Identification of New MmpL3 Inhibitors by Untargeted and Targeted Mutant Screens Defines MmpL3 Domains with Differential Resistance

Abstract

The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosis. M. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus. Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.

Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

“…Since the strains with two mutations now had relatively high-level resistance to both of our original compounds (Ͼ50 M) (Tables 1 and 2), we were unable to attempt further rounds of resistant strain isolation to IDR-0334448 or IDR-0033216. However, the spectrum of cross-resistance to SQ109 and AU1235 was variable (Table 2), as might be expected if compounds have unique interactions with MmpL3 (23). Therefore, we made use of the observation that the strains were not fully resistant to these compounds and conducted a third round of resistant mutant isolation.…”

Physicochemical properties and Mycobacterium tuberculosis transporters: keys to efficacious antitubercular drugs?

“…Mtb also possesses export carrier proteins such as mycobacterial membrane protein large 3 (MmpL3), 64 the target of SQ109 (ref. 73) and other molecules, [74][75][76] involved in the export of essential molecules to the periplasm, 11 with studies unravelling its likely substrates utilized in cell wall biogenesis. 77 Understanding the nuances and interplay of these various transport mechanisms could pave the way for targeted combination therapies and new drugs less prone to drug-drug interactions through their interference with host transporters and metabolising enzymes.…”

Mycobacterial Cell Wall: A Source of Successful Targets for Old and New Drugs

“…With the success of SQ109 and the essentiality of mmpl3 in M. tuberculosis, novel inhibitors of Mmpl3 have been actively sought. Inhibition of Mmpl3 is not specific to the chemical structure of SQ109 and numerous chemical scaffolds are being identified as Mmpl3 inhibitors [185,190,191].…”

“…Academic and pharmaceutical laboratories are developing new inhibitors of enzymes involved in mycolic acid biosynthesis. Inhibitors of KasA [213], InhA [83,[90][91][92], Pks13 [214][215][216][217] and Mmpl3 [185,190,191,218,219] are being tested.…”