Computational screening of putative small-molecule binders of Mycobacterium tuberculosis Pup using pharmacophore modelling, molecular docking and molecular dynamics simulation

Abstract

The proteasome has recently gained attention as a potential drug target for tuberculosis (TB). Proteasome inhibitors like Bortezomib and Carfilzomib have demonstrated significant anti-TB activity in various cell lines and animal models of TB infection. However, their severe side effects limit their suitability as therapeutic agents against Mycobacterium tuberculosis. This highlights the need to develop inhibitors targeting enzymes that lack human counterparts Therefore, this study aims to target another protein known as Prokaryotic ubiquitin like protein (Pup) of this proteasome pathway as there is no functional analogue of it in the humans. With this scenario, the present work focuses on the computer-aided identification of novel Pup inhibitors (PupIs) via ligand-based 3-D pharmacophore modelling. The 3D structure of M. tuberculosis Pup protein, complexed with the amino-terminal coiled coil of the M. tuberculosis proteasomal ATPase Mpa (PDB ID: 3M91), was retrieved, and the B chain was extracted for further analysis. A dataset of known proteasome inhibitors from CHEMBL was screened through molecular docking. Two small molecules, Mol 14 and Mol 15, with binding energies of -6.3 kcal/mol and -7.3 kcal/mol, were identified and used to construct pharmacophore queries. These queries were validated and screened against external databases to identify potential hits. Further docking studies, pharmacokinetic predictions, toxicity assessments, and molecular dynamics (MD) simulations were conducted.  Three promising inhibitors—Mol 006, Mol 265, and Mol 280—demonstrated strong binding affinities (-6.5 to -5.7 kcal/mol). MD simulations validated the stability of Mol 280 (−6.5 kcal/mol) in complex with the B chain of 3M91, with RMSD values of 3.98 Å (protein alone) and 3.55 Å (complexed with the inhibitor Mol 280), suggesting its potential as a novel PupI. These findings support further development of Mol 280 as a therapeutic candidate against MDR-TB.