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

Rational Design and In Silico Evaluation of a Dual-Action Antimicrobial Peptide Targeting Efflux Pump and Membrane Integrity in Pseudomonas aeruginosa.

Discovery of Novel Antimicrobial Peptides from Casein via Integrated In Silico and Experimental Approaches: Identification and Mechanistic Insights

A Comprehensive In-Silico Pipeline for the Discovery of Non-toxic, Stable Antimicrobial Peptides From Databases for Targeting Multi-Drug Resistant K. pneumoniae

The rapid emergence of multidrug-resistant Klebsiella pneumoniae has significantly reduced the effectiveness of conventional antibiotics, highlighting the need for alternative therapeutic strategies. This study employed a comprehensive in silico pipeline to identify antimicrobial peptides (AMPs) targeting the essential DNA replication initiator protein DnaA. A total of 28,361 peptide sequences were collected from publicly available AMP databases and sequentially filtered based on peptide length, net charge, GRAVY score, instability index, antimicrobial activity, toxicity, hemolytic potential, aggregation propensity, sequence similarity and favorable amphipathic properties. Four peptides satisfied all selection criteria and were subjected to structural prediction, membrane-binding analysis, protein–DNA docking, protein–peptide docking, and Normal Mode Analysis. Protein–DNA docking identified the functional DNA-binding residues of DnaA, while peptide docking demonstrated that all four peptides interacted within this region. Peptide 3 exhibited the strongest predicted interaction, with a binding energy of −61.8±5.1, a buried surface area of 1151.7±30.8 Å2, and seven hydrogen bonds with key DnaA residues. Normal Mode Analysis further supported the structural stability of the peptide–protein complexes. These findings identify four promising AMP candidates targeting DnaA and provide a computational framework for peptide prioritization against multidrug-resistant K. pneumoniae. However, the proposed interactions remain computational predictions and require experimental validation.

In vitro evaluation of novel consensus-sequence-generated Brevinin-2 antimicrobial peptides

ABSTRACT Antibiotic-resistant bacteria are a global public health threat that is becoming increasingly difficult to address with conventional therapeutics. Consequently, there is much interest in studying alternative biologics that circumvent antibiotic resistance. The Brevinin-2 family of antimicrobial peptides (AMPs) is a group of naturally occurring molecules that have the potential for high activity and low toxicity. Herein, we investigated the potential of a consensus-sequence-driven approach to Brevinin-2 peptide synthesis and evaluated their action against a panel of multidrug-resistant (MDR) bacteria, including carbapenemase-resistant Escherichia coli. We used the positional frequency of amino acids to generate novel synthetic peptides representative of the Brevinin-2 family. Four templates—G30, G33, S33, and G37—were synthesized by standard fluorenylmethyloxycarbonyl (FMOC) chemistry, purified by reverse-phase fast protein liquid chromatography (RP-FPLC), and tested for antibacterial activity and hemolytic toxicity. The results demonstrated a broadly applicable chemical peptide synthesis pipeline for the Brevinin-2 family with a high degree of purity (>90%). Two peptides—S33 and G33—exhibited activity consistent with potential selectivity toward gram-positive and -negative bacteria, respectively, while G37 displays broad-spectrum activity, with growth of a Class B carbapenemase-resistant E. coli inhibited at 16 µM and a Class A carbapenemase-resistant K. pneumoniae inhibited at 64 µM. G37 acts rapidly, slowing growth within 30 min and fully killing targeted bacteria within 150 min. Although moderate levels of hemolytic toxicity pose a challenge for future development, the consensus-sequence approach toward novel Brevinin-2 AMP discovery via alignment combined with in vitro antimicrobial analysis shows promise to test and initially validate other peptides. IMPORTANCE The escalating threat of antimicrobial resistance (AMR) demands innovative therapeutic strategies beyond traditional antibiotics. This study demonstrates a systematic, consensus-sequence-driven approach to designing antimicrobial peptides (AMPs) from the naturally occurring Brevinin-2 family, offering a replicable framework for accelerated drug discovery. Our novel peptide G37 exhibits bactericidal activity against carbapenemase-resistant Escherichia coli within 150 min while maintaining low hemolytic activity at therapeutic concentrations. The potential preferential activity shown by peptides S33 and G33 for gram-positive versus gram-negative bacteria provides prospective valuable insights into structure-activity relationships that can guide further peptide optimization. By combining computational sequence analysis, standardized solid-phase peptide synthesis, and comprehensive in vitro validation, this work establishes a streamlined pipeline for Brevinin-2 peptide development. This methodology addresses the urgent need for alternative antimicrobials while providing a scalable approach to combat multidrug-resistant (MDR) pathogens. The escalating threat of antimicrobial resistance (AMR) demands innovative therapeutic strategies beyond traditional antibiotics. This study demonstrates a systematic, consensus-sequence-driven approach to designing antimicrobial peptides (AMPs) from the naturally occurring Brevinin-2 family, offering a replicable framework for accelerated drug discovery. Our novel peptide G37 exhibits bactericidal activity against carbapenemase-resistant Escherichia coli within 150 min while maintaining low hemolytic activity at therapeutic concentrations. The potential preferential activity shown by peptides S33 and G33 for gram-positive versus gram-negative bacteria provides prospective valuable insights into structure-activity relationships that can guide further peptide optimization. By combining computational sequence analysis, standardized solid-phase peptide synthesis, and comprehensive in vitro validation, this work establishes a streamlined pipeline for Brevinin-2 peptide development. This methodology addresses the urgent need for alternative antimicrobials while providing a scalable approach to combat multidrug-resistant (MDR) pathogens.

