There are two cysteine residues in TBNAT, which further supports the interpretation of a reaction with the cysteine residues exposed during denaturation in the presence of excess piperidinol. The cysteine residues 2-Pyridylethylamine dihydrochloride following denaturation become equivalent to those in glutathione and cysteine itself. It is also interesting to note that the free cysteine can to react with these compounds, although it does require a long incubation time. This demonstrates that activation of these compounds to form reactive PVK in the presence of the enzyme is compatible with reaction through the active site cysteine. The search for novel drug targets against M. tuberculosis has been escalated recently under the pressure of the emergence of extensively drug resistant strains. Arylamine N-acetyltransferase is one of the novel targets that plays an important role in cell wall synthesis and intracellular survival of mycobacteria within the macrophage. From a previous HTS, the piperidinol scaffold was identified as a selective prokaryotic NAT inhibitor that shows good antimycobacterial activity. In order to explore this scaffold as a possible lead for anti-tubercular therapies, a series of inhibitors was tested for their activity against TBNAT and MMNAT and for their antimycobacterial activity. In addition to inhibiting NAT activity, the compounds were 17-PA potent against M. tuberculosis with an MIC below 17 mM. The data do not preclude the presence of additional targets within M. tuberculosis. However, the concept of poly-pharmacy in which one drug has multiple targets is an extremely useful asset in drug design, particularly for antimicrobials where resistance is a major consideration. A novel mechanism of NAT inhibition by the piperidinols was revealed by MS-analysis, and from the 3D-structure of the MMNAT-1 complex. The mechanism of inactivation of NAT involves the formation of PVKs that form an adduct with the active site cysteine. This mechanism was also observed with acyclic Mannich bases considered for the drug design of antimalarial agents. Drug leads that exhibit activation followed by covalent modification of targets have been proposed to be beneficial in developing new TB therapies. This approach has indeed been historically successful with the front-line anti-tubercular drug isoniazid and the related drug ethionamide both retrospectively shown to be prodrugs that require activation to inhibit mycolic acid synthesis. The activated intermediates for those agents form a covalent adduct with the biological molecule NAD. Specific covalent enzyme inactivators have gained recent interest in drug design as being usually associated with lower doses and a longer duration of action, as well as avoiding resistance. The possible toxicity associated with such a mechanism requires the careful design of highly selective agents.