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

UNLABELLED: 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.