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, optimisation 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 not 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 the transpeptidase activities of Ldts and PBPs in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; though under the tested conditions a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, inhibition of both PBPs and Ldts was observed in 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 optimising PBPs and Ldt transpeptidase inhibition, particularly of Ldts, to improve β-lactam efficacy versus mycobacteria.