New antitubercular prodrugs with novel mechanisms of action

Tuberculosis (TB) is a contagious and deadly disease caused by Mycobacterium tuberculosis bacillus, which has reached pandemic proportions. In 2015, it was estimated globally that there were 10.4 million new cases of TB and 1.8 million deaths, 400.000 of whom were HIV-positive. In recent years, the control of TB is hampered by the emergence and the spread of M. tuberculosis drug-resistant strains (MDR-TB, XDR-TB, TDR-TB). Considering the extremely dangerous threat to public health represented by these M. tuberculosis drug-resistant strains, it is urgently needed the development of anti-TB compounds with novel mechanisms of action and new cellular targets. Recently, in this direction, three new antitubercular promising prodrugs, the thienopyrimidine TP11126053 (TP053) and the thiophenecarboxamides 7947882 and 7904688, have been identified. TP053, with an MIC in vitro of 0.125 μg/mL is active against both replicating and non-replicating M. tuberculosis bacilli. By microbiological, genetic and biochemical studies, it was characterized as a prodrug activated by the reduced form of the DsbA-like mycoredoxin Rv2466c. The 7947882 and 7904688, both with an MIC value of 0.5 µg/mL, emerged from the phenotypic screening of a National Institute of Allergy and Infectious Diseases (NIAD) set of chemical compounds. They showed an efficient inhibitory activity against M. tuberculosis H37Rv in its growing, latent and intracellular conditions. With a multidisciplinary approach, the 7947882 and 7904688 were characterized as prodrugs activated by the monooxigenase EthA that affect the CTP synthetatse PyrG. The active metabolite of 7947882 was identified and characterized as direct inhibitor of PyrG and PanK. In this work we studied in depth the mechanism of action and of resistance of these three prodrugs. Firstly, it was demonstrated that the M. tuberculosis Rv0579 protein is involved in the mechanism of resistance to TP053 by microbiological and biochemical approaches, even if its physiological role still remains unclear. In addition, the mechanism of action of TP11126053 was clarified by metabolomic, meta-proteomic, biochemical and microbiological methodologies. In fact, the activated compound globally affects the mycobacterial growth by NO release mainly affecting the protein synthesis. For the last two prodrugs, 7947882 and 7904688, it was demonstrated that the panthotenate kinase PanK represents their second target, as well as PyrG. Consequently, these molecules are two multitargeting compounds targeting PyrG and panK. In order to find further multitargeting compounds, it has been developed a phenotypic target-based screening leading to the identification of two further promising agents, affecting both targets. Finally, two putative active metabolites of 7904688 prodrug have been identified by a metabolomic approach and their characterization is still under investigation.