In-silico identification and experimental validation of possible inhibitor for the bifunctional protein GlmU of...

Antimicrobial resistance renders numerous antibiotics ineffective, resulting in persistent infections and increased mortality rates. This makes identifying novel therapeutic targets imperative, necessitating the investigation of vital bacterial mechanisms. The bifunctional protein N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in Acinetobacter baumannii. GlmU catalyzes the synthesis of an intermediate that enters the peptidoglycan and lipopolysaccharide synthesis, which is essential for bacterial survival. In this study, the GlmU monomer was modeled using Phyre2, refined with GalaxyWeb, and confirmed through PSVS, ensuring a reliable 3D structure. Binding sites on GlmU were identified using PUResNetV2.0, revealing two key sites corresponding to uridyltransferase and acetyltransferase activities. A GlmU trimer was constructed, and molecular docking of 55 potential inhibitors was performed against both the monomer and trimer. 3,3'-methylenebis-(4-hydroxy-coumarin) emerged as the most promising inhibitor, with strong interactions at both binding sites and the trimer. Molecular dynamics simulations confirmed the stability of the GlmU-3,3'-methylenebis-(4-hydroxy-coumarin) complex. Mutation analysis of key interacting residues further highlighted their importance in maintaining the protein's stability. Experimental validation of lead confirmed its effect on bacterial growth, peptidoglycan content, lipid accumulation, carbohydrate concentration, reactive oxygen species (ROS) production, protein carbonylation, and biofilm formation in A. baumannii. All these results suggest the therapeutic potential of this molecule against A. baumannii via targeting GlmU protein.