Molecular Basis of Class A (cid:2) -Lactamase Inhibition by Relebactam

(cid:2) -Lactamase production is the major (cid:2) -lactam resistance mechanism in Gram-negative bacteria. (cid:2) -Lactamase inhibitors (BLIs) efﬁcacious against serine (cid:2) -lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae . We show that relebactam inhibits ﬁve clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum (cid:2) -lactamases L2 (inhibition constant 3 (cid:3) M) and CTX-M-15 (21 (cid:3) M) and the carbapenemases KPC-2, -3, and -4 (1 to 5 (cid:3) M). Against puriﬁed class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC 50 ]). MIC assays indicate relebactam po-tentiates (cid:2) -lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae , with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across ﬁve clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efﬁcacy of BLI– (cid:2) -lactam combinations. in triplicate, in cation-adjusted Mueller- Hinton broth (Sigma) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (47). Experiments were performed in microtiter plates (Corning) containing medium with ceftazidime, imi- penem, or aztreonam with inhibitor (4 mg liter (cid:2) 1 avibactam [MedChemExpress] or relebactam [MedChemExpress] dissolved in dimethyl sulfoxide). Plates were incubated overnight at 37°C for 18 to 24 h, and the absorbance at 600 nm was read using a POLARstar Omega (BMG LabTech) plate reader. Protein puriﬁcation and crystallization. The L2 (cid:2) -lactamase was puriﬁed and crystallized as described previously (25). The mature polypeptide (codons 28 to 290) of CTX-M-15 in the expression vector pOPINF (48) was expressed in SoluBL21 (DE3) E. coli cells (Genlantis) and grown in 2xYT medium supplemented with 50 (cid:3) g/ml carbenicillin to produce N-terminally His-tagged CTX-M-15. Three liters of culture was incubated at 37°C until reaching an optical density at 600 nm (OD 600 ) of 0.8 and subsequently grown at 18°C overnight with 0.75 mM IPTG to induce protein expression. Cells were harvested by spectrometry of relebactam fragmentation in KPC variants. To investigate modiﬁcations to enzymes relebactam, 3 with at Mass spectra were positive ion mode by integrated autosampler/solid-phase extraction