Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT, 59812, United States.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717, United States.
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 = CHCH, CHCHF, OCH(CH), OCH(CH) (CHF)] 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 ∼ 10-10 Mmin) and underwent spontaneous and 2-PAM-mediated reactivation within 30 min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (k ∼ 10-10 Mmin), 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.
在这项研究中，通过体外动力学和质谱研究了代表 VX 和沙林的拟态物和氟代拟态物的 Me-P(O)(OPNP)(OR)[PNP=对硝基苯基；R=CHCH、CHCHF、OCH(CH) 、OCH(CH)(CHF)]对 HuAChE 和 HuBChE 的抑制机制。体外测量表明，VX 和氟代 VX 拟态物是 HuAChE 和 HuBChE 的相对较强抑制剂(k∼10-10Mmin)，并在 30 分钟内自发和 2-PAM 介导的重新激活。沙林拟态物是 HuAChE 和 HuBChE 的较弱抑制剂(k∼10-10Mmin)，通常不会自发重新激活，尽管使用 2-PAM 在 18 小时时可以部分重新激活 HuAChE 加合物。通过 Q-TOF 和 MALDI-TOF 质谱分析确定了拟态物对 HuAChE 和 HuBChE 的抑制机制。用胰蛋白酶消化拟态物加合物，并通过 Q-TOF 鉴定含有 OP 修饰丝氨酸的活性位点肽，其代表附着的 OP 部分的质量增加分别为三重和四重电荷离子。相应地，通过 MALDI-TOF 鉴定代表 OP 加合物肽质量增加的胰蛋白酶肽的单同位素离子。质谱分析验证了附着在 HuAChE 或 HuBChE 上的 OP 部分是 MeP(O)(OR)-O-丝氨酸肽(失去 PNP 离去基团)的身份，其机制与化学战剂中的机制一致。VX 修饰肽随时间的 MALDI-TOF MS 分析表明，加合物与母体肽(重新激活)的比例稳定降低，而沙林拟态修饰肽在实验过程中(24 小时)基本保持完整。总体而言，拟态物上氟原子的存在略微改变了抑制和重新激活的速率常数，但抑制机制(PNP 基团的排出)没有改变。
