Inhibitor Report for: Cyclosarin

The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.

The purpose of this study was to compare the therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6 (1-(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium )-2-butene dibromide), with presently used oximes (pralidoxime, obidoxime, methoxime) and H-oximes (HI-6, HLo-7) by in vitro and in vivo methods. In vitro, methoxime seems to be the most efficacious reactivator of GF agent-inhibited acetylcholinesterase because the phosphorylation of acetylcholinesterase by GF agent markedly increases its affinity for the enzyme. The oxime BI-6 is more efficacious than other presently used oximes (pralidoxime, obidoxime) but its reactivating efficacy does not reach the efficacy of H-oximes tested. On the other hand, obidoxime and pralidoxime appear to be very poor reactivators of GF agent-inhibited acetylcholinesterase because the phosphonylation of acetylcholinesterase by GF agent markedly decreases their affinity to the enzyme. In vivo, H oximes (HI-6, HLo-7) are the most efficacious antidotes for the treatment of acute poisoning with GF agent in rats while the presently used oximes such as pralidoxime and obidoxime are practically ineffective. BI-6 and methoxime are more efficacious than pralidoxime and obidoxime, nevertheless their therapeutic efficacy does not reach the efficacy of H oximes. Our results show that the ability of oximes to reactivate GF agent-inhibited acetylcholinesterase in vitro usually corresponds to their therapeutic effects against GF agent in vivo.

The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythrocyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5-60 min by obidoxime, pralidoxime, HI 6 or HL 7 (10 and 30 microM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HL 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HL 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HL 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.

Organophosphorus nerve agents (OPNAs) are highly toxic compounds inhibiting cholinergic enzymes in the central and autonomic nervous systems and neuromuscular junctions, causing severe intoxications in humans. Medical countermeasures and efficient decontamination solutions are needed to counteract the toxicity of a wide spectrum of harmful OPNAs including G, V and Novichok agents. Here, we describe the use of engineered OPNA-degrading enzymes for the degradation of various toxic agents including insecticides, a series of OPNA surrogates, as well as real chemical warfare agents (cyclosarin, sarin, soman, tabun, VX, A230, A232, A234). We demonstrate that only two enzymes can degrade most of these molecules at high concentrations (25 mM) in less than 5 min. Using surface assays adapted from NATO AEP-65 guidelines, we further show that enzyme-based solutions can decontaminate 97.6% and 99.4% of 10 gm(-)(2) of soman- and VX-contaminated surfaces, respectively. Finally, we demonstrate that these enzymes can degrade ethyl-paraoxon down to sub-inhibitory concentrations of acetylcholinesterase, confirming their efficacy from high to micromolar doses.

The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue SCPEP1 as potential targets of organophosphates (OP). CatA could be stably inhibited by low microM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 microM followed by VR (KI = 2.8 microM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 x 10(3) M(-1)sec(-1) and 1.2 x 10(3) M(-1)sec(-1), respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KI(DFP) = 13 microM and 11 microM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure-activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k(i)), for the spontaneous dealkylation (k(a)) and reactivation (k(s)) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLo 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure-activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLo 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure-activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.

The purpose of this study was to compare the therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6 (1-(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium )-2-butene dibromide), with presently used oximes (pralidoxime, obidoxime, methoxime) and H-oximes (HI-6, HLo-7) by in vitro and in vivo methods. In vitro, methoxime seems to be the most efficacious reactivator of GF agent-inhibited acetylcholinesterase because the phosphorylation of acetylcholinesterase by GF agent markedly increases its affinity for the enzyme. The oxime BI-6 is more efficacious than other presently used oximes (pralidoxime, obidoxime) but its reactivating efficacy does not reach the efficacy of H-oximes tested. On the other hand, obidoxime and pralidoxime appear to be very poor reactivators of GF agent-inhibited acetylcholinesterase because the phosphonylation of acetylcholinesterase by GF agent markedly decreases their affinity to the enzyme. In vivo, H oximes (HI-6, HLo-7) are the most efficacious antidotes for the treatment of acute poisoning with GF agent in rats while the presently used oximes such as pralidoxime and obidoxime are practically ineffective. BI-6 and methoxime are more efficacious than pralidoxime and obidoxime, nevertheless their therapeutic efficacy does not reach the efficacy of H oximes. Our results show that the ability of oximes to reactivate GF agent-inhibited acetylcholinesterase in vitro usually corresponds to their therapeutic effects against GF agent in vivo.

