# Development of new M. tuberculosis MmpL3 inhibitors

> **NIH NIH R21** · MICHIGAN STATE UNIVERSITY · 2020 · $191,999

## Abstract

Identifying new antibiotics is critical for the long-term control of multi-drug resistant tuberculosis (TB).
Several new classes of compounds that kill Mycobacterium tuberculosis (Mtb) have been discovered in recent
years, including inhibitors of the MmpL3 mycolic acid transporter. MmpL3 is a common target of small
molecule inhibitors of mycobacterial growth identified by whole cell, phenotypic screens. It is a mycolic acid
flippase that moves trehalose monomycolate (TMM) to the pseudoperiplasmic space, from where TMM is
further modified to trehalose dimycolate (TDM) and incorporated into the mycomembrane. Mtb and M.
smegmatis mmpL3 knockdown strains provide genetic evidence that mmpL3 is essential for survival both in
vitro and in a mouse TB infection model. This essentiality makes MmpL3 an attractive therapeutic target and
supports efforts to characterize small molecules targeting MmpL3. Multiple MmpL3 inhibitors also exhibit
synergistic interactions with TB antibiotics, 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 function by inhibiting MmpL3 function. These compounds are bactericidal both in
vitro and against intracellular Mtb in primary murine macrophages. Mutations in mmpL3 provided resistance to
these compounds. Mtb cells treated with these compounds were shown to accumulate TMM and have reduced
levels of TDM, further supporting their targeting MmpL3 activity. Cluster analysis of cross-resistance profiles,
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.
The combined study of multiple mutants and new compounds provides new insights into structure-function
interactions of MmpL3 and its inhibitors and will enable the development of new classes of Mtb inhibitors.
 The goal of this proposal is to conduct exploratory studies to develop new, more durable compounds that
inhibit MmpL3. Compounds will be optimized for potency and pharmacokinetic (PK) properties and tested for
efficacy in an acute mouse model of Mtb infection (Aim 1). Additionally, we will conduct studies examining the
frequency of resistance of Mtb treated with combinations of MmpL3 inhibitors (Aim 2). We hypothesize that
specific combinations of inhibitors, based on differences in their interactions with MmpL3, may reduce the
frequency of resistance or select for resistant mutants with reduced fitness. This finding would support the
development a more durable, hybrid MmpL3 inhibitor sharing the combined activity of the paired treatments.
New chemical matter and proof-of-concept data generated in these studies will derisk further development of a
new MmpL3 inhibitor that can treat MDR-TB.
OVERALL IMPACT: This project will optimize novel Mtb inhibitors, define their function in vivo a...

## Key facts

- **NIH application ID:** 9871233
- **Project number:** 1R21AI148909-01
- **Recipient organization:** MICHIGAN STATE UNIVERSITY
- **Principal Investigator:** Robert B Abramovitch
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $191,999
- **Award type:** 1
- **Project period:** 2020-03-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9871233

## Citation

> US National Institutes of Health, RePORTER application 9871233, Development of new M. tuberculosis MmpL3 inhibitors (1R21AI148909-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9871233. Licensed CC0.

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