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

Abstract

22combination studies of the inhibitors revealed interactions that were specific to the clades 40 identified in the cross-resistance profiling. Additionally, modeling of resistance substitutions to 41 the MmpL3 crystal structure revealed clade specific localization of the residues to specific 42 domains of MmpL3, with the clades showing differential resistance. Several compounds 43 exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, 44supporting their further development. The combined study of multiple mutants and novel 45 compounds provides new insights into structure-function interactions of MmpL3 and small 46 molecule inhibitors. 47 the last decade, several of these screens have identified MmpL3 as the proposed target for 53 diverse small molecule inhibitors including AU1235, BM212, C215, DA-5, E11, 54 indolecarboxamides, HC2091, PIPD1, Rimonabant, Spiro, THPP and 55 SQ109 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 . MmpL3 is an essential flippase responsible for transporting 56 acetylated-trehalose monomycolate (TMM) synthesized in the cytoplasm to the pseudo-57 periplasmic space 13, 14, 15, 16, 17 . These TMMs are then converted into trehalose dimycolate (TDM) 58by the Ag85 complex in the cell envelope 18 . MmpL3 is essential as evidenced by a pre-existing 59 rescue allele being required to generate an mmpL3 knockout 2, 14, 17, 19, 20, 21 , lack of mutants in 60 high-throughput transposon mutagenesis screens 22, 23 , and studies that show rapid killing in vitro 61 and in vivo in acute infection models when mmpL3 expression is conditionally inhibited 14, 19 . This 62 makes MmpL3 an attractive target for drug development, with one of its inhibitors, SQ109, 63 currently in clinical trials 24 . 64MmpL3 inhibitors fall into diverse classes of chemical scaffolds 25, 26, 27 , making it hard to 65 computationally predict potential MmpL3 inhibitors based on chemical scaffolds. However, given 66 the frequent finding of MmpL3 as a target, it is reasonable to expect that many new hits in a 67 high throughput screen (HTS) may be acting against MmpL3. MmpL3 inhibitors have been 68 identified by the isolation and sequencing of resistant mutants with single nucleotide variations 69 (SNVs) mapping to the coding region of mmpL3, which is time-consuming and costly. Efforts to 70 discover MmpL3 inhibitors using targeted approaches include generating hypomorphs, where a 71 mmpL3 knock down strain showed enhanced sensitivity to MmpL3 inhibitors, including AU1235 72 14 . However, this strain was also shown to be sensitive to isoniazid (INH) an inhibitor of InhA of 73 the FAS-II pathway involved in mycolic acid synthesis, suggesting that while a mmpL3 74 knockdown strain has robust screening potential for inhibitors of mycolic acid synthesis, 75 maturation, and transport, such strains are not specific enough to identify inhibitors that 76 selectively target MmpL3. 77An alternative approach, employed in this study, is to use a pool of mmpL3 resistant 78 mutants to discover potential MmpL3 ...

Cryo-EM structure and resistance landscape of M. tuberculosis MmpL3: An emergent therapeutic target

“…These mutations likely weaken the affinity of antitubercular drugs targeting the transmembrane site, as has been demonstrated for a handful of Msmg MmpL3 variants by MST ( Zhang et al., 2019 ). This conclusion is also consistent with proposals that the magnitude and spectrum of the resistance phenotype given by an MmpL3 mutant correlates with its proximity to this pocket ( Williams et al., 2019 ). However, our analysis reveals that a significant proportion of RPs are located >10 Å away from the drug pocket, including some within the LMNG binding site (asterisks in Figure 2 A), suggesting that there is at least one additional, and as yet uncharacterized, MmpL3 resistance mechanism; likely involving the PD and its cavity.…”

MtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates

“…In Mycobacteria, two independentlysynthesized fatty acids, the short alpha branch (C24-C26) and long meromycolate chain (C50-C60), produced by the single FAS-I enzyme and FAS-II complex (12,13), respectively, are modified then condensed by the cytoplasmic polyketide synthase, Pks13 (14), to form a 2alkyl 3-keto fatty acid which is then reduced by CmrA to generate the mature, hydroxylated mycolic acid (15). These fatty acids are conjugated to trehalose to form TMM which is then bound and flipped to the periplasm by the integral membrane lipid transporter MmpL3 (16,17), the target of several classes of new antimycobacterial inhibitors (18,19). While structurally diverse classes of MmpL3 inhibitors have been reported, several of these compounds were subsequently found to indirectly inhibit MmpL3, by disrupting the transmembrane electrochemical proton gradient (20).…”

Pipeline of anti‐Mycobacterium abscessus small molecules: Repurposable drugs and promising novel chemical entities

“…Compound HC2091 (Figure 10) was originally identified as an inhibitor of M. tuberculosis in vitro (bactericidal activity) and in macrophages (bacteriostatic activity) by Zheng et al 207 It was reported in the same study that the compound inhibits the mycolic acid transporter, MmpL3. In another screening, HC2091 displayed almost 97% growth inhibition of M. abscessus at 20 μM (MIC 50 ~6.25 μM) in vitro 208 . In addition, William et al noted that HC2091 is not cytotoxic and exhibits good solubility and favorable microsome stability.…”