# Defining the Mechanism of Action and Resistance of New Mycobacterium abscessus MmpL3 Inhibitors

## TL;DR

This paper introduces new inhibitors targeting MmpL3 in Mycobacterium abscessus, showing promising antibacterial activity and insights into resistance mechanisms.

## Contribution

The study identifies new MmpL3 inhibitors with low resistance frequency and reveals structure-driven resistance patterns through experimental and computational analysis.

## Key findings

- New MmpL3 inhibitors show comparable in vitro activity to standard treatments and are effective against Mab in macrophages and biofilms.
- Inhibitors exhibit additive or synergistic effects with standard antibiotics and have low eukaryotic cytotoxicity.
- Computational simulations correlate with biological cross-resistance patterns, revealing structural-functional associations in resistance.

## Abstract

Mycobacterium abscessus (Mab) is
difficult to treat due to intrinsic and acquired resistance to diverse
antibiotics. Among the intrinsic resistance factors is the mycomembrane,
a complex structure that limits permeability to several classes of
antibiotics. Here, we report new inhibitors of MmpL3, an essential
transporter required to build the mycomembrane. Several of the MmpL3
inhibitors have comparable activity in vitro to standard-of-care treatments,
exhibit both time- and dose-dependent bactericidal activity, have
low eukaryotic cytotoxicity, and are efficacious against Mab growing
in macrophages or in biofilms. The inhibitors had varying activities
against a panel of 30 different multidrug-resistant clinical isolates
and are additive or synergistic with standard-of-care antibiotics,
suggesting they could be included in combination therapy. The inhibitors
also exhibit a low frequency of resistance, with some of the isolated
mutants displaying differential patterns of sensitivity and resistance
to the different MmpL3 inhibitors and putative fitness defects. Cross-resistance
profiles of 15 structurally related inhibitors against 16 different
MmpL3 resistant mutants demonstrate structure-driven clustering patterns
of the inhibitors, where those carrying a similar scaffold cluster
together and different MmpL3 amino-acid substitutions account for
these differences. Cross-resistance profiles were also simulated computationally,
showing significant correlation between the computationally calculated
parameters and the biological patterns of cross-resistance and emphasizing
specific structural–functional associations driving resistance
or susceptibility. These inhibitors and their analogs hold promise
for clinical translation, and the established structural–functional
associations provide mechanistic insights into the function of MmpL3,
resistance and susceptibility of MmpL3 inhibitors, and fitness costs
associated with MmpL3 resistance.