Anoz-Carbonell, E. | bioRxiv Science

bioRxiv2025

Exploiting Vitamin B6 Dependency: BVL3572S Inhibits HisC and AlaA to Kill Mycobacterium tuberculosis

Tuberculosis remains the leading cause of death from a single infectious agent worldwide, and the growing prevalence of multi-drug resistant Mycobacterium tuberculosis (Mtb) underscores the urgent need for antibiotics with novel mechanisms of action. Here, we characterize BVL3572S, a hydroxamic acid-containing compound that is bactericidal and potently inhibits the growth of both extracellular and intracellular Mtb. Integrated transcriptomic, genetic, and biochemical analyses identified the pyridoxal phosphate (PLP)-dependent aminotransferases HisC (Rv1600) and AlaA (Rv0337c; formerly AspC) as the primary molecular targets of BVL3572S, thereby simultaneously impacting L-histidine and L-alanine biosynthesis. Spontaneous resistance mutants harbored mutations in hisC or alaA. Target engagement was further supported by overexpression studies: AlaA overexpression increased resistance in the presence of L-His whereas HisC overexpression paradoxically increased susceptibility. X-ray crystallography revealed a covalent adduct between PLP and BVL3572S within the HisC active site. The short occupancy of this adduct suggests a futile cycle that sequesters PLP. Isotopic labeling revealed widespread perturbation of amino acid biosynthesis, consistent with PLP starvation. The stepwise resistance observed upon supplementation with L-His and L-Ala together or with PLP alone suggests inhibition of multiple targets. Genome-scale CRISPRi and Tn-seq analyses additionally indicated disruptions in central metabolism, cell envelope integrity, and redox balance, possibly due to PLP depletion cascades. Consistent with its inhibition of AlaA, BVL3572S displayed strong synergy with D-cycloserine, a second-line antitubercular drug targeting D-alanine synthesis and impacting peptidoglycan synthesis, highlighting the potential of this compound in combination therapy. Collectively, our findings establish BVL3572S as a promising lead compound acting through a previously unexploited, multitarget mechanism that induces broad metabolic stress in Mtb, offering a novel therapeutic strategy against drug-resistant tuberculosis.

microbiology↗

bioRxiv2020

In silico discovery and biological validation of ligands of FAD synthase, a promising new antimicrobial target

New treatments for diseases caused by antimicrobial-resistant microorganisms can be developed by identifying unexplored therapeutic targets and by designing efficient drug screening protocols. In this study, we have screened a library of compounds to find ligands for the flavin-adenine dinucleotide synthase (FADS) -a potential target for drug design against tuberculosis and pneumonia- by implementing a new and efficient virtual screening protocol. The protocol has been developed for the in silico search of ligands of unexplored therapeutic targets, for which limited information about ligands or ligand-receptor structures is available. It implements an integrative funnel-like strategy with filtering layers that increase in computational accuracy. The protocol starts with a pharmacophore-based virtual screening strategy that uses ligand-free receptor conformations from molecular dynamics (MD) simulations. Then, it performs a molecular docking stage using several docking programs and an exponential consensus ranking strategy. The last filter, samples the conformations of compounds bound to the target using MD simulations. The MD conformations are scored using several traditional scoring functions in combination with a newly-proposed score that takes into account the fluctuations of the molecule with a Morse-based potential. The protocol was optimized and validated using a compound library with known ligands of the Corynebacterium ammoniagenes FADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17 in silico filtered molecules were tested experimentally. We identified five inhibitors of the activity of the flavin adenylyl transferase mononucleotide of the FADS, and some of them were able to inhibit growth of three bacterial species: Corynebacterium ammoniagenes, Mycobacterium tuberculosis, and Streptococcus pneumoniae, where the former two are human pathogens. Overall, the results show that the integrative VS protocol is a cost-effective solution for the discovery of ligands of unexplored therapeutic targets. Author summaryDeveloping cures for antimicrobial-resistant microorganisms is a pressing necessity. Addressing this problem requires the discovery of novel therapeutic targets -for example, bacterial proteins with no human homologues- and the development of cost-effective drug screening protocols. In this work, we tackled the problem on both sides. We developed an efficient and successful integrative computational protocol for screening inhibitory-molecules for unexplored targets. We used it to discover five novel inhibitors of flavin-adenine dinucleotide synthase (FADS), a promising protein target of pathogens causing tuberculosis and pneumonia.

biophysics↗