The inhibition reactivation and mechanism of VX- sarin- fluoro-VXand fluoro-sarin surrogates following their interaction with HuAChE andHuBuChE.

摘要

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 activesite-containing peptide bearing the OP-modified serine identified by Q-TOF astriply- and quadruply-charged ions representing the respective increase in massof the attached OP moiety. Correspondingly, monoisotopic ions of the trypticpeptides representing the mass increase of the OP-adducted peptide wasidentified by MALDI-TOF. The mass spectrometry analyses validated the identityof 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 withchemical warfare agents. MALDI-TOF MS analyses of the VX-modified peptidesversus time showed a steady reduction in adduct versus parent peptide(reactivation), whereas the sarin-surrogate-modified peptides remained largelyintact over the course of the experiment (24 h). Overall, the presence of afluorine atom on the surrogate modestly altered the rate constants of inhibitionand reactivation, however, the mechanism of inhibition (ejection of PNP group)did not change.