Ashima Saxena

Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase

Amino acid sequence alignments of cholinesterases revealed that 6 of 14 aromatic amino acid residues lining the active center gorge of acetylcholinesterase are replaced by aliphatic amino acid residues in butyrylcholinesterase. The Y337 (F330) in mammalian acetylcholinesterase, which is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridines and one end of bisquaternary compounds such as BW284C51 and decamethonium. Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Inhibition studies of (-)-huperzine A with human butyrylcholinesterase mutants, where A328 (KI = 194.6 microM) was modified to either F (KI = 0.6 microM, as in Torpedo acetylcholinesterase) or Y (KI = 0.032 microM, as in mammalian acetylcholinesterase), confirmed previous observations made with acetylcholinesterase mutants that this residue is important for binding huperzine A. Inhibition studies of ethopropazine with butyrylcholinesterase mutants, where A328 (KI = 0.18 microM) was modified to either F (KI = 0.82 microM) or Y (KI = 0.28 microM), suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active site gorge showed that the poor inhibitory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to accommodate the bulky inhibitor molecule. The volume of the butyrylcholinesterase active site gorge is approximately 200 A3 larger than that of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations.

Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200mg will be required to protect a human against 2×LD(50) of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at ∼24h and had a mean residence time of 78-103h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5×LD(50) of soman or 8×LD(50) of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents.

Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators

Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.

Bioscavenger for protection from toxicity of organophosphorus compounds.

Current antidotal regimens for organophosphorus compound (OP) poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and postexposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride (Gray, 1984). Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits, or, in many cases, permanent brain damage (Dunn and Sidell, 1989). These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets. Several studies over the last two decades have demonstrated that exogenously administered human serum butyrylcholinesterase (Hu BChE) can be used successfully as a safe, efficacious, and single prophylactic treatment to counteract the toxicity of OPs. It also has potential use for first responders (civilians) reacting to terrorist nerve gas release, pesticide overexposure, or succinylcholine-induced apnea. A dose of 200 mg of Hu BChE in humans is envisioned as a prophylactic treatment that can protect from exposure of 2-5 x LD50 of nerve agents (Ashani, 2000).

In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity

A novel approach for treating organophosphorus (OP) poisoning is the use of enzymes, both stoichiometric and catalytic, as bioscavengers to sequester these compounds in circulation before they reach their physiological targets. Human serum butyrylcholinesterase and a recombinant form of this enzyme produced in the milk of transgenic goats have completed Phase I clinical trials as stoichiometric bioscavengers for the protection of humans against OP nerve agents. However, a major limitation of the first generation bioscavenger is the 1:1 stoichiometry between the enzyme and the OP. Therefore, efforts are underway to develop the second generation catalytic bioscavenger, which will neutralize/hydrolyze multiple OP molecules. To avoid any complications related to adverse immune reactions, three enzymes from human (Hu) sources are being considered for development as catalytic bioscavengers: (1) prolidase; (2) paraoxonase 1 (PON1); (3) senescence marker protein-30 (SMP-30). Towards this effort, native or recombinant (r) forms of candidate catalytic bioscavengers were isolated and characterized for their ability to hydrolyze G-type nerve agents at concentrations of 10muM and 1mM. Results show that mammalian enzymes were significantly less efficient at hydrolyzing nerve agents as compared to bacterial organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA). Recombinant Hu prolidase was the most efficient and the only mammalian enzyme that hydrolyzed all four G-type nerve agents. On the other hand, both rHu PON1 and Mo SMP-30 showed 10-fold lower activity towards sarin compared to rHu prolidase and did not hydrolyze tabun. Based on these results, Hu prolidase appears to be the most promising candidate for further development: (1) it can be easily expressed in E. coli; (2) of the three candidate enzymes, it is the only enzyme that hydrolyzes all four G-type agents. Efforts to improve the catalytic efficiency of this enzyme towards OP nerve agents are underway.

Exploiting Protein Fluctuations at the Active-Site Gorge of Human Cholinesterases: Further Optimization of the Design Strategy to Develop Extremely Potent Inhibitors

Protein conformational fluctuations are critical for biological functions, although the relationship between protein motion and function has yet to be fully explored. By a thorough bioinformatics analysis of cholinesterases (ChEs), we identified specific hot spots, responsible for protein fluctuations and functions, and those active-site residues that play a role in modulating the cooperative network among the key substructures. This drew the optimization of our design strategy to discover potent and reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase (hAChE and hBuChE) that selectively interact with specific protein substructures. Accordingly, two tricyclic moieties differently spaced by functionalized linkers were investigated as molecular yardsticks to probe the finest interactions with specific hot spots in the hChE gorge. A number of SAR trends were identified, and the multisite inhibitors 3a and 3d were found to be the most potent inhibitors of hBuChE and hAChE known to date.

Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning

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.

Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases.

Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2-3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.

Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase.

We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.

Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Göttingen minipigs exposed to sarin vapor

Human serum butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a prophylactic countermeasure against organophosphorus nerve agents. This study was designed to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to a lethal dose of sarin (GB) vapor. Male Göttingen minipigs were subjected to: air exposure, GB vapor exposure, or pretreatment with Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection 24 h prior to exposure to 4.1 mg/m(3) of GB vapor for 60 min. Electrocardiograms (ECG), electroencephalograms (EEG), and pupil size were recorded throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB present. Untreated animals exposed to GB vapor exhibited cardiac abnormalities and generalized seizures, ultimately succumbing to respiratory failure. Pretreatment with 3.0 or 6.5 mg/kg of Hu BChE delayed blood gas and acid-base disturbances and the onset of cardiac and neural toxic signs, but failed to increase survivability. Pretreatment with 7.5 mg/kg of Hu BChE, however, completely prevented toxic signs, with blood chemistry and ECG and EEG parameters indistinguishable from control during and after GB exposure. GB bound in plasma was 200-fold higher than plasma from pigs that did not receive Hu BChE, suggesting that Hu BChE scavenged GB in blood and prevented it from reaching other tissues. Thus, prophylaxis with Hu BChE alone not only increased survivability, but also prevented cardiac abnormalities and neural toxicity in minipigs exposed to a lethal dose of GB vapor.