Indolcarboxamide Is a Preclinical Candidate for Treating Multidrug-Resistant Tuberculosis

Abstract

New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.

Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3

“…The identification and characterization of MmpL3 inhibitors have thus far rested on the whole-genome sequencing of spontaneous resistant mutants and the metabolic labeling with [1,2-14 C]acetate of inhibitor-treated cells to monitor the transfer of mycolic acids to their cell envelope acceptors (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)17). Because the development and further optimization of MmpL3 inhibitors would greatly benefit from less cumbersome approaches, and because of the technical difficulty of developing an in vitro transport assay for this protein, we next sought to determine whether the M. tuberculosis mc 2 6206 MmpL3-DUC knockdown could be used in the development of a target-based whole-cell screening assay.…”

“…Whether the identified inhibitors inhibit MmpL3 directly or through an indirect mechanism involving the dissipation of the proton motive force from which MmpL3 derives its energy remains to be determined on a case-to-case basis (17,18). Nonetheless, SQ109 is undergoing phase II clinical trials (19), and at least two other series of MmpL3 inhibitors are currently under development for their potential to reduce treatment duration and kill MDR isolates while lacking untoward effects on the human cytochrome P450 enzymes that metabolize antivirals (4,9,13,17,19).…”

“…To date, whole-cell screening strategies are the ones that have led to the identification of the greatest number of novel inhibitors of potential therapeutic interest (2)(3)(4). Intriguingly, in the last 4 years, the screening of compound libraries against M. tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants has identified multiple small molecules, including the TB drug candidate SQ109, as inhibitors of the mycolic acid transporter, MmpL3 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). MmpL3 is an integral inner membrane transporter of the resistance, nodulation, and division (RND) superfamily, involved in the export of mycolic acids under the form of trehalose monomycolate (TMM) to the periplasmic space, where this glycolipid can then serve as a mycolic acid donor in the building of the mycobacterial outer membrane, otherwise known as the mycomembrane (5,15,16).…”

Novel Insights into the Mechanism of Inhibition of MmpL3, a Target of Multiple Pharmacophores in Mycobacterium tuberculosis

“…While the apparent promiscuity of MmpL3 as a drug target may owe to unusual vulnerability or druggability, the diversity of the chemical scaffolds identified and important differences in their spectra of activity led us to question their direct mechanism of action on the transporter. In particular, the fact that SQ109, BM212, and THPP compounds display inhibitory activity against a broad spectrum of bacterial and fungal pathogens that are devoid of mycolic acids (7,20,21), while AUs and indolecarboxamides specifically target mycobacteria (17,19), suggests that the three first compounds had at least one broader-spectrum target in M. tuberculosis or killed the bacterium through a different mechanism, impacting MmpL3 only indirectly. Another important difference between the MmpL3 inhibitors resides in their activities against nonreplicating M. tuberculosis bacilli.…”

“…Surprisingly, in the last 2 years, the whole-cell-based screening of compound libraries against M. tuberculosis in culture coupled to the whole-genome sequencing of spontaneous resistant mutants identified a variety of pharmacophores that apparently shared the same mode of action as SQ109. These inhibitors include the 1,5-diarylpyrrole derivative BM212 and analogs (10,11), the benzimidazole C215 (12), tetrahydropyrazolo [1,5-a]pyrimidine-3-carboxamides (THPPs) (13,14), N-benzyl-6=,7=-dihydrospiro[piperidine-4,4=-thieno [3,2-c]pyrans (13), indolecarboxamides (15)(16)(17)(18), and adamantyl ureas (AUs) (19) (Fig. 1).…”