HC2091 Kills Mycobacterium tuberculosis by Targeting the MmpL3 Mycolic Acid Transporter

Abstract

Tuberculosis, caused by the intracellular pathogen , is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills in a time- and dose-dependent manner and inhibits growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating , thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.

Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance

“…Additionally, TDM significantly decreased in cultures treated with HC2169 and HC2184 as well as the positive control SQ109 (Figure 2a and Supplemental Figure 3a). These results are consistent with previously described MmpL3 inhibitors 1, 2, 3, 4, 6, 7, 8, 9, 26 and support that these four compounds inhibit MmpL3 activity.…”

“…The amount of resistance conferred by the mixed mmpL3 mutant strains against each compound varied, with some compounds, like HC2032, HC2138, and HC2169 losing nearly all activity in the mutant background (Supplemental Figure 5) as indicated by the high Relative EC 50 (fold difference between mmpL3 mutant pool and WT) values of >36, >20, and >44 (Table 1). Despite the high activity of SQ109 in the WT background, the relative EC 50 was only 2 (Table 1); however, this observation is consistent with previous studies which only report marginal increases in MIC values in mmpL3 mutant backgrounds 8, 9, 21, 26 . Included in our hits were two urea-based compounds HC2138 and HC2169 (Figure 1 and Figure 3b).…”

“…Previously, two HTS were conducted, targeting the two component regulatory systems, DosRST and PhoPR 28, 29, 30 . In addition to inhibitors targeting these pathways, a series of compounds was identified that inhibited Mtb growth independent of the targeted pathways 9, 28, 30 . A series of high throughput assays were then conducted to prioritize these compounds (Supplemental Figure 1) including confirming hits, testing for eukaryotic cytotoxicity in primary murine bone marrow-derived macrophages (BMMΦ, ≤10% cytotoxicity), and testing for the ability of the compounds to inhibit Mtb growth inside BMMΦ (≥25% growth inhibition).…”

“…We therefore hypothesized that by pooling unique mmpL3 mutant strains into a single mixed culture we could overcome limitations of cross resistance variability. For the targeted phenotypic screen, we directly compared percent growth inhibition (%GI) of either WT or a mixed mmpL3 mutant pool consisting of twenty-four unique mmpL3 mutant strains, including three strains previously described as resistant to HC2091 9 (see Supplemental Table 2). The cultures were treated with 20μM of each of the 163-Mtb growth inhibitors as well as DMSO (negative control), Rifampin (RIF, positive control), Bedaquiline (BDQ), Clofazimine (CFZ), INH, para-aminosalicylic acid (PAS), H 2 O 2 , or SQ109 for a total of 171 different treatments (Supplemental Figure 4a and 4b).…”

“…In efforts to identify new tuberculosis (TB) antibiotics, whole cell-based phenotypic screens have been conducted against the pathogen Mycobacterium tuberculosis (Mtb). Over the last decade, several of these screens have identified MmpL3 as the proposed target for diverse small molecule inhibitors including AU1235, BM212, C215, DA-5, E11, indolecarboxamides, HC2091, NITD-349, PIPD1, Rimonabant, Spiro, TBL-140, THPP and SQ109 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 . MmpL3 is an essential flippase responsible for transporting acetylated-trehalose monomycolate (TMM) synthesized in the cytoplasm to the pseudo-periplasmic space 13, 14, 15, 16, 17 .…”