The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE

Chem Biol Interact. 2018 Aug 1:291:220-227. doi: 10.1016/j.cbi.2018.06.019. Epub 2018 Jun 18.


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.

MeSH terms

  • Acetylcholinesterase / metabolism*
  • Butyrylcholinesterase / metabolism*
  • Chemical Warfare Agents / toxicity
  • Cholinesterase Reactivators / pharmacology*
  • Enzyme Activation / drug effects
  • Fluorescence
  • Humans
  • Kinetics
  • Organophosphorus Compounds / toxicity
  • Organothiophosphorus Compounds / toxicity*
  • Paraoxon / toxicity
  • Sarin / toxicity*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Chemical Warfare Agents
  • Cholinesterase Reactivators
  • Organophosphorus Compounds
  • Organothiophosphorus Compounds
  • VX
  • Sarin
  • Acetylcholinesterase
  • Butyrylcholinesterase
  • Paraoxon