Targeting mycobacterial transpeptidases: evaluating the roles of Ldt and PBP inhibition in suppressing Mycobacterium smegmatis

β-lactams demonstrate promising in vitro activity against Mycobacterium species and are being explored for tuberculosis treatment; however, evidence of their in vivo efficacy versus Mycobacterium tuberculosis remains limited. To achieve broad clinically relevant potency, optimization of the classical β-lactam scaffolds or development of new or non-β-lactam inhibitors for mycobacterial transpeptidases is likely required. In mycobacteria, potential targets of β-lactams include l,d -transpeptidases (Ldts) and penicillin-binding proteins (PBPs). Reports suggest that dual inhibition of Ldts and PBPs may be necessary to achieve effective anti-mycobacterial activity, yet the specific contributions of Ldt and PBP inhibition to the β-lactam antibacterial mechanisms are poorly understood. We used fluorogenic substrate mimics to investigate the effects of β-lactams and reported Ldt _Mt2 inhibitors on Mycobacterium smegmatis ( Msm ), assessing their impacts on Ldt and PBP transpeptidase activities in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; under the tested conditions, a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, apparent inhibition of both PBPs and Ldts was observed with all active inhibitors, though β-lactams manifest increased potency of PBP inhibition. The combination of the β-lactams meropenem and faropenem with selected Ldt _Mt2 inhibitors manifested an additive inhibitory effect against Msm . Our results highlight the importance of further optimizing β-lactam efficacy versus mycobacterial PBPs and Ldt transpeptidases.