Identification of novel pyrazolo[4,3-c]pyridine and diazepane derivatives as potent inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase B

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis ( M.tb ), continues to pose a critical global health threat as a leading infectious cause of mortality. Therapeutic efficacy is increasingly compromised by the emergence of multidrug-resistant strains and the limitations of existing regimens, which necessitate treatment durations of six months or longer. Protein tyrosine phosphatase B from Mtb (PtpB- Mtb ) has been recognized as a critical virulence factor, representing a promising target for novel antitubercular therapies due to its unique structural and functional properties. In this study, a comprehensive structure-based virtual screening approach was employed to identify novel small-molecule scaffolds with inhibitory potential against PtpB- Mtb . The ChemBridge compound library was curated and filtered for drug-like properties, followed by hierarchical molecular docking and molecular dynamics simulations to prioritize candidates with high predicted affinity and stability within the PtpB- Mtb active site. Quantum mechanical calculations further characterized the electronic properties of top hits. Recombinant PtpB- Mtb was expressed and purified to homogeneity, and in vitro enzymatic assays were performed to evaluate the inhibitory potency and selectivity of shortlisted compounds. Two derivatives bearing pyrazolo[4,3-c]pyridine and 1,4-diazepane ring nuclei demonstrated significant inhibition of PtpB- Mtb activity, exhibiting IC₅₀ values of 14.4 µM and 32.6 µM, respectively. Biolayer interferometry confirmed strong and specific binding to PtpB-Mtb, with dissociation constants (K d ) of 0.012 µM and 0.57 µM. The integrated workflow presented herein highlights the potential of these novel scaffolds as starting points for the development of selective, cell-permeable PtpB- Mtb inhibitors, offering a promising avenue for next-generation anti-tubercular drug discovery.