Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([(11)C]POX) and profiling in live rats is reported. Naive rats intravenously injected with [(11)C]POX showed a rapid decrease in parent tracer to ~ 1% with an increase in radiolabeled serum proteins to 87%, and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 min reflecting covalent modification of proteins by [(11)C]POX. Ex vivo biodistribution and imaging profiles in naive rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection, and persisted over the 60 min experiment. This showed for the first-time that even low POX exposures (~200 ng of tracer) can rapidly enter brain. Rats given an LD(50) dose of non-radioactive paraoxon at the LD(50) 20 or 60 min prior to [(11)C]POX tracer revealed protein pools were blocked. Blood radioactivity at 20 min were markedly lower than naive levels due to rapid protein modification by non-radioactive POX, however by 60 min, the blood radioactivity returned to near naive levels. Live rat tissue imaging-derived radioactivity values were 10-37% of naive levels in non-radioactive POX pretreated rats at 20 min, but by 60 min the AUC values had recovered to 25-80% of naive. The live rat imaging supported blockade by non-radioactive POX pretreatment at 20 min and recovery of proteins by 60-min. Significance Statement Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX etc. To study the distribution and penetration of POX into the CNS and other tissues, a positron emission tomography (PET) tracer analog, [(11)C]POX, was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.
Organophosphorus esters (OPs) were originally developed as pesticides but were repurposed as easily manufactured, inexpensive, and highly toxic chemical warfare agents. Acute OP toxicity is primarily due to inhibition of acetylcholinesterase (AChE), an enzyme in the central and peripheral nervous system. OP inhibition of AChE can be reversed using oxime reactivators but many show poor CNS penetration, indicating a need for new clinically viable reactivators. However, challenges exist on how to best measure restored AChE activity in vivo and assess the reactivating agent efficacy. This work reports the development of molecular imaging tools using radiolabeled OP analog tracers that are less toxic to handle in the laboratory, yet inhibit AChE in a similar fashion to the actual OPs. Carbon-11 and fluorine-18 radiolabeled analog tracers of VX and sarin OP agents were prepared. Following intravenous injection in normal Sprague-Dawley rats (n = 3-4/tracer), the tracers were evaluated and compared using noninvasive microPET/CT imaging, biodistribution assay, and arterial blood analyses. All showed rapid uptake and stable retention in brain, heart, liver, and kidney tissues determined by imaging and biodistribution. Lung uptake of the sarin analog tracers was elevated, 2-fold and 4-fold higher uptake at 5 and 30 min, respectively, compared to that for the VX analog tracers. All tracers rapidly bound to red blood cells (RBC) and blood proteins as measured in the biodistribution and arterial blood samples. Analysis of the plasma soluble activity (nonprotein/cell bound activity) showed only 1-6% parent tracer and 88-95% of the activity in the combined solid fractions (RBC and protein bound) as early as 0.5 min post injection. Multivariate analysis of tracer production yield, molar activity, brain uptake, brain area under the curve over 0-15 min, and the amount of parent tracer in the plasma at 5 min revealed the [(18)F]VX analog tracer had the most favorable values for each metric. This tracer was considered the more optimal tracer relative to the other tracers studied and suitable for future in vivo OP exposure and reactivation studies.
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.
Title: Inhibition of Acetylcholinesterases by Stereoisomeric Organophosphorus Compounds Containing Both Thioester and p-Nitrophenyl Leaving Groups Talley TT, Chao CK, Berkman CE, Richardson RJ, Thompson CM Ref: Chemical Research in Toxicology, 33:2455, 2020 : PubMed
Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP R(P)R(C) isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest S(P)S(C) isomer. eeAChE inhibited by the R(P)R(C)- or R(P)S(C)-MSNP isomer underwent spontaneous reactivation -10- to 20-fold faster than the enzyme inhibited by S(P)R(C)- and S(P)S(C)-MSNP, and only 4% spontaneous reactivation was observed from the S(P)R(C)-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by R(P)R(C)- or S(P)R(C)-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the R(P)S(C)- or S(P)S(C)-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by R(P)S(C)- and S(P)S(C)-MSNPs than inhibition by either R(C) isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by R(P)R(C)- and S(P)R(C)-MSNPs was 2.5- to 5-fold faster than inhibition by R(P)S(C)- or S(P)S(C)-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from S(P)-isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by isomalathion.
Title: Comparison of the reactivation rates of acetylcholinesterase modified by structurally different organophosphates using novel pyridinium oximes Bharate SB, Chao CK, Thompson CM Ref: Environ Toxicol Pharmacol, 71:103218, 2019 : PubMed
A novel panel of oximes were synthesized, which have displayed varying degree of reactivation ability towards different organophosphorus (OP) modified cholinesterases. In the present article, we report a comparative reactivation profile of a series of quaternary pyridinium-oximes for electric eel acetylcholinesterase (EEAChE) inhibited by the organophosphorus (OP) inhibitors methyl paraoxon (MePOX), ethyl paraoxon (POX; paraoxon) and diisopropyl fluorophosphate (DFP) that are distinguishable as dimethoxyphosphoryl, diethoxyphosphoryl and diisopropoxyphosphoryl AChE-OP-adducts. Most of the 59-oximes tested led to faster and more extensive reactivation of MePOX- and POX-inhibited EEAChE as compared to DFP-modified EEAChE. All were effective reactivators of three OP-modified EEAChE conjugates showing 18-21% reactivation for DFP-inhibited AChE and >/=45% reactivation for MePOX- and POX-inhibited EEAChE. Oximes 7 and 8 showed kr values better than pralidoxime (1) for DFP-inhibited EEAChE. Reactivation rates determined at different inhibition times showed no significant change in kr values during 0-90min incubation with three OPs. However, a 34-72% decrease in kr for MePOX and POX and > 95% decrease in kr for DFP-inhibited EEAChE was observed after 24h of OP-exposure (aging).
