Rv2466c Mediates the Activation of TP053 To Kill Replicating and Non-replicating Mycobacterium tuberculosis

Abstract

The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 Å resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a β-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti-mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053

“…Initial velocities recorded at different TP053 concentrations in the presence of the reductant 1 mM dithiothreitol (DTT) and catalytic amounts of Rv2466c were consistent with Michaelis-Menten kinetics (k cat ϭ 0.0403 s Ϫ1 ; K m ϭ 6.489 M) indicating that Rv2466c catalyzes the reduction of TP053 by DTT. This conclusion was supported by the observation that Rv2466c in its oxidized state and in the absence of DTT was unable to metabolize TP053 (9). The rate-limiting step in the catalytic cycle of Rv2466c, in the presence of 1 mM DTT, is the reduction of TP053 by reduced Rv2466c, and not the reduction of oxidized Rv2466c by DTT.…”

“…Interestingly, we have recently discovered a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating bacilli ( Fig. 1) (9). Structure-activity relationship analysis demonstrated that a NO 2 group at position C6 is essential for anti-TB activity as any alternative substitution, including NH 2 , carbonitrile, carboxylate, or carboxamide, resulted in an inactive TP compound.…”

“…To elucidate the mechanism of action of these TP derivatives, spontaneous resistant mutants of M. tuberculosis H37Rv to TP053 were isolated (9). This strategy had proven useful to study antibiotic resistance in M. tuberculosis and other human bacterial pathogens (10,11).…”

“…1). In combination with structure-activity relationship studies, it is suggested that the nitro group is being reduced to generate toxic anion radicals and/or a variety of other highly reactive compounds including nitroso and hydroxylamine derivatives (9), as these products are extremely toxic, readily damage unknown cellular targets including proteins, membrane lipids, and DNA and likely leading to M. tuberculosis death (9).…”

“…The crystal structure of reduced Rv2466c has been solved recently, revealing a unique homodimer in which a ␤-strand is swapped between the thioredoxin domains of each subunit, and an ␣-helical subdomain inserted into each thioredoxin domain (9). A large, mostly hydrophobic groove harbors a redox activesite motif CPWC, typical of the thioredoxin superfamily of oxidoreductases (13)(14)(15)(16).…”