The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythrocyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5-60 min by obidoxime, pralidoxime, HI 6 or HL 7 (10 and 30 microM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HL 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HL 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HL 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.

Cyclohexylmethylphosphonofluoridate (cyclosarin) is a highly toxic organophosphate, which was shown to be rather resistant to conventional oxime therapy. To give more insight into the inhibition, reactivation and aging kinetics, human acetyl-(AChE) and butyrylcholinesterase (BChE) were inhibited by cyclosarin (k2 of 7.4 and 3.8 x 10(8) M(-1) min(-1), respectively; pH 7.4, 37 degrees C) and reactivated with obidoxime, pralidoxime and three experimental oximes. The new oxime HL 7 (1-[[[4-aminocarbonyl)-pyridinio]-methoxy]-methyl]-2,4-bis-[ (hydroxyimino)methyl] pyridinium dimethanesulphonate) was shown to be superior to the other oximes. At oxime concentrations anticipated to be relevant in humans, obidoxime and pralidoxime were extremely weak reactivators of AChE. Aging velocity of BChE was almost fourfold higher compared to AChE (ka of 0.32 h(-1) and 0.08 h(-1), respectively). A substantial spontaneous reactivation was observed with AChE. These results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning. By contrast, HL 7 was shown to be an extremely potent reactivator of human AChE and BChE, which supports its position as a broad-spectrum oxime.

Changes in serum biochemical and hematological parameters were studied in 20 male rhesus monkeys following acute poisoning by the organophosphate nerve agent cyclohexylmethylphosphonofluoridate (CMPF or GF). Animals were challenged with 5 x LD50 GF (233 micrograms/kg, IM) following pretreatment with pyridostigmine (0.3-0.7 mg/kg per 24 h) and treated with atropine (0.4 mg/kg, IM) and either 2-PAM (25.7 mg/kg, IM) or H16 (37.8 mg/kg, IM) at the onset of clinical signs or at 1 min after exposure. Muscle fasciculations, tremors, or convulsions occurred in 19 of 20 animals. Serum biochemical and hematologic parameters were analyzed 2 days and 7 days after exposure and compared to pre-exposure baseline values. Significant increases in creatine kinase (CK), lactate dehydrogenase (LD), aspartate transaminase (AST), alanine transaminase (ALT) and potassium ion (K+), associated with damage to striated muscle and metabolic acidosis, occurred in both oxime-treated groups 2 days after exposure. Total protein, albumin, red blood cell (RBC) count, hemoglobin concentration (Hb) and hematocrit (Hct), were decreased in both oxime-treated groups at 7 days. The results demonstrate that animals exposed to a single high dose of GF and treated with standard therapy exhibit changes in serum biochemical and hematological indices directly and indirectly associated with their clinical presentations.

Cyclohexylmethylphosphonofluoridate (CMPF) is an organophosphate cholinesterase inhibitor with military significance. The purpose of these studies was 1) to determine the acute toxicity of CMPF in the male rhesus monkey, 2) to evaluate the efficacy of pyridostigmine (PYR) pretreatment plus atropine and oxime (2-PAM or H16) treatment, and 3) to evaluate the pathological consequences of acute poisoning. An i.m. LD50 of CMPF was estimated using an up-and-down dose selection procedure and 12 animals. The 48-h and 7-day LD50 was 46.6 micrograms/kg, i.m. In the protection experiments, pyridostigmine (0.3-0.7 mg/kg/24 h) was administered by surgically implanted osmotic minipumps for 3-12 days resulting in 21-65% inhibition of erythrocyte acetylcholinesterase activity. Animals were challenged with 5 x L50 CMPF (233 micrograms/kg) and treated with atropine (0.4 mg/kg) and either 2-PAM (25.7 mg/kg) or HI6 (37.8 mg/kg) at the onset of signs or 1 min after challenge. Osmotic pumps were removed within 30 min after agent challenge. Pyridostigmine, atropine, and either 2-PAM or H16 were completely effective against CMPF, saving ten of ten animals in each group. In comparison, three of five animals challenged with 5 x LD50 of soman and treated with atropine and 2-PAM survived 7 days. The primary histologic lesions in the acute toxicity group were neuronal degeneration/necrosis and spinal cord hemorrhage. The CMPF treated groups (total of 20 animals) had minimal nervous system changes with no significant lesion difference resulting from the different oxime therapies. The primary non-neural lesions were degenerative cardiomyopathy and skeletal muscle degeneration which occasionally progressed to necrosis and mineralization.