The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC₅₀ value of 0.59 μg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25–0.5 μg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10⁻⁵ or 10⁻¹ μg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

Introduction: With the increasingly serious problem of bacterial drug resistance caused by NDM-1, it is an important strategy to find effective inhibitors to assist β-lactam antibiotic treatment against NDM-1 resistant bacteria. In this study, PHT427 (4-dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide) was identified as a novel NDM-1 inhibitor and restored the susceptibility of meropenem against Enterobacteriaceae producing NDM-1.Methods: We used a high throughput screening model to find NDM-1 inhibitor in the library of small molecular compounds. The interaction between the hit compound PHT427 and NDM-1 was analyzed by fluorescence quenching, surface plasmon resonance (SPR) assay, and molecular docking analysis. The efficacy of the compound in combination with meropenem was evaluated by determining the FICIs of Escherichia coli BL21(DE3)/pET30a(+)-bla_NDM–1 and Klebsiella pneumoniae clinical strain C1928 (producing NDM-1). In addition, the mechanism of the inhibitory effect of PHT427 on NDM-1 was studied by site mutation, SPR, and zinc supplementation assays.Results: PHT427 was identified as an inhibitor of NDM-1. It could significantly inhibit the activity of NDM-1 with an IC₅₀ of 1.42 μmol/L, and restored the susceptibility of meropenem against E. coli BL21(DE3)/pET30a(+)-bla_NDM–1 and K. pneumoniae clinical strain C1928 (producing NDM-1) in vitro. The mechanism study indicated that PHT427 could act on the zinc ions at the active site of NDM-1 and the catalytic key amino acid residues simultaneously. The mutation of Asn220 and Gln123 abolished the affinity of NDM-1 by PHT427 via SPR assay.Discussion: This is the first report that PHT427 is a promising lead compound against carbapenem-resistant bacteria and it merits chemical optimization for drug development.

Background Bedaquiline (BDQ), a first-in-class diarylquinoline compound, was approved for therapy of multidrug-resistant tuberculosis (MDR-TB) by the FDA in 2012. Previous studies have shown that the addition of BDQ to the preferred regimen resulted in faster sputum-culture conversion, but more deaths compared with the placebo group. Since the mechanism of BDQ is related to ATP synthase, and ATP synthase is closely linked to aging-related diseases, we hypothesized that BDQ may cause mitochondrial dysfunction, leading to cellular apoptosis and senescence. Methods The Cell Counting Kit-8 (CCK-8) assay is used to assess the viability of cells in the presence or absence of bedaquiline treatment. We used flow cytometry to detect Annexin V-PI and ROS levels in different groups of cells. TMRM staining is performed to examine the changes in mitochondrial membrane potential of the cells. Western blot is used to measure the expression levels of proteins associated with aging and apoptosis. The β-Galactosidase kit is used for staining to examine the proportion of senescent cells in different groups. Results In the current study, we evaluated the apoptosis and senescence induction effects of BDQ in human embryonic lung fibroblasts MRC-5 cells and rat cardiomyocytes H9C2 cells and explored the potential molecular mechanisms. The results demonstrated that BDQ reduced the cell viability in a dose- and time-dependent manner. In addition, BDQ induced cellular apoptosis and senescence, and increased Reactive Oxygen Species (ROS) level. Conclusions Our results revealed that BDQ can cause cellular apoptosis and senescence for the first time, with the aim of optimizing anti-TB drug regimens in anticipation of better outcomes.