Underexplored Ligand‐Binding Features of FabI From Staphylococcus aureus and Escherichia coli: A Comparative Pharmacophoric Modeling and Surface Mapping Approach

Computational Identification of Potential Inhibitors Targeting the YjfP Catalytic Triad in Klebsiella Pneumoniae Through Virtual Screening, Docking, and Molecular Dynamics Simulation

Structure-based identification of potential inhibitors of essential Staphylococcus aureus enzymes with in vitro antibacterial screening

Staphylococcus aureus is a Gram-positive bacterium responsible for a wide range of clinical disorders. The emergence of multidrug-resistant strains continues to complicate the management of infections caused by this pathogen. Multiple-target-directed ligands (MTDLs) represent a polypharmacological drug discovery strategy that may reduce the likelihood of resistance emergence by simultaneously disrupting multiple essential bacterial pathways. In the present study, a structure-based in silico screening approach was employed to identify compounds with predicted interactions against five essential S. aureus enzymes, namely MurB, DNA gyrase B, dihydrofolate reductase, dihydropteroate synthase, and tyrosyl-tRNA synthetase. The identified multitarget hits were subjected to computational pharmacokinetic evaluation and molecular mechanics/generalized Born surface area calculations to estimate binding free energies. The selected compounds were subsequently evaluated through in vitro antibacterial assays, of which four candidates exhibited inhibitory activity against S. aureus, with two compounds demonstrating minimum inhibitory concentrations of 4 µg/mL against the reference strain. These two compounds also exhibited their most favorable computational binding profiles against MurB, with docking scores of − 8.884 and − 6.781 kcal/mol and MM/GBSA binding free energies of − 97.801 and − 72.404 kcal/mol. Accordingly, their MurB complexes were subjected to 100 ns molecular dynamics simulations, which showed stable protein–ligand complexes with RMSD values remaining below 3 Å throughout the simulation period. Overall, this study presents a structure-based screening workflow that identified potential inhibitory hits supported by in vitro antibacterial evaluation.

Characterization of beddomeilactone amide as bacterial DNA gyrase inhibitor: in vitro evaluation and computational insights

Structure-based discovery of phospholipase A1 (PlaF) inhibitors in Pseudomonas aeruginosa using integrated in silico and experimental approaches

Phospholipase A1 (PlaF) is a membrane-bound virulence factor in Pseudomonas aeruginosa, involved in glycerophospholipid remodeling and host–pathogen signaling. Inhibiting PlaF represents a promising strategy to reduce the virulence and pathogenesis of multidrug-resistant P. aeruginosa. In this study, a library of 114 small molecules spanning seven chemical classes was evaluated using an integrated multi-step screening workflow. Compounds were subjected to drug-likeness filtering, molecular docking, free energy analysis, antimicrobial susceptibility testing, in silico mutational analysis, and 100 ns molecular dynamics simulations. Among the tested scaffolds, two lead candidates—B4 and C2—showed strong binding affinity to PlaF, structural stability, and favorable toxicity profiles. B4 is a methyl 3-methoxythiophene derivative with a fluorinated benzamido group, while C2 is a thioureido-substituted compound. These findings support B4 and C2 as promising scaffolds for PlaF inhibition and hold potential for advancing anti-virulence drug discovery against P. aeruginosa, offering a novel therapeutic approach to combat antibiotic resistance.

The Activity of Antimicrobial Peptides (AMPs) Identified Via an Integrated in Silico and Pilot in Vitro Screening Approach Towards ESKAPE Pathogens.

Antimicrobial resistance (AMR), driven largely by ESKAPE(E) pathogens, represents a critical global health challenge. The increasing prevalence of multidrug-resistant (MDR) bacteria and the declining effectiveness of conventional antibiotics have created an urgent need for innovative therapeutic agents with novel mechanisms of action. In this study, we aimed to establish a stability-centric computational discovery pipeline to identify and characterize novel antimicrobial peptides (AMPs) with potent and selective bactericidal activity against ESKAPE pathogens, and to validate the lead candidates through experimental in vitro assays. A library of approximately 3,000 AMPs was retrieved from public databases and subjected to in silico screening for physicochemical properties and predicted toxicity. Following filtration, 200 peptides were selected as ligands for molecular docking against essential target proteins from ESKAPE pathogens. Based on docking scores, binding pocket occupancy, and ligand-protein interaction, the top 10 peptides against each bacterium were shortlisted. The four top consensus hits of Vespid Chemotactic Peptide VT1 (VCP-VT1), Taromycin A, CN-AMP1, and Alliumin were selected based on their superior inhibitory profiles. Subsequent peptide-protein docking analyses confirmed their binding modes and interaction patterns. These four candidates were advanced to peptide-protein interaction profiling, followed by pilot in vitro minimum bactericidal concentration (MBC) determination and cytotoxicity assessment on a mammalian cell line. Hierarchical consensus scoring identified VCP-VT1, Taromycin A, CN-AMP1, and Alliumin as the top-ranked binding AMPs. VCP-VT1 was the most potent peptide in preliminary MBC assays, with modest activity against Gram-positive Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus, and Gram-negative Acinetobacter baumannii and Pseudomonas aeruginosa (MBC50 of 50 µM, MBC > 90 of 100 µM). All peptides showed minimal cytotoxicity against a cultured human cell line and non-toxic ADMET parameters in silico. This reductionist validation of a stability-pre-filtered computational shortlist substantiated VCP-VT1 as a priority bactericidal lead AMP. Its favourable preliminary efficacy and safety margins would warrant further pharmacokinetic optimization and eventual in vivo efficacy studies against recalcitrant multidrug-resistant (MDR) pathogens. This integrated computational-experimental framework provided a generally applicable strategy for accelerating the discovery of peptide-based therapeutics to combat the growing threat of AMR.

Discovery of isokurarinone as an ATCase-engaging lead with potent activity against methicillin-resistant Staphylococcus aureus

ABSTRACT The global proliferation of methicillin-resistant Staphylococcus aureus (MRSA) persists as a significant contributor to challenging infections, highlighting the urgent necessity for therapies that utilize novel mechanisms. Aspartate transcarbamoylase (ATCase), which catalyzes the initial committed step of de novo pyrimidine biosynthesis, represents a promising metabolic target with potential relevance to MRSA fitness and persistence. Through structure-based virtual screening and experimental validation, we identified the flavonoid isokurarinone as a compound targeting ATCase, demonstrating potent anti-MRSA activity. Docking and molecular dynamics simulations, along with surface plasmon resonance and differential scanning fluorimetry, provided evidence for direct binding, which was accompanied by a decrease in pyrB expression and a reduction in ATCase activity. Non-targeted metabolomics revealed a disruption of pyrimidine nucleotide homeostasis, coinciding with impaired membrane integrity, reduced proton motive force, decreased intracellular ATP levels, and increased oxidative stress. Isokurarinone also showed an additive interaction with vancomycin in vitro, inhibited biofilm formation and altered the expression of virulence-associated genes. Safety evaluation showed cell-type-dependent cytotoxicity in mammalian cells, while no obvious acute oral toxicity was observed in mice at a single dose of 2 g/kg. In a murine model of MRSA-infected wounds, isokurarinone accelerated wound closure, reduced bacterial burden, and attenuated local inflammatory mediators. Collectively, these findings support the notion of ATCase as a metabolism-guided target for MRSA and nominate isokurarinone as a promising lead scaffold for therapeutic development.