity in the plasma. The recommended substrate for both enzymes is 1.0 mM acetylthiocholine. The protocol is compared with other two recommended protocols for the activity measurements of the two enzymes using the Ellman method. The paper discusses the advantages and disadvantages of each and concludes with a proposal for an international agreement between laboratories for the evaluation of a standardized protocol.
ESTHER : Reiner_2000_Arh.Hig.Rada.Toksikol_51_13
PubMedSearch : Reiner_2000_Arh.Hig.Rada.Toksikol_51_13
PubMedID: 11059068