Synergistic Interactions of MmpL3 Inhibitors with Antitubercular Compounds
            <i>In Vitro</i>

Li, Wei; Sánchez-Hidalgo, Andrea; Jones, Victoria; Moura, Vinicius Calado Nogueira de; North, E. Jeffrey; Jackson, Mary

doi:10.1128/aac.02399-16

Cited by 57 publications

(66 citation statements)

References 23 publications

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“…In order to test this hypothesis in whole cell Mtb, we used the recently described dia gonal m easurement of n -way d rug interactions (DiaMOND) approach 37 . RIF was included as a control for these assays, as this drug has been shown to be synergistic when tested with other MmpL3 inhibitors such as AU1235 and SQ109 38, 39 . The results of DiaMOND, shown in Figure 5, identified synergistic interactions (FIC 2 < 1.0) between all combinations of MmpL3 inhibitors and RIF.…”

Section: Resultsmentioning

confidence: 99%

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

Williams

Haiderer

Coulson

et al. 2019

Preprint

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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 ...

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Section: Resultsmentioning

confidence: 99%

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

Williams

Haiderer

Coulson

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Shortly thereafter, the pyrrole derivative BM212 (La Rosa et al, 2012) and the adamantyl urea AU1235 (Grzegorzewicz et al, 2012) with antitubercular activity were found to target mmpL3 as resistant mutants harboured mutated mmpL3 alleles. MmpL3 inhibitors act also synergistically with other antitubercular drugs, which is of interest to efforts that aim to reduce the duration of TB treatments (Li et al, 2017).The divergent scaffolds from which these different inhibitors were derived as well as their broad-spectrum activity against non-mycolate producing bacteria and fungal species, suggested that MmpL3 was probably not the direct target of these inhibitors. Li et al (2014) have shown that SQ109 causes dissipation of the PMF in M. tuberculosis, thus disrupting MmpL3 function.…”

Section: Mmpl3 As An Attractive Antimycobacterial Drug Targetmentioning

confidence: 99%

“…The continuing rise of MDR tuberculosis and the emergence of difficultto-treat NTM infections warrant the need for new drugs with new modes of action that will shorten treatment duration. In this regard, MmpL3 inhibitors may have significant potential (Li et al, 2017). Since the export of TMM requires the action of the essential TmaT mycolyl acyltransferase, MmpL interactors such as TmaT may represent potential drug targets that remain to be explored.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

The diverse family of MmpL transporters in mycobacteria: from regulation to antimicrobial developments

Viljoen

Dubois

Girard-Misguich

et al. 2017

Molecular Microbiology

110

119

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SummaryMycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance-Nodulation-Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surfaceexposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid-growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non-tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/ functional information are also provided to explain the molecular basis underlying the proton-motive force driven lipid transport. Overall, this review emphasises the Janus-face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.

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“…It is hypothesized that several inhibitors indirectly modulate MmpL3 activity by perturbing PMF, which is required for MmpL3 lipid export activity (22). Multiple MmpL3 inhibitors also exhibit synergistic interactions with TB antibiotics, further supporting interest in this target (27). We previously performed a high-throughput phenotypic screen of a library of Ͼ200,000 compounds for inhibitors of the M. tuberculosis PhoPR and DosRST twocomponent regulatory systems (28,29), and as part of these screens, we also identified compounds that inhibited M. tuberculosis growth.…”

mentioning

confidence: 99%

HC2091 Kills Mycobacterium tuberculosis by Targeting the MmpL3 Mycolic Acid Transporter

Zheng

Williams

Coulson

et al. 2018

Antimicrob Agents Chemother

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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.

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Synergistic Interactions of MmpL3 Inhibitors with Antitubercular Compounds In Vitro

Cited by 57 publications

References 23 publications

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

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

The diverse family of MmpL transporters in mycobacteria: from regulation to antimicrobial developments

HC2091 Kills Mycobacterium tuberculosis by Targeting the MmpL3 Mycolic Acid Transporter

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