Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom.
Ineos Oxford Institute for Antimicrobial Research, University of Oxford, Oxford OX1 3TA, United Kingdom.
Proc Natl Acad Sci U S A. 2022 May 3;119(18):e2117310119. doi: 10.1073/pnas.2117310119. Epub 2022 Apr 29.
β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.
β-内酰胺类抗生素是最重要的一类抗菌药物，但由于耐药性的出现，其应用受到了越来越多的限制，其中最重要的机制是丝氨酸β-内酰胺酶（SBL）催化水解。通过与青霉素衍生的 SBL 抑制剂（SBLi）联合使用，可以显著扩大β-内酰胺类抗菌药物的抗菌谱，即使用青霉素硫代磺酰胺类的他唑巴坦和舒巴坦作为 SBLi，它们是通过酰化亲核丝氨酸起作用的基于机制的抑制剂。最近，新型 SBLi 恩美曲妥珠单抗（enmetazobactam）已完成临床试验。对恩美曲妥珠单抗抑制 SBL 机制的生物物理研究表明，它可以抑制所有 SBL 类别的代表，而不会发生实质性的支架断裂，这与之前关于他唑巴坦和舒巴坦抑制 SBL 的报道形成了对比。因此，我们使用变性和非变性条件下的质谱、X 射线晶体学和 NMR 光谱学重新研究了他唑巴坦和舒巴坦的作用机制。结果表明，先前报道的青霉素硫代磺酰胺衍生的酰基-酶复合物的广泛断裂并未对 SBL 抑制产生实质性贡献。除了观察到先前鉴定的抑制剂诱导的 SBL 修饰外，结果还表明，青霉素硫代磺酰胺与 SBL 长时间反应会导致亲核丝氨酸脱水，形成脱氢丙氨酸残基，然后发生反应，形成以前未观察到的赖氨酰丙氨酸交联。该结果阐明了广泛临床应用的 SBLi 的作用机制，揭示了关于 SBLi 作用机制的质谱研究解释的局限性，并将为通过反应形成水解稳定的酰基-酶复合物的新型 SBLi 的开发提供信息。
