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

Abstract

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.