# Chemical biology studies of MmpL3 inhibition and resistance in mycobacteria

> **NIH NIH R01** · MICHIGAN STATE UNIVERSITY · 2024 · $747,825

## Abstract

MmpL3 (Mycobacterium membrane protein Large 3) is a common target of inhibitors of mycobacterial growth
identified by whole cell, phenotypic, high throughput screens. MmpL3 is a mycolate flippase that moves trehalose
monomycolate (TMM) to the pseudoperiplasmic space, from where TMM is modified to trehalose dimycolate
(TDM) and incorporated into the mycomembrane. Mycobacterium tuberculosis (Mtb) and M. smegmatis mmpL3
knockdown strains show that mmpL3 is essential for survival both in vitro and in mice. This phenotype makes
MmpL3 an attractive therapeutic target and supports efforts to characterize molecules targeting MmpL3. Multiple
MmpL3 inhibitors exhibit synergistic interactions with TB drugs, further supporting interest in this target.
Using an innovative combination of untargeted and targeted mutant screens, we have identified ten new and
distinct scaffolds that inhibit MmpL3 function. These compounds are bactericidal both in vitro and against
intracellular Mtb in primary murine macrophages. The inhibitors are mycobacteria specific with several showing
activity against the non-tuberculous mycobacterial (NTM) species M. abscessus (Mab), including the HC2099
and HC2091 series. Pilot structure activity relationship (SAR) studies involving the synthesis of over 100 analogs
of HC2099 and HC2091 have identified analogs with whole cell Mtb half-maximal efficacies of ~80 nM and ~280
nM, respectively. Several analogs exhibited high solubility, stability in microsomes and no cytotoxicity in
macrophages, supporting their further development. For example, MSU-43085, an analog of HC2099, is orally
bioavailable, active against Mtb in an acute murine model of infection and has activity comparable to standard
of care drugs against Mab in vitro and in macrophages. Therefore, these series will be valuable tools to
understand inhibitor-MmpL3 structure-function interactions and as leads for new TB drug development.
Our library of MmpL3 inhibitors and mutants also will enable our team to define mechanisms of resistance in
MmpL3. Cluster analysis of cross resistance profiles, generated by dose response experiments for each
combination of 13 MmpL3 inhibitors against 24 different mmpL3 mutants, defined two clades of inhibitors and
two clades of resistant mutants. Pairwise combination studies of the inhibitors revealed antagonistic, synergistic
and additive interactions that were specific to the identified clades. Modeling of resistance substitutions to the
MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3. These
findings support our hypothesis that combinations of MmpL3 inhibitors or rationally designed molecules can be
employed to reduce the frequency of resistance.
The overall goals of this study are to: 1) optimize new MmpL3 inhibitors to define inhibitor MmpL3 interactions
and generate proof-of-concept data showing efficacy in vivo (Aim 1); and 2) define mechanisms of resistance in
MmpL3 to devise strategi...

## Key facts

- **NIH application ID:** 10887650
- **Project number:** 5R01AI173285-02
- **Recipient organization:** MICHIGAN STATE UNIVERSITY
- **Principal Investigator:** Robert B Abramovitch
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $747,825
- **Award type:** 5
- **Project period:** 2023-07-13 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10887650

## Citation

> US National Institutes of Health, RePORTER application 10887650, Chemical biology studies of MmpL3 inhibition and resistance in mycobacteria (5R01AI173285-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10887650. Licensed CC0.

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