Sergei D. Varfolomeev

Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents

Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km > 106 M−1min−1) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.

Synthesis and study of the photochromic behavior of 3-[6′-nitro-1,3,3-trimethylspiro(indolino-2,2′-[2H]-chromen-5-yl)]propenoic acid and its ethyl ester

A new photochromic probe containing a spacer with the reactive carboxyl terminal group at the C5 atom of the photochrome molecule was synthesized. The spectral and kinetic study of the behavior of the new photochromic probes in toluene and ethanol was performed.

Comparison of anti-aggregatory activities of 5-phenyl-3-(3-pyridyl)isoxazole and 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole

Two bioisosteric analogs, 5-phenyl-3-(3-pyridyl)isoxazole and 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole, were synthesized so as to compare their antiaggregatory activities, to determine a pharmacologically active fragment in molecules of this type, and to explore the mechanisms of action of potential antiag-gregatory compounds belonging to the class of 3,5-substituted isoxazoles. Antiaggregatory activities of these compounds were studied in vitro using three aggregation inducers, such as arachidonic acid, adenosine diphosphate, and adrenaline. It was shown that 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole and 5-phenyl-3-(3-pyridyl)isoxazole completely suppressed platelet aggregation induced by arachidonic acid and the second wave of platelet aggregation induced by adrenaline or adenosine diphosphate. The antiaggregatory activity of substituted isoxasole was 1.1–1.5 times higher than that of substituted oxadiazole. In contrast to the isoxazole analog, 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole in concentrations of 300–400 μM partially suppressed the first wave of aggregation induced by adenosine diphosphate. It was demonstrated that both compounds were not thrombin inhibitors in vitro at concentrations up to 250 μM. Thus, introduction of a nitrogen atom into the C4-position of the isoxazole ring changes the molecule properties. It suggests that the pharmacophoric fragment of the molecule should be the whole isoxazole or 1,2,4-oxadiazole ring but not a part of the ring as was supposed previously.