New insights into the mechanism of action of the thienopyrimidine antitubercular prodrug TP053

The thienopyrimidine TP053 is an antitubercular prodrug active against both replicating and non-replicating Mycobacterium tuberculosis cells, which requires activation by the mycothiol-dependent nitroreductase Mrx2. Investigation of the mechanism of action of TP053 revealed that Mrx2 releases nitric oxide from this drug both in the enzyme assays with purified Mrx2 and in mycobacterial cultures, which can explain its activity against non-replicating bacilli, similar to pretomanid activated by the nitroreductase Ddn. In addition, we identified a highly reactive metabolite, 2-(4-mercapto-6-(methylamino)-2-phenylpyrimidin-5-yl)ethan-1-ol, which can contribute to the antimycobacterial effects on replicating cells. In summary, we explained the mechanism of action of TP053 on both replicating and non-replicating M. tuberculosis and report a novel activity for Mrx2, which in addition to Ddn, represents another example of nitroreductase releasing nitric oxide from its substrate. These findings are particularly relevant in the context of drugs targeting non-replicating M. tuberculosis, which were shown to be killed by increased levels of nitric oxide.