Makafe, G. G. | Search bioRxiv

bioRxiv2026

Induced alanine auxotrophy as a therapeutic strategy against Mycobacterium tuberculosis

New antitubercular agents acting through previously unexploited mechanisms are urgently needed. Using a drug-repurposing platform, we identified TI-374, a hydroxamic acid containing compound that inhibits Mycobacterium tuberculosis (Mtb) with sub-micromolar potency. Systems analysis, resistance mapping, supplementation assays, and biochemical studies showed that TI-374 inhibits two PLP-dependent aminotransferases, AlaA and HisC1. However, its activity is driven primarily by irreversible inhibition of AlaA, whereas HisC1 inhibition is only partially reversible, revealing differential reversibility between the two targets. Optimization yielded TI-801, a low-nanomolar AlaA inhibitor. Both compounds remained active against intracellular Mtb in a macrophage infection model, where alanine supplementation did not rescue growth, indicating that host-derived alanine is unlikely to bypass AlaA inhibition. Genetic deletion of alaA attenuated Mtb survival in a murine infection model. Together, these findings support AlaA as a host-relevant metabolic vulnerability in Mtb and TI-801 as a mechanistic chemical probe for its validation as an antitubercular target.

pharmacology and toxicology↗

bioRxiv2025

Mouse Models Uniformly Featuring Human-like Lesions Harboring Drug-tolerant Mycobacterium tuberculosis

Mouse models have been key to studies of tuberculosis pathogenesis and drug efficacy, but many, such as those employing BALB/c mice, fail to reproduce the full range of heterogenous microniches observed in well-structured human lesions, which feature hypoxic caseous cores of necrotic debris surrounded by infected foamy macrophages. The granuloma presents a variety of environments differing in levels of oxygen, ions, nutrients, and intra versus extracellular residence, which determine the physiological state of the infecting bacillus and its susceptibility to immune or drug control. Recently, alternative mouse strains such as C3HeB/FeJ have allowed the study of infection and treatment in the context of these varied environments but exhibit substantial inconsistency in development of human-like lesions, both within and between individual mice. Building on the observation that inducible nitric oxide synthase (Nos2)-deficient mice consistently develop hypoxic necrotic lesions, we have established two simplified models with infection by the aerosol route. The first uses the slightly attenuated M. tuberculosis R1Rv strain, which produces a progressive infection that is contained at a high stable burden by an adaptive immune response. In the second model, vaccination with the attenuated {Delta}RD1, pantothenate auxotroph mc2 6230 protects from an otherwise lethal infection with virulent M. tuberculosis Erdman. This model reflects most contemporary tuberculosis infections, which take place in the context of a pre-existing immune response from vaccination. Both variations uniformly develop well-structured hypoxic necrotic lesions harboring drug tolerant bacteria. These refined models will be useful in studies of M. tuberculosis infection and treatment.

microbiology↗