DOI: 10.14457/tu.the.2020.944
title: Challenges in design and modification of novel short α-helical peptide as an alternative therapeutic agent

description: Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics for the treatment of drug-resistant infections. AMPs can be rationally designed by modulating physicochemical determinants to favor desired biological parameters and turned into novel therapeutics. In this study, key structural and physicochemical parameters, in combination with rational engineering, were utilized to design novel short α-helical peptides inspired by cathelicidin and aurein natural peptides. By comparing homologous sequences and abstracting the conserved residue type, sequence templates of cathelicidin (P0) and aurein (A0) were obtained. Two peptide derivatives, P7 and A3, were generated by amino acid substitution based on their residue composition and distribution. In order to enhance antimicrobial activity, a hybrid analog of P7A3 was designed. The results demonstrated that P7A3 had higher antibacterial activity than the parental peptides with unexpectedly high hemolytic activity. Strikingly, C-terminal truncation of hybrid peptides containing only the α-helical segment (PA-18) and shorter derivatives confer potent antimicrobial activity with reduced hemolytic activity in a length-dependent manner. Among all derivatives, two peptide candidates, P7 and PA-13, showed remarkable broad-spectrum antibacterial activity with a favorable safety profile. This study supports the use of rational design in development of new antibacterial agents. Both peptides tended to form an α-helical structure upon interacting with membrane-mimicking environments. Therefore, the action mode at the membrane-level was investigated by monitoring the fluorescence intensity of TAMRA-labelled peptides and membrane probes. Likewise, localization, cell morphological and ultrastructural changes induced by these peptides, were observed using confocal, scanning and transmission electron microscopy, respectively. The results indicated that P7 and PA-13 killed targeted bacteria through binding to bacterial membranes, penetration and then accumulation in bacterial cytoplasm. This induced membrane depolarization, permeabilization, and subsequent leakage of intracellular contents and cell death. The pH- and thermo-stable P7 peptide showed enhanced antibacterial activity toward drug-resistant foodborne Salmonella Typhimurium and its monophasic variant. Additionally, P7 mostly maintained their killing activity in the presence of extreme conditions including diverse salts, and widely pHs and heat (even at 100°C) demonstrating the potential application in food processing and preservation. Notably, the peptide did not induce bacterial resistance in vitro. The shortest hybrid peptide derivative, PA-13, showed remarkable antibacterial activity against clinically isolated multidrug-resistant Pseudomonas aeruginosa and anti-inflammatory response via LPS neutralization with dose-dependent, inhibiting, LPS-mediated Toll-like receptor activation. This revealed the therapeutic potency of a novel hybrid peptide PA-13.In order to improve enzyme stability of PA-13 for translating into therapeutic applications, chitosan-dextran sulfate nanoparticles (CS-DS NPs) network was designed for protecting peptide from enzymatic degradation. Successfully, PA-13 loaded nano-network mostly eradicated targeted bacteria in trypsin treated condition, while PA-13 alone had no killing activity. Additionally, the nano-network showed rheological properties required to serve as a promising formulation for topical applications.