Discovering novel inhibitors of RfaH from Klebsiella pneumoniae to combat antimicrobial resistance.

RfaH is a crucial protein involved in anti-termination of transcription, which is necessary for spreading virulence in certain types of bacteria, such as Klebsiella pneumoniae and Escherichia coli. RfaH works by interacting directly with RNA polymerase and ribosomes, which activates the production of certain components needed for the bacteria's survival. Targeting RfaH offers a novel approach to hindering bacterial transcription and virulence. In this study, we performed computational screening of the IMPPAT 2.0 database consisting of 17,967 natural compounds, which were filtered based on Lipinski's RO5 filter, selecting only those that had druglike properties. We performed virtual screening on the remaining 11,708 druglike phytochemicals and selected those having strong binding affinity and specificity, leading to the identification of top hits. These hits were further evaluated based on their pharmacokinetic features like PAINS filter, pharmacokinetic properties, pan assay interference, and interaction analysis. Finally, three phytochemicals, Withanone, Withametelin B, and Ixocarpanolide were identified as potential inhibitors for RfaH, having appreciable affinity of - 9.0, - 9.0 and - 8.8 kcal/mol specificity towards the binding pocket of RfaH. An all-atom molecular dynamic simulation was carried out for 500 ns to examine the structural flexibility and dynamic stability of RfaH and RfaH-ligand complexes, which revealed that complexes maintained stability throughout the given duration. All the selected compounds have shown drug-like properties as predicted from ADMET analysis and their physicochemical parameters. These compounds selectively bind to the crucial binding sites of RfaH and interact with important residues, preventing its binding with RNAP which can further be exploited as potential lead molecules against RfaH, providing a promising therapeutic avenue for combating antibiotic resistance.