Covalent Fragment-Based Drug Discovery Targeting Novel Allosteric Sites of Mycobacterium tuberculosis InhA: An Integrated Structural and Synthetic Approach

Enoyl-ACP reductase InhA of Mycobacterium tuberculosis is an established antitubercular drug target inhibited by isoniazid following activation by the KatG enzyme. Resistance to isoniazid most commonly arises through KatG mutations, which abolish prodrug activation rather than modifying InhA itself, making direct InhA inhibitors independent of KatG activation an important therapeutic strategy. Covalent fragment-based drug discovery targeting allosteric sites distal from the isoniazid binding site represents a frontier approach to circumventing current resistance. This study applied covalent FBDD to identify and develop novel allosteric InhA inhibitors as potential direct antitubercular agents. A targeted covalent fragment library of 320 electrophilic fragments was screened against recombinant InhA by competitive mass spectrometry and thermal shift assays. Crystal structures of fragment-InhA complexes were determined to map binding modes. Selected fragment hits were elaborated by structure-guided synthetic chemistry using a grow-link-optimise strategy. Final lead compounds were evaluated for whole-cell antimycobacterial activity against H37Rv, isoniazid-resistant clinical strains, and MDR-TB isolates. Enzyme kinetic analysis characterised the mode of InhA inhibition. Eight covalent fragment hits were confirmed, with four mapping to a previously uncharacterised allosteric pocket adjacent to the substrate binding groove. Fragment elaboration produced two lead compounds with InhA inhibition IC50 below 500 nM and whole-cell MIC against isoniazid-resistant isolates below 2 micrograms per millilitre, with no cross-resistance to isoniazid. These results deliver original structural and chemical evidence validating the targeted allosteric pocket of InhA as a druggable site for resistance-breaking antitubercular drug development. Keywords: fragment-based drug discovery, InhA, tuberculosis, covalent inhibitors, allosteric site.