Novel C5α-substituted carbapenems enhance <i>Mycobacterium abscessus</i> killing via selective target binding and reduced hydrolysis by Bla _Mab

<i>Mycobacterium abscessus</i> (<i>Mab</i>) poses significant clinical challenges and underscores the urgent need for safer and more effective treatments, including β-lactams. Among currently available carbapenems, imipenem is widely used to treat <i>Mab</i> infections by combining with other antibiotics. Commercial carbapenems share a common scaffold with C2 modifications, whereas this study focuses on novel carbapenem candidates with C5α modifications. We evaluated their antibacterial activity against <i>Mab</i> ATCC 19977 and clinical isolates, as well as their acylation of peptidoglycan target receptors (L,D-transpeptidases [LDTs] and penicillin-binding proteins [PBPs]) and the β-lactamase enzyme Bla_Mab. <i>In vitro</i> studies of two C5α-modified carbapenems, JDB/NA-1-157 and JDB/NA-1-208, revealed distinct antibacterial effects. JDB/NA-1-157 demonstrated potent bacterial killing with low minimum inhibitory concentrations (MICs; 0.125-8 mg/L) and near-complete eradication within 5 days, surpassing the efficacy of the standard-of-care regimen (amikacin + clarithromycin + imipenem). In contrast, JDB/NA-1-208 exhibited poor bacterial killing, with high MICs (16-256 mg/L) and limited efficacy in time-kill studies. However, JDB/NA-1-208 showed synergistic killing when combined with other β-lactams. Mechanistically, JDB/NA-1-208 is not a substrate for Bla_Mab, while JDB/NA-1-157 is, albeit with low catalytic efficiency. This is supported by the observation that the addition of avibactam did not enhance synergy with JDB/NA-1-157. The substantial bacterial killing effect of JDB/NA-1-157 is attributed to its high binding affinity for PBP-B, PBP-lipo, PonA2, D,D-carboxypeptidase, and LDT1-2. These findings highlight the potential of novel C5α-modified carbapenems, particularly JDB/NA-1-157, as promising therapeutic candidates for <i>Mab</i> infections.