Computational Design, Synthesis and Biological Evaluation of Novel Peptidomimetic Inhibitors of Mycobacterium tuberculosis ClpP1P2 Protease Complex

Tuberculosis caused by Mycobacterium tuberculosis remains a major infectious disease cause of death in Nigeria, and the escalating prevalence of drug-resistant strains demands urgent development of antibiotics acting on novel targets. The caseinolytic protease complex ClpP1P2 of M. tuberculosis performs essential protein quality control functions and is non-homologous to human proteases, making it an attractive antibacterial target with a favourable selectivity profile. This study employed structure-based computational design, synthesis, and biological evaluation of peptidomimetic inhibitors targeting the ClpP1P2 active site. Virtual screening of a focused peptidomimetic library against the ClpP1P2 crystal structure was conducted using Glide docking and free energy perturbation calculations to prioritise fifteen compounds for synthesis. Target compounds were synthesised by solid-phase peptide synthesis incorporating non-natural amino acids and electrophilic warhead functionalities for covalent active site engagement. All synthesised compounds were characterised by HPLC purity assessment, ESI-MS, and NMR. Enzymatic inhibition was assessed using a fluorogenic casein substrate assay. Whole-cell antimycobacterial activity was determined against H37Rv, MDR-TB, and XDR-TB clinical isolates. Two lead compounds showed ClpP1P2 inhibition IC50 values below 200 nM and whole-cell MIC against MDR-TB below 1 microgram per millilitre. Selectivity against human ClpP was confirmed at concentrations 200-fold above the antimycobacterial MIC. This study provides original structure-activity relationship insights for ClpP1P2 peptidomimetic inhibitors and validates the target for Nigerian-context antituberculosis drug discovery. Keywords: tuberculosis, ClpP1P2, peptidomimetics, drug resistance, structure-based design.