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.
Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid.
