Antimicrobial peptides (AMps) are promising alternatives to classical antibiotics for the treatment of drug-resistant infections. Due to their versatility and unlimited sequence space, AMps can be rationally designed by modulating physicochemical determinants to favor desired biological parameters and turned into novel therapeutics. in this study, we utilized key structural and physicochemical parameters, in combination with rational engineering, to design novel short α-helical hybrid peptides inspired by the well-known natural peptides, cathelicidin and aurein. 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, PA-13, showed remarkable broad-spectrum antibacterial activity, especially against Pseudomonas aeruginosa with no toxicity. PA-13 maintained antimicrobial activity