Past assassinations and terrorist attacks demonstrate the need for a more effective antidote against nerve agents and other organophosphates (OP) that cause brain damage through inhibition of acetylcholinesterase (AChE). Our lab has invented a platform of phenoxyalkyl pyridinium oximes (US patent 9,277,937) that demonstrate the ability to cross the blood-brain barrier in in vivo rat tests with a sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP) and provide evidence of brain penetration by reducing cessation time of seizure-like behaviors, accumulation of glial fibrillary acidic protein (GFAP), and hippocampal neuropathology, as opposed to the currently approved oxime, 2-pyridine aldoxime methyl chloride (2-PAM). Using two of the novel oximes (Oximes 1 and 20), this project examined whether gene expression changes might help explain this protection. Expression changes in the piriform cortex were examined using polymerase chain reaction arrays for inflammatory cytokines and receptors. The hippocampus was examined via quantitative polymerase chain reaction for the expression of immediate-early genes involved in brain repair (Bdnf), increasing neurotoxicity (Fos), and apoptosis control (Jdp2, Bcl2l1, Bcl2l11). In the piriform cortex, NIMP significantly stimulated expression for the macrophage inflammatory proteins CCL4, IL-1A, and IL-1B. Oxime 20 by itself elicited the most changes. When it was given therapeutically post-NIMP, the largest change occurred: a 310-fold repression of the inflammatory cytokine, CCL12. In the hippocampus, NIMP increased the expression of the neurotoxicity marker Fos and decreased the expression of neuroprotective Bdnf and antiapoptotic Bcl2l1. Compared with 2-PAM, Oxime 20 stimulated Bcl2l1 expression more and returned expression closer to the vehicle control values.
The efficiency of human butyrylcholinesterase (BChE) as a stoichiometric bioscavenger of nerve agents is well established. However, wide use is currently limited by production and purification costs. Aiming at identifying an alternative human protein bioscavenger, we looked for an original scaffold candidate by virtual screening of the Protein Data Bank for functional similarity using the "Surfing the Molecules" software (sumo-pbil.ibcp.fr) and a search model based on the BChE active site topology. Besides the expected acetylcholinesterase and butyrylcholinesterase, we identified a set of bile salt activated lipases structures, among which the human pancreatic lipase (hBAL) that shares 34% identity with BChE. We produced the recombinant enzyme in mammalian cells, purified it, and measured the inhibition constants for paraoxon and surrogates of VX, sarin and tabun. We solved the X-ray structure of apo hBAL and conjugates with paraoxon and the surrogates at resolutions in the 2-A range. These structures allow the assessment of hBAL for scavenging nerve agents. They revealed that hBAL has inverted stereoselectivity for the surrogates of nerve agent compared to human cholinesterases. We observed a remarkable flip of the catalytic histidine driven by the chelation of Zn(2+). Dealkylation of the conjugate, aka aging, was solely observed for paraoxon.
Title: The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE Chao CK, Balasubramanian N, Gerdes JM, Thompson CM Ref: Chemico-Biological Interactions, 291:220, 2018 : PubMed
In this study, the mechanisms of HuAChE and HuBChE inhibition by Me-P(O) (OPNP) (OR) [PNP = p-nitrophenyl; R = CH(2)CH(3), CH(2)CH(2)F, OCH(CH(3))(2), OCH(CH(3)) (CH(2)F)] representing surrogates and fluoro-surrogates of VX and sarin were studied by in vitro kinetics and mass spectrometry. The in vitro measures showed that the VX- and fluoro-VX surrogates were relatively strong inhibitors of HuAChE and HuBChE (k(i) - 10(5)-10(6) M(-1)min(-1)) and underwent spontaneous and 2-PAM-mediated reactivation within 30 min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (k(i) - 10(4)-10(5) M(-1)min(-1)), and in general did not undergo spontaneous reactivation, although HuAChE adducts were partially reactivatable at 18 h using 2-PAM. The mechanism of HuAChE and HuBChE inhibition by the surrogates was determined by Q-TOF and MALDI-TOF mass spectral analyses. The surrogate-adducted proteins were trypsin digested and the active site-containing peptide bearing the OP-modified serine identified by Q-TOF as triply- and quadruply-charged ions representing the respective increase in mass of the attached OP moiety. Correspondingly, monoisotopic ions of the tryptic peptides representing the mass increase of the OP-adducted peptide was identified by MALDI-TOF. The mass spectrometry analyses validated the identity of the OP moiety attached to HuAChE or HuBChE as MeP(O) (OR)-O-serine peptides (loss of the PNP leaving group) via mechanisms consistent with those found with chemical warfare agents. MALDI-TOF MS analyses of the VX-modified peptides versus time showed a steady reduction in adduct versus parent peptide (reactivation), whereas the sarin-surrogate-modified peptides remained largely intact over the course of the experiment (24 h). Overall, the presence of a fluorine atom on the surrogate modestly altered the rate constants of inhibition and reactivation, however, the mechanism of inhibition (ejection of PNP group) did not change.
