# Genetic and Cheminformatic Characterization of Mycobacterium tuberculosis Inhibitors Discovered in the Molecular Libraries Small Molecule Repository

## TL;DR

This study identifies and characterizes small molecules that inhibit Mycobacterium tuberculosis, offering insights into their mechanisms and potential for drug development.

## Contribution

The study introduces a combined cheminformatics and mutant screening approach to characterize antitubercular compounds from the MLSMR library.

## Key findings

- 101 isoniazid analogs lose activity against katG mutants, confirming their dependence on KatG.
- Eight isoniazid analogs retain partial activity against katG mutants, suggesting alternative mechanisms.
- Nitrofuranyl benzothiazoles show enhanced activity against mmpL3 and katG mutants, indicating collateral sensitivity.

## Abstract

High-throughput screening (HTS) of small molecules is
a starting
point for many drug development pipelines, including tuberculosis.
These screens often result in multiple hits whose mechanisms of action
remain unknown. From our initial HTS of the Molecular Libraries Small
Molecule Repository (MLSMR), we cherry-picked 935 compounds that inhibited
the growth of Mycobacterium tuberculosis and set out to provide an early assessment of their antimycobacterial
properties and mechanism of action. To characterize the MLSMR Mtb
growth inhibitors, a combination of cheminformatics and targeted mutant
screening against mutants in katG, hadAB, and a mixed pool of mmpL3 mutants was used to
characterize the hits. As a validation of this approach, we identified
101 isoniazid analogs that predictably lose all their antimycobacterial
activities against the katG mutant. Interestingly,
eight isoniazid analogs retain part of their activity against the
mutant, suggesting an alternative KatG-independent mechanism. This
approach also identified new compounds belonging to already known
scaffolds that target HadAB or MmpL3. Additionally, we explored the
nitro-containing compounds in our data set and discovered nitrofuranyl
benzothiazoles that show enhanced activity against the mmpL3 and katG mutants, a phenomenon known as collateral
sensitivity. Overall, this study will serve as an important resource
for further follow-up studies of antitubercular small molecules in
the MLSMR library and provide a well-characterized training set for
artificial intelligence-driven antimycobacterial drug discovery.