Lang Pauline A, de Munnik Mariska, Oluwole Abraham O, Claridge Timothy D W, Robinson Carol V, Brem Jürgen, Schofield Christopher J
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom.
Ineos Oxford Institute for Antimicrobial Research, Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom.
Chembiochem. 2024 Nov 18;25(22):e202400280. doi: 10.1002/cbic.202400280. Epub 2024 Sep 13.
Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC-MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.
克拉维酸是一种在医学上具有重要意义的丝氨酸β-内酰胺酶（SBLs）抑制剂。我们报告了利用变性和非变性质谱（MS）研究克拉维酸抑制代表性的安布勒A类（TEM-116）、C类（大肠杆菌AmpC）和D类（OXA-10）SBLs的机制。与对青霉烷砜的观察结果相似，大多数结果支持一种涉及酰基酶复合物形成的机制，随后恶唑烷环打开，但没有有效的后续支架片段化（在pH 7.5时）。这一观察结果与之前的质谱研究形成对比，之前的研究将克拉维酸支架片段化产物鉴定为主要的SBL结合产物。在本文研究的所有SBLs中，酸性条件促进了片段化，而酸性条件常用于液相色谱-质谱分析。然而，在中性条件下，TEM-116与克拉维酸长时间反应时观察到缓慢的片段化。虽然我们的结果表明克拉维酸支架片段化在体内对SBL抑制可能并不关键，但开发能片段化形成稳定共价复合物的抑制剂仍具有重要意义。
