Hybrid virtual screening identifies dipyrazole carboxamide derivatives as novel direct InhA inhibitors with antitubercular activity

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 8; p. 130827
• Main Authors: Punkvang, Auradee, Pakamwong, Bongkochawan, Phusi, Naruedon, Thongdee, Paptawan, Chayajarus, Kampanart, Sangswan, Jidapa, Pangjit, Kanjana, Suttisintong, Khomson, Leanpolchareanchai, Jiraporn, Hongmanee, Poonpilas, Santanirand, Pitak, Spencer, James, Mulholland, Adrian J., Sureram, Sanya, Kittakoop, Prasat, Pungpo, Pornpan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2025
• Subjects: Antitubercular Agents - chemistry; Antitubercular Agents - pharmacology; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Drug Evaluation, Preclinical; Humans; InhA inhibitor; M. tuberculosis; Microbial Sensitivity Tests; Molecular Docking Simulation; Molecular Dynamics Simulation; Mycobacterium tuberculosis - drug effects; Mycobacterium tuberculosis - enzymology; Oxidoreductases - antagonists & inhibitors; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Pyrazoles - chemistry; Pyrazoles - pharmacology; Structure-Activity Relationship; Tuberculosis; Virtual screening; mM; INH; DTT; ns; SAR; kDa; MTT; GPU; PDB; COVID-19; μM; NADH; Ni–NTA; IC50; MD; InhA inhibitor; E. coli; ADME; Virtual screening; RMSD; Molecular dynamics simulation; SDS–PAGE; EDTA; μg/mL; M. tuberculosis; TB; MABA; MWCO; Tuberculosis; PMSF; DD-CoA; DNA; LB; RESP; XP; IPTG; HPC; PAINS; GAFF; InhA; nm
• DOI: 10.1016/j.bbagen.2025.130827
• ISSN: 0304-4165; 1872-8006

Direct inhibitors of M. tuberculosis enoyl-acyl carrier protein reductase (M. tuberculosis InhA) remain effective against variants with mutations associated with isoniazid resistance. In our previous study, structure-based virtual screening was employed to discover such inhibitors. However, most identified hits exhibited limited antimycobacterial activity, with minimum inhibitory concentration (MIC) values of >100 μg/mL. To address this challenge, we refined our virtual screening strategy by integrating ligand- and structure-based virtual screening approaches. The efficacy of this hybrid virtual screening approach was validated through biological assays measuring MIC and half-maximal inhibitory concentration (IC50) for the inhibition of M. tuberculosis growth and InhA activity, respectively. Among 14 identified hits, compounds 3 and 10, classified as dipyrazole carboxamide derivatives, were validated as promising lead candidates, with MIC values of 25 and 50 μg/mL and IC50 values of 10.60 ± 0.56 and 5.08 ± 0.30 μM, respectively. The relatively low hit-to‑lead conversion rate (14 %) is ascribed to our observation that nine of the identified hits, including compounds 3 and 10, showed some level of precipitation in the MIC assay medium. Molecular dynamics simulations show that the dipyrazole carboxamide moiety in compounds 3 and 10 forms essential hydrogen bonds with nicotinamide adenine dinucleotide (oxidized form) (NAD+) in the InhA binding pocket. Notably, both compounds 3 and 10 exhibit favorable safety profiles, with no toxicity observed in Caco-2 cells at concentrations up to 100 μg/mL. Consequently, we believe that these compounds present promising starting points for further lead optimization and development of novel antitubercular agents. [Display omitted] •Hybrid virtual screening identified potent direct M. tuberculosis InhA inhibitors.•Compounds 3 and 10 showed low MICs and strong InhA binding interactions.•Lead compounds exhibited no cytotoxicity in Caco-2 cells up to 100 μg/mL.