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.
2-Pyridinealdoxime methiodide (2-PAM) is a widely used antidote for the treatment of organophosphorus (OP) exposure that reactivates the target protein acetylcholinesterase. Carbon-11 2-PAM was prepared to more fully understand the in vivo mode of action, distribution, and dynamic qualities of this important countermeasure. Alkylation of 2-pyridinealdoxime with [(11)C]CH3I provided the first-in-class [(11)C]2-PAM tracer in 3.5% decay corrected radiochemical yield from [(11)C]CH3I, >99% radiochemical purity, and 4831 Ci/mmol molar activity. [(11)C]2-PAM tracer distribution was evaluated by ex vivo biodistribution and in vivo dynamic positron emission tomography (PET) imaging in naive (OP exposure deficient) rats. Tracer alone and tracer coinjected with a body mass-scaled human therapeutic dose of 30 mg/kg nonradioactive 2-PAM demonstrated statistically similar tissue and blood distribution profiles with the greatest uptake in kidney and significantly lower levels in liver, heart, and lung with lesser amounts in blood and brain. The imaging and biodistribution data show that radioactivity uptake in brain and peripheral organs is rapid and characterized by differential tissue radioactivity washout profiles. Analysis of arterial blood samples taken 5 min after injection showed approximately 82% parent [(11)C]2-PAM tracer. The imaging and biodistribution data are now established, enabling future comparisons to outcomes acquired in OP intoxicated rodent models.
Title: Novel Organophosphate Ligand O-(2-Fluoroethyl)-O-(p-Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability and Analysis of the Inhibition and Reactivation of Cholinesterases Chao CK, Ahmed SK, Gerdes JM, Thompson CM Ref: Chemical Research in Toxicology, 29:1810, 2016 : PubMed
The organophosphate O-(2-fluoroethyl)-O-(p-nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-18 radiolabeled organophosphate inhibitor ([18F]1) of acetylcholinesterase (AChE). In rats, [18F]1 localizes in AChE rich regions of the brain and other tissues where it likely exists as the (CH3)(18FCH2CH2O)P(O)-AChE adduct (ChE-1). Characterization of this adduct would define the inhibition mechanism and subsequent postinhibitory pathways and reactivation rates. To validate this adduct, the stability (hydrolysis) of 1 and ChE-1 reactivation rates were determined. Base hydrolysis of 1 yields p-nitrophenol and (CH3) (FCH2CH2O)P(O)OH with pseudo first order rate constants (kobsd) at pH 7.4 (PBS) of 3.25 x 10-4 min-1 (t1/2 = 35.5 h) at 25 degrees C and 8.70 x 10-4 min-1 (t1/2 = 13.3 h) at 37 degrees C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; ki = 7.5 x 105 M-1 min-1), electric eel acetylcholinesterase (EEAChE) (ki = 3.0 x 106 M-1 min-1), and human serum butyrylcholinesterase (HuBChE; 1.95 x 105 M-1 min-1). Spontaneous and oxime-mediated reactivation rates for the (CH3) (FCH2CH2O)P(O)-serine ChE adducts using 2-PAM (10 muM) were (a) HuAChE 8.8 x 10-5 min-1 (t1/2 = 131.2 h) and 2.41 x 10-2 min-1 (t1/2 = 0.48 h), (b) EEAChE 9.32 x 10-3 min-1 (t1/2 = 1.24 h) and 3.33 x 10-2 min-1 (t1/2 = 0.35 h), and (c) HuBChE 1.16 x 10-4 min-1 (t1/2 = 99.6 h) and 4.19 x 10-2 min-1 (t1/2 = 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo is the formation of (CH3)(FCH2CH2O)P(O)-serine adducts and monoacid (CH3)(FCH2CH2O)P(O)OH from hydrolysis and reactivation.
Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena occurs. In this report, we prepared a panel of beta-substituted ethoxy and gamma-substituted propoxy phosphonoesters of the type p-NO(2)PhO-P(X)(R)[(O(CH(2))(n)Z] (R=Me, Et; X=O, S; n=2, 3; Z=halogen, OTs) and examined the inhibition of three AChEs by select structures in the panel. The beta-fluoroethoxy methylphosphonate analog (R=Me, Z=F, n=2) was the most potent anti-AChE compound comparable (ki approximately 6 x 10(6)M(-1)min(-1)) to paraoxon against EEAChE. Analogs with Z=Br, I, or OTs were weak inhibitors of the AChEs, and methyl phosphonates (R=Me) were more potent than the corresponding ethyl phosphonates (R=Et). As expected, analogs with a thionate linkage (PS) were poor inhibitors of the AChEs.
