In Silico Identification of Conserved Hypothetical Proteins as Novel Drug Targets and Phytochemical Inhibitors against Clostridium perfringens

Abstract

The growing global threat of antimicrobial resistance (AMR) necessitates the urgent identification of new drug targets against pathogens such as the Gram-positive, spore-forming anaerobe Clostridium perfringens, a major cause of diarrhea, enterotoxemia, and myonecrosis in humans and animals. We propose that conserved hypothetical proteins (HPs)—a largely uncharacterized component of the C. perfringens proteome—represent an overlooked resource for novel therapeutics. Using a stringent subtractive proteomics and in silico pipeline, we analyzed 2661 C. perfringens proteins. We focused on 1179 HPs, identifying 496 conserved across strains. Sequential filtering based on physicochemical properties, subcellular localization, and non-homology to the human gut microbiome yielded 22 promising non-homologous HPs suitable as drug targets. To identify lead compounds, we screened 69 drug-like phytochemical ligands with known antibacterial activity. Molecular docking revealed strong inhibitory interactions for multiple ligand–protein pairs, with binding energies as low as –10.6 kcal/mol (e.g., (+)-galbacin, tuberosin, and (–)-asarinin against specific HPs). These findings strongly support the utility of conserved hypothetical proteins as a rich, untapped source of new antimicrobial targets. Our study demonstrates that in silico analyses can rapidly accelerate drug discovery against fast-evolving pathogens by prioritizing novel targets and corresponding lead compounds for subsequent experimental validation. As a critical next step, these candidate proteins and their top-scoring ligand interactions should undergo experimental validation through biochemical assays, structural characterization, and in vitro inhibition studies to confirm their therapeutic potential.