MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14782/m_MTBLS14782_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14782/s_MTBLS14782.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14782/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14782/a_MTBLS14782_LC-MS_positive_reverse-phase.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14782<p>Metabolites were identified by extracting their exact masses with a tolerance of 10 ppm.</p>MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phase<p>Chromatographic separation of amino acids was carried out on a SUPELCO PFP column (150 × 2.1 mm, 5 µm) with a HS F5 guard column (20 × 2.1 mm, 5 µm). The column was maintained at 30 °C, and the flow rate was set at 0.25 mL/min. The solvent system consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The gradient was adapted from the method of Boudah et al. and was as follows: 0 min, 2% B; 2 min, 2% B; 10 min, 5% B; 16 min, 35% B; 20 min, 100% B; 24 min, 100% B. The column was then equilibrated for 6 min at the initial conditions before the next sample was analyzed. The injection volume was 5 µL, and the autosampler temperature was maintained at 4 °C.</p>Exploiting Vitamin B6 Dependency: BVL3572S Inhibits HisC and AlaA to Kill <i>Mycobacterium tuberculosis</i>. 10.1101/2025.10.27.684782.Hanna KULYKTBIM. tuberculosis H37Rvmass spectrometry assay<p>The cultures were centrifuged (3,500 x g, 5 min) and the pellet resuspended in HCl 6 N before a 10 h incubation at 100 °C. Samples were then lyophilized and the mass of the lyophilized product recorded.</p>M. tuberculosis H37Rvhttps://www.ebi.ac.uk/metabolights/MTBLS14782Alain Baulard. Univ. Lille, CNRS, Inserm, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France. alain.baulard@inserm.fr.Hanna KULYK. MetaboHUB-MetaToul, National Infrastructure of Métabolomiques & Fluxomics (ANR-11-INBS-0010), 31077 Toulouse, France Institut National des Sciences Appliquées - Toulouse Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse, France Institut National des Sciences Appliquées - Toulouse. Toulouse Biotechnology Institute, Bio & Chemical Engineering Université de Toulouse - CNRS 5504 - INRAE 792 - INSA TBI - INSA Toulouse 135 avenue de Rangueil 31077 Toulouse CEDEX 04. hbarbier@insa-toulouse.fr.Zainab Edoo. Univ. Lille, CNRS, Inserm, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France. zainab.edoo@inserm.fr.<p>Data processing was performed with Skyline software.</p><p>Values were normalized to untreated controls to compute percent change in 13C labeling under drug treatment and further adjusted using the optical density difference between treated and untreated cultures and using the mass of the lyophilized products.</p>Dosehbarbier@insa-toulouse.fr<p><em>M. tuberculosis</em> H37Rv was cultured up to an OD600 nm of 0.3 in Sauton medium containing NH4Cl (instead of aspartate) as the nitrogen source supplemented with glycerol 0.2%, Tween 80 0.05% and OAD 10%. The culture was treated with BVL3572S (1 µg/ml) or DMSO for 2 h at 37 °C before addition of 13C-glycerol 0.2% and further incubated for 16 h. The OD600 nm of the cultures was between 0.41 to 0.43 (in the presence of treatment) and 0.46 (without treatment).</p>MetabolomicsThermo Scientific Vanquish UHPLC SystemFluxomicscentroid spectrumM. tuberculosis H37RvMycobacterium tuberculosistargeted analysisThermo Scientific Orbitrap Exploris 120Multi-drug resistant Mycobacterium tuberculosisIon chromatography-high resolution mass spectrometry (IC-HRMS)Antitubercular therapyIsotopic profilingexperimental sampleThermo Scientific Vanquish UHPLC SystemFluxomicscentroid spectrumM. tuberculosis H37RvMycobacterium tuberculosistargeted analysisMulti-drug resistant Mycobacterium tuberculosisThermo Scientific Orbitrap Exploris 120Ion chromatography-high resolution mass spectrometry (IC-HRMS)Isotopic profilingAntitubercular therapyexperimental sample<p>Mass detection was performed in positive electrospray ionization (ESI+) mode. The mass spectrometer settings were as follows: spray voltage, 3.5 kV; capillary temperature, 325 °C; desolvation temperature, 350 °C; and maximum injection time set to Auto. Nitrogen was used as sheath gas (50 arbitrary units) and as auxiliary gas (10 arbitrary units). The automatic gain control (AGC) target was set to 1 × 10^6, and a resolution of 60,000 was used over the m/z 50–750 range. MS analyses were performed in Full Scan mode. Data acquisition was carried out using Thermo Scientific Xcalibur software. </p>NorvValSerIleLysGlnPheProTyrCystineGluTrpHisOHProlineGlySarcosineAlaArgLeuMetAsnThrAspfalseExploiting Vitamin B6 Dependency: BVL3572S Inhibits HisC and AlaA to Kill Mycobacterium tuberculosis<p>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.</p>2026-06-232026-06-17MTBLS14782MTBLC16977MTBLC16467MTBLC17196MTBLC17053MTBLC16283MTBLC18050MTBLC16015MTBLC15428MTBLC15971MTBLC17191MTBLC15603MTBLC18019MTBLC16643MTBLC18314MTBLC18095MTBLC17295MTBLC17203MTBLC15611MTBLC17115MTBLC16857MTBLC16828MTBLC17895MTBLC16414CHEBI:16977CHEBI:16467CHEBI:17196CHEBI:17053CHEBI:16283CHEBI:18050CHEBI:16015CHEBI:15428CHEBI:15971CHEBI:17191CHEBI:15603CHEBI:18019CHEBI:16643CHEBI:18314CHEBI:18095CHEBI:17295CHEBI:17203CHEBI:15611CHEBI:17115CHEBI:16857CHEBI:16828CHEBI:17895CHEBI:16414