Relebactam is a Potent Inhibitor of the KPC-2 {beta}-Lactamase and Restores the Susceptibility of Imipenem Against KPC-Producing Enterobacteriaceae [PublishAheadOfPrint]

The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core similar to avibactam, however relebactam's R1 side chain also includes a piperidine ring compared to the carboxyamide of avibactam. Here, we investigated the inactivation of Klebsiella pneumoniae carbapenemase (KPC-2), the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae. Minimal inhibitory concentration (MIC) measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae (K. pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Citrobacter koseri, and Escherichia coli) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/L). Relebactam inhibited KPC-2 with a k₂/K value of 24,750 M^-1s^-1 and demonstrated a slow k_off of 0.0002 s^-1. Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2-relebactam acyl-enzyme complex was stable for up to 24 hours. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation has been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that in comparison to the KPC-2-avibactam, the positioning of active site water molecules is less favorable for desulfation. In the acyl-complexes, the water molecules are within 2.5-3 Å of the avibactam sulfate, however more than 5-6 Å from the relebactam sulfate. As a result, we propose that the KPC-2-relebactam acyl-complex is more stable than the KPC-2-avibactam acyl-enzyme complex. The clinical implications of this difference are not currently known.

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