AbstractA new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC ₁₇ exhibited the best extracellular antitubercular activity (MIC₅₀ = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC ₁₇ may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.