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Publication Abstract from PubMed

beta-Lactamases hydrolyze beta-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) beta-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum beta-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cbeta closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of beta-lactamase inhibition by these agents. IMPORTANCE beta-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing beta-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to beta-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining beta-lactams with beta-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to beta-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of beta-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.

Penicillanic Acid Sulfones Inactivate the Extended-Spectrum beta-Lactamase CTX-M-15 through Formation of a Serine-Lysine Cross-Link: an Alternative Mechanism of beta-Lactamase Inhibition.,Hinchliffe P, Tooke CL, Bethel CR, Wang B, Arthur C, Heesom KJ, Shapiro S, Schlatzer DM, Papp-Wallace KM, Bonomo RA, Spencer J mBio. 2022 Jun 28;13(3):e0179321. doi: 10.1128/mbio.01793-21. Epub 2022 May 25. PMID:35612361^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.