Reactivator Report for: RS194B

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticide exposures; significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include admin-istration of atropine with an oxime, to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently-used oximes either reactivate poorly, do not readily cross the blood brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure eg RS194B have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticide or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these studies and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos, which require oxidation by P450 in the liver to convert inactive thions to the active toxic oxon forms, and once again demonstrated its efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 hours was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

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.

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

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticide exposures; significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include admin-istration of atropine with an oxime, to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently-used oximes either reactivate poorly, do not readily cross the blood brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure eg RS194B have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticide or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these studies and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos, which require oxidation by P450 in the liver to convert inactive thions to the active toxic oxon forms, and once again demonstrated its efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 hours was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

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.

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

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.

Oxime antidotes regenerate organophosphate-inhibited acetylcholinesterase (AChE). Although they share a common mechanism of AChE reactivation, the rate and amount of oxime that enters the brain are critical to the efficacy, a process linked to the oxime structure and charge. Using a platform based on the organophosphate [(18) F]-VXS as a positron emission tomography tracer for active AChE, the in vivo distribution of [(18) F]-VXS was evaluated after an LD50 dose (250 mug/kg) of the organophosphate paraoxon (POX) and following oximes as antidotes. Rats given [(18) F]-VXS tracer alone had significantly higher radioactivity (two- to threefold) in the heart and lung than rats given LD50 POX at 20 or 60 min prior to [(18) F]-VXS. When rats were given LD50 POX followed by 2-PAM (cationic), RS194b (ionizable), or monoisonitrosoacetone (MINA) (neutral), central nervous system (CNS) radioactivity returned to levels at or above untreated naive rats (no POX), whereas CNS radioactivity did not increase in rats given the dication oximes HI-6 or MMB-4. MINA showed a significant, pairwise increase in CNS brain radioactivity compared with POX-treated rats. This new in vivo dynamic platform using [(18) F]-VXS tracer measures and quantifies peripheral and CNS relative changes in AChE availability after POX exposure and is suitable for comparing oxime delivery and AChE reactivation in rats.

The deleterious effects of nerve agents over the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) turned these compounds into the most dangerous chemical weapons known. Among the antidotes in use today against these agents, oximes in combination with other drugs are the only treatment with any action. HI-6 and 2-PAM are cationic oximes proved to be effective for the reactivation of AChE inhibited by the nerve agents VX and sarin (GB). However, when it comes to reactivation of AChE inside the central or peripheral nervous systems, charged molecules present low diffusion due to low penetration through the blood-brain barrier. Uncharged oximes appear as an interesting alternative to solve this problem, but the development and enhancement of more efficient uncharged oximes capable of reactivating human AChE is still necessary. Given the limitations for in vivo and in vitro experimental studies with nerve agents, modeling is an important tool that can contribute to a better understanding of factors that may affect the efficiency of uncharged oximes. In order to investigate the interaction and behavior of cationic and uncharged oximes, we performed here molecular docking, molecular dynamics simulations, and binding energies calculations of the known cationic oximes HI-6 and 2-PAM plus four uncharged oximes found in the literature, complexed with human AChE (HssACHE) conjugated with the nerve agents VX and GB. The uncharged oximes showed different behaviors, especially RS194B, which presented stability inside AChE-VX, but presented free binding energy lower than cationic oximes, suggesting that structural alterations could favor its interactions with these complexes. In contrast, HI-6 and 2-PAM showed higher affinities with more negative binding energy values and larger contribution of the amino acid Asp74, demonstrating the importance of the quaternary nitrogen to the affinity and interaction of oximes with AChE-GB and AChE-VX conjugates.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

The neutral oxime reactivator RS194B with a seven-membered ring has shown better efficacy towards the tabun-inhibited AChE than that of RS69N with a six-membered ring and RS41A with a five-membered ring. The difference in the efficacy of these reactivators has remained unexplored. We report here the origin of the difference of efficacy of these reactivators based on the conformational analysis, quantum chemical calculations and steered molecular dynamics (SMD) simulations. The conformational analysis using B3LYP/6-31G(d) level of theory revealed that RS41A and RS194B are more stable in gauche conformation due to the gauche effect (-N-C-C-N- bonds) whereas RS69N prefers anti-conformation. The SMD simulations show that RS194B retains in more stable gauche conformation inside the active gorge of AChE during different time intervals that experiences more hydrogen bonding, hydrophobic interactions with the catalytic anionic site (CAS) residues and weaker interactions with the peripheral anionic site (PAS) residues compared to RS41A and RS69N. In an effort to design an even superior reactivator, RS194B-S has been chosen with a subtle change in the geometry of RS194B by replacing the carbonyl oxygen with the sulfur atom. The newly designed reactivator RS194B-S can also be a promising candidate to reactivate tabun-inhibited AChE.