Title: Novel brain-penetrating oximes for reactivation of cholinesterase inhibited by sarin and VX surrogates Chambers JE, Meek EC, Chambers HW Ref: Annals of the New York Academy of Sciences, 1374:52, 2016 : PubMed
Current oxime reactivators for organophosphate-inhibited cholinesterase (ChE) do not effectively cross the blood-brain barrier and therefore cannot restore brain ChE activity in vivo. Our laboratories have studied highly relevant sarin and VX surrogates, which differ from their respective nerve agents only in the leaving group and thereby leave ChE phosphylated with the same chemical moiety as sarin and VX. Our laboratories have developed novel substituted phenoxyalkyl pyridinium oximes that lead to reduced ChE inhibition in the brains of rats challenged with a high sublethal dosage of the sarin surrogate, whereas 2-PAM did not, using a paradigm designed to demonstrate brain penetration. In addition, treatment of rats with these novel oximes is associated with attenuation of seizure-like behavior compared to rats treated with 2-PAM, providing additional evidence that the oximes penetrate the blood-brain barrier. Further, some of the oximes provided 24-h survival superior to 2-PAM, and shortened the duration of seizure-like behavior when rats were challenged with lethal dosages of the sarin and VX surrogates, providing additional support for the conclusion that these oximes penetrate the brain.
Title: Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase Coban A, Carr RL, Chambers HW, Willeford KO, Chambers JE Ref: Toxicol Lett, 248:39, 2016 : PubMed
Because testing of nerve agents is limited to only authorized facilities, our laboratory developed several surrogates that resemble nerve agents because they phosphylate the acetylcholinesterase (AChE) with the same moiety as the actual nerve agents. The inhibition kinetic parameters were determined for AChE by surrogates of cyclosarin (NCMP), sarin (NIMP, PIMP and TIMP) and VX (NEMP and TEMP) and other organophosphorus compounds derived from insecticides. All compounds were tested with rat brain and a subset was tested with mouse brain and purified human erythrocyte AChE. Within the compounds tested on all AChE sources, chlorpyrifos-oxon had the highest molecular rate constant followed by NCMP and NEMP. This was followed by NIMP then paraoxon and DFP with rat and mouse brain AChE but DFP was a more potent inhibitor than NIMP and paraoxon with human AChE. With the additional compounds tested only in rat brain, TEMP was slightly less potent than NEMP but more potent than PIMP which was more potent than NIMP. Methyl paraoxon was slightly less potent than paraoxon but more potent than TIMP which was more potent than DFP. Overall, this study validates that the pattern of inhibitory potencies of our surrogates is comparable to the pattern of inhibitory potencies of actual nerve agents (i.e., cyclosarin>VX>sarin), and that these are more potent than insecticidal organophosphates.
Title: Testing of novel brain-penetrating oxime reactivators of acetylcholinesterase inhibited by nerve agent surrogates Chambers JE, Chambers HW, Meek EC, Pringle RB Ref: Chemico-Biological Interactions, 203:135, 2013 : PubMed
A critical need for combating the effects of organophosphate (OP) anticholinesterases, such as nerve agents, is the current lack of an effective oxime reactivator which can penetrate the blood-brain barrier (BBB), and therefore reactivate inhibited acetylcholinesterase (AChE) in the brain. Our laboratories have synthesized and have initiated testing of novel phenoxyalkyl pyridinium oximes (patent pending) that are more lipophilic than currently approved oximes. This is a preliminary report on these novel oximes which have been tested in vitro in rat brain homogenates with highly relevant surrogates for sarin (phthalimidyl isopropyl methylphosphonate; PIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). The oximes demonstrated a range of 14-76% reactivation of rat brain AChE in vitro. An in vivo testing paradigm was developed in which the novel oxime was administered at the time of maximal brain AChE inhibition (about 80%) (1h) elicited by nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate). This paradigm, with delayed administration of oxime to a time when brain AChE was starting to recover, was designed to minimize reactivation/reinhibition of peripheral AChE during the reactivation period which would decrease the availability of the surrogate for entry into the brain; this paradigm will allow proof of concept of BBB penetrability. The initial studies of these oximes in vivo with the sarin surrogate NIMP have indicated reactivation of up to about 25% at 30min after oxime administration and substantial attenuation of seizure behavior from some of the oximes. Therefore these novel oximes have considerable potential as brain-protecting therapeutics for anticholinesterases.
