The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE
Chih-Kai Chao, Narayanaganesh Balasubramanian, John M. Gerdes, Charles M. Thompson
In this study, the mechanisms of HuAChE and HuBChE inhibition by Me-P(O) (OPNP) (OR) [PNP?=?p-nitrophenyl; R?=?CH2CH3, CH2CH2F, OCH(CH3)2, OCH(CH3) (CH2F)] 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 (ki???105-106?M?1min?1) and underwent spontaneous and 2-PAM-mediated reactivation within 30?min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (ki???104-105?M?1min?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.
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