Commercial carbapenem antibiotics, including Meropenem, Ertapenem, and Imipenem, are currently being used to treat multidrug resistant and extensively drug resistant tuberculosis. Carbapenems are essential last-line agents for serious Gram-negative and Gram-positive infections, and, like other β-lactam antibiotics, are irreversible inhibitors of serine D,D-transpeptidases involved in peptidoglycan (PG) biosynthesis. In contrast to most species, however, mycobacteria also utilize non-homologous cysteine L,D-transpeptidases (Ldts) to crosslink the stem peptides of PG, and, unlike most classes of β-lactam antibiotics, carbapenems have been shown to form long-lived acyl-enzymes with those cysteine Ldts. Structurally, current commercial carbapenems are C2 modifications of a common scaffold and structure activity relationships were optimized for broad spectrum activity against 20^th century pathogens and resilience toward 20^th century β-lactamases. This study describes the evaluation of atypical (i.e. non-C2 modified) carbapenems against Mycobacterium tuberculosis and the nontuberculous mycobacterial species, including Mycobacterium abscessus, and their acylation profiles against the class 2 Ldts from M. tuberculosis (Ldt_Mt2), M. abscessus (Ldt_Mab2), Mycobacterium chelonae (LdtB), and Mycobacterium smegmatis (LdtB). In vitro evaluation of these C5 modified carbapenems against these pathogens revealed compounds with standalone (i.e. in the absence of β-lactamase inhibitors) minimum inhibitory concentrations (MICs) superior to Meropenem-Clavulanate (for M. tuberculosis) and Meropenem-Avibactam (for M. abscessus). These atypical carbapenems acylate class 2 Ldts, but interestingly, degrade differently on these enzymes; high resolution mass spectrometry data reveal these drugs acylate class 2 Ldts as well as Meropenem, but subsequently undergo fragmentation to a Δm = +86 Da on Ldt_Mt2, which is atypical of a carbapenem. Conversely, the same compounds acylate nontuberculous Ldts, but do not readily degrade to the Δm = +86 Da species in all cases. Mechanistic rationale for the divergence of the nonhydrolytic fragmentation of these compounds from that of commercial carbapenems is proposed, and active site differences among orthologous Ldts supporting the differential degradation patterns are highlighted. This series of compounds illustrates the potential of novel C5 modified carbapenems as promising therapeutic candidates for treatment of mycobacterial infections.

Phil Mortimer

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