Four nonvolatile nerve agent surrogates, 4-nitrophenyl ethyl dimethylphosphoramidate (NEDPA, a tabun surrogate), 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate), and two sarin surrogates, phthalimidyl isopropyl methylphosphonate (PIMP) and 4-nitrophenyl isopropyl methylphosphonate (NIMP), were synthesized and tested as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. These surrogates were designed to phosphorylate cholinesterases with the same moiety as their respective nerve agents, making them highly relevant for the study of cholinesterase reactivators. Surrogates were characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. NEMP, PIMP, and NIMP were potent inhibitors of rat brain, skeletal muscle, diaphragm, and serum AChE as well as human erythrocyte AChE and serum BuChE in vitro. PIMP was determined to degrade quickly in aqueous solution, making it useful for in vitro assays only, and NEDPA was not a potent inhibitor of AChE or BuChE in vitro; therefore, these two surrogates were not tested in subsequent in vivo studies. Sublethal dosages (yielding about 80% brain AChE inhibition) were determined for both the stable sarin surrogate, NIMP (0.325 mg/kg ip), and the VX surrogate, NEMP (0.4 mg/kg ip), in adult male rats. Time course studies indicated the time to peak brain AChE inhibition for both NIMP and NEMP to be 1 h postexposure. Both surrogates yielded severe cholinergic signs. These dosages did not require the addition of atropine to prevent lethality, and the rate of AChE aging was slow, making these surrogates useful for reactivation studies both in vitro and in vivo. The surrogates synthesized in this study are potent yet safer to test than nerve agents and are useful tools for initial screening of nerve agent oxime therapeutics.
In order to enhance the enzymatic detoxification rate of organophosphorus (OP) nerve agents we have searched for more active variants of recombinant mammalian paraoxonase (PON1). We have previously identified three key positions in PON1 that affect OP hydrolysis: Leu69, Val346 and His115, that significantly enhance the hydrolysis of cyclosarin (GF), soman, chlorpyrifos-oxon (ChPo), O-isopropyl-O-(p-nitrophenyl)methylphosphonate (IMP-pNP) and diisopropyl fluorophosphate (DFP). GC/FPD analysis compared to residual AChE inhibition assay displayed stereoselective hydrolysis of GF, soman and IMP-pNP, indicating that wild type PON1 and its variant V346A are more active toward the less toxic P(+) optical isomer. In order to obtain new PON1 variants with reversed stereoselectivity, displaying augmented activity toward the more toxic isomer P(-) of nerve agents, we synthesized new asymmetric fluorogenic OPs (Flu-OPs). Six Flu-OPs were prepared containing either ethyl (E), cyclohexyl (C) or pinacolyl (P) alkyl radicals attached to methyl-phosphonyl (MP) moiety analogous to the structure of VX, GF and soman, respectively. The fluorescent moieties are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy-9,9-dimethyl-9H-acridin-2-one (DDAO). The kinetics of AChE and BChE inhibition by these new Flu-OPs display k(i) values 8.5x10(4) to 8.5x10(7) and 5x10(4) to 2x10(6)M(-1)min(-1), respectively. EMP-MeCyC and EMP-DDAO are the most active inhibitors of AChE whereas CMP-MeCyC and CMP-DDAO are better inhibitors of BChE than AChE, indicating accommodation of bulky cyclohexyl group inside the active site of BChE. PMP-MeCyC and PMP-DDAO are the least active inhibitors of both AChE and BChE. CMP-MeCyC and CMP-DDAO were significantly detoxified only by the five-site mutations PON1 variant L69V/S138L/S193P/N287D/V346A. Degradation kinetics of Flu-OPs measured by increase in absorbance of the released fluorogenic group was fit by a two exponential function, indicating faster hydrolysis of the less toxic optical isomer. Interestingly, wt PON1 caused only 50% degradation of racemic EMP-MeCyC, CMP-MeCyC and CMP-DDAO indicating complete hydrolysis of P(+) isomer. This remarkable stereoselectivity was used for the enzymatic separation of the P(-) isomer of CMP-MeCyC. The bimolecular rate constant k(i) for human AChE inhibition by the isolated P(-) isomer of CMP-MeCyC is five-fold larger than that of its P(+) isomer. The marked preference of wt PON1 toward P(+) stereo-isomer of CMP-MeCyC and CMP-DDAO renders their P(-) stereo-isomers suitable for the selection of new OP hydrolase variants with reversed stereoselectivity.
We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.
Title: New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes Ohta H, Ohmori T, Suzuki S, Ikegaya H, Sakurada K, Takatori T Ref: Pharm Res, 23:2827, 2006 : PubMed
INTRODUCTION: A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). RESULTS AND DISCUSSION: This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. CONCLUSION: Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.