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

This study presents a structure-based screening workflow that identified potential inhibitory hits supported by in vitro antibacterial evaluation that may reduce the likelihood of resistance emergence by simultaneously disrupting multiple essential bacterial pathways.

An integrative computational and experimental evaluation of natural compounds with potential to target LuxS in Klebsiella pneumoniae

Klebsiella pneumoniae (KPn) has emerged as a major global health concern due to increasing antimicrobial resistance (AMR). The development of new antimicrobial agents is essential to address multidrug-resistant and emerging hypervirulent strains associated with increased morbidity and mortality worldwide. In this study, an integrative computational–experimental approach was employed to characterise the anti-quorum sensing and anti-biofilm activities of selected natural compounds targeting the LuxS/autoinducer-2 (AI-2) system in KPn. Six compounds were initially screened using homology modelling, molecular docking, pharmacophore analysis, and molecular dynamics (MD) simulations. Resveratrol, quercetin, and epigallocatechin gallate showed favourable binding to LuxS. Although quercetin (ΔG_bind = − 45.47 kcal/mol) and epigallocatechin gallate (− 43.04 kcal/mol) exhibited stronger predicted affinities than resveratrol (− 39.22 kcal/mol), resveratrol demonstrated superior complex stability (RMSD: 1.8 ± 0.2 Å) and persistent interactions with key residues (Tyr63, Trp68). Experimental validation demonstrated that resveratrol exhibited significant antimicrobial activity (MIC/MBC = 32 µg/mL), significantly inhibited quorum sensing-associated phenotypes, and reduced KPn-Acinetobacter baumannii (ABa) dual-species biofilm formation inhibition by 54.7%. These findings identify resveratrol as a promising anti-virulence candidate and highlight the utility of integrative computational and experimental strategies for the identification of compounds targeting quorum sensing and biofilm formation inhibition in multidrug-resistant pathogens. Graphical abstract showing natural compounds investigated in this study and their interaction with the LuxS protein, a key regulator of quorum sensing in Klebsiella pneumoniae Graphical abstract showing natural compounds investigated in this study and their interaction with the LuxS protein, a key regulator of quorum sensing in Klebsiella pneumoniae