Fatalities from organophosphate (OP) insecticide result from both occupational and deliberate exposure; significantly impacting human health. Like nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with a pyridinium aldoximes e.g. pralidoxime, to reactivate the OP-inhibited AChE. However, commonly used oximes inefficiently cross the blood brain barrier and are rapidly cleared and their benefit is debated. Recent findings have demonstrated the ability of a novel zwitterionic, centrally acting, brain penetrating oxime (RS194B) to reverse severe symptoms and rapidly reactivate sarin-inhibited AChE in macaques but has not been tested following OP pesticide poisoning. The severe symptoms following a lethal dose of inhaled paraoxon (100ug/kg), which mimicked those in insecticide poisoned individuals, were rapidly reversed in macaques by post-exposure IM administration of 80mg/kg of RS194B. This occurred with a concomitant reactivation of AChE to 40-100% in <1hr and BChE (40% in 8hr). These findings will be used to develop a macaque model with RS194B as a post-exposure treatment for insecticide poisoning and generate efficacy data for approval under the FDA Animal rule.

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

Organophosphorus-based (OP) nerve agents represent some of the most toxic substances known to mankind. The current standard of care for exposure has changed very little in the past decades, and relies on a combination of atropine to block receptor activity and oxime-type acetylcholinesterase (AChE) reactivators to reverse the OP binding to AChE. Although these oximes can block the effects of nerve agents, their overall efficacy is reduced by their limited capacity to cross the blood-brain barrier (BBB). RS194B, a new oxime developed by Radic et al. (J. Biol. Chem., 2012) has shown promise for enhanced ability to cross the BBB. To fully assess the potential of this compound as an effective treatment for nerve agent poisoning, a comprehensive evaluation of its pharmacokinetic (PK) and biodistribution profiles was performed using both intravenous and intramuscular exposure routes. The ultra-sensitive technique of accelerator mass spectrometry was used to quantify the compound's PK profile, tissue distribution, and brain/plasma ratio at four dose concentrations in guinea pigs. PK analysis revealed a rapid distribution of the oxime with a plasma t1/2 of approximately 1 h. Kidney and liver had the highest concentrations per gram of tissue followed by lung, spleen, heart and brain for all dose concentrations tested. The Cmax in the brain ranged between 0.03 and 0.18% of the administered dose, and the brain-to-plasma ratio ranged from 0.04 at the 10 mg/kg dose to 0.18 at the 200 mg/kg dose demonstrating dose dependent differences in brain and plasma concentrations. In vitro studies show that both passive diffusion and active transport contribute little to RS194B traversal of the BBB. These results indicate that biodistribution is widespread, but very low quantities accumulate in the guinea pig brain, indicating this compound may not be suitable as a centrally active reactivator.

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

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

The currently fielded pre-hospital therapeutic regimen for the treatment of organophosphorus (OP) poisoning in the United States (U.S.) is the administration of atropine in combination with an oxime antidote (2-PAM Cl) to reactivate inhibited acetylcholinesterase (AChE). Depending on clinical symptoms, an anticonvulsant, e.g., diazepam, may also be administered. Unfortunately, 2-PAM Cl does not offer sufficient protection across the range of OP threat agents, and there is some question as to whether it is the most effective oxime compound available. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection at the FDA approved human equivalent dose (HED) of 2-PAM Cl against tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX, and the pesticides paraoxon, chlorpyrifos oxon, and phorate oxon. Male Hartley guinea pigs were subcutaneously challenged with a lethal level of OP and treated at approximately 1min post challenge with atropine followed by equimolar oxime therapy (2-PAM Cl, HI-6 DMS, obidoxime Cl2, TMB-4(Trimedoxime), MMB4-DMS, HLo-7 DMS, MINA, and RS194B) or therapeutic-index (TI) level therapy (HI-6 DMS, MMB4-DMS, MINA, and RS194B). Clinical signs of toxicity were observed for 24h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellman's method. When the oxime is standardized against the HED of 2-PAM Cl for guinea pigs, the evidence from clinical observations, lethality, quality of life (QOL) scores, and cholinesterase reactivation rates across all OPs indicated that MMB4 DMS and HLo-7 DMS were the two most consistently efficacious oximes.

