Targeting of essential mycobacterial replication enzyme DnaG primase revealed Mitoxantrone and Vapreotide as novel mycobacterial growth inhibitors**

Abstract Tuberculosis (TB) is the second leading cause of mortality after COVID‐19, with a global death toll of 1.6 million in 2021. The escalating situation of drug‐resistant forms of TB has threatened the current TB management strategies. New therapeutics with novel mechanisms of action are urgently required to address the current global TB crisis. The essential mycobacterial primase DnaG with no structural homology to homo sapiens presents itself as a good candidate for drug targeting. In the present study, Mitoxantrone and Vapreotide, two FDA‐approved drugs, were identified as potential anti‐mycobacterial agents. Both Mitoxantrone and Vapreotide exhibit a strong Minimum Inhibitory Concentration (MIC) of ≤25μg/ml against both the virulent (M.tb‐H37Rv) and avirulent (M.tb‐H37Ra) strains of M.tb. Extending the validations further revealed the inhibitory potential drugs in ex vivo conditions. Leveraging the computational high‐throughput multi‐level docking procedures from the pool of ~2700 FDA‐approved compounds, Mitoxantrone and Vapreotide were screened out as potential inhibitors of DnaG. Extensive 200 ns long all‐atoms molecular dynamic simulation of DnaGDrugs complexes revealed that both drugs bind strongly and stabilize the DnaG during simulations. Reduced solvent exposure and confined motions of the active centre of DnaG upon complexation with drugs indicated that both drugs led to the closure of the active site of DnaG. From this study‘s findings, we propose Mitoxantrone and Vapreotide as potential anti‐mycobacterial agents, with their novel mechanism of action against mycobacterial DnaG.