Organophosphates (OP) inhibit acetylcholinesterase (AChE, EC 3.1.1.7), both in peripheral tissues and central nervous system (CNS), causing adverse and sometimes fatal effects due to the accumulation of neurotransmitter acetylcholine (ACh). The currently used therapy, focusing on the reactivation of inhibited AChE, is limited to peripheral tissues because commonly used quaternary pyridinium oxime reactivators do not cross the blood brain barrier (BBB) at therapeutically relevant levels. A directed library of thirty uncharged oximes that contain tertiary amine or imidazole protonable functional groups that should cross the BBB as unionized species was tested as tabun-hAChE conjugate reactivators along with three reference oximes: DAM (diacetylmonoxime), MINA (monoisonitrosoacetone), and 2-PAM. The oxime RS150D [N-((1-(3-(2-((hydroxyimino)methyl)-1H-imidazol-1-yl)propyl)-1H-1,2,3-triazol-4-y l)methyl)benzamide] was highlighted as the most promising reactivator of the tabun-hAChE conjugate. We also observed that oximes RS194B [N-(2-(azepan-1-yl)ethyl)-2-(hydroxyimino)acetamide] and RS41A [2-(hydroxyimino)-N-(2-(pyrrolidin-1-yl)ethyl)acetamide], which emerged as lead uncharged reactivators of phosphylated hAChE with other OPs (sarin, cyclosarin and VX), exhibited only moderate reactivation potency for tabun inhibited hAChE. This implies that geometry of oxime access to the phosphorus atom conjugated to the active serine is an important criterion for efficient reactivation, along with the chemical nature of the conjugated moiety: phosphorate, phosphonate, or phosphoramidate. Moreover, modification of the active center through mutagenesis enhances the rates of reactivation. The phosphoramidated-hAChE choline-binding site mutant Y337A showed three-times enhanced reactivation capacity with non-triazole imidazole containing aldoximes (RS113B, RS113A and RS115A) and acetamide derivative (RS194B) than with 2PAM.

A library of more than 200 novel uncharged oxime reactivators was used to select and refine lead reactivators of human acetylcholinesterase (hAChE) covalently conjugated with sarin, cyclosarin, VX, paraoxon and tabun. N-substituted 2-hydroxyiminoacetamido alkylamines were identified as best reactivators and reactivation kinetics of the lead oximes, RS41A and RS194B, were analyzed in detail. Compared to reference pyridinium reactivators, 2PAM and MMB4, molecular recognition of RS41A reflected in its Kox constant was compromised by an order of magnitude on average for different OP-hAChE conjugates, without significant differences in the first order maximal phosphorylation rate constant k2. Systematic structural modifications of the RS41A lead resulted in several-fold improvement with reactivator, RS194B. Kinetic analysis indicated Kox reduction for RS194B as the main kinetic constant leading to efficient reactivation. Subtle structural modifications of RS194B were used to identify essential determinants for efficient reactivation. Computational molecular modeling of RS41A and RS194B interactions with VX inhibited hAChE, bound reversibly in Michaelis type complex and covalently in the pentacoordinate reaction intermediate suggests that the faster reactivation reaction is a consequence of a tighter RS194B interactions with hAChE peripheral site (PAS) residues, in particular with D74, resulting in lower interaction energies for formation of both the binding and reactivation states. Desirable in vitro reactivation properties of RS194B, when coupled with its in vivo pharmacokinetics and disposition in the body, reveal the potential of this oxime design as promising centrally and peripherally active antidotes for OP toxicity.

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K(ox)) and maximum reactivation rate (k(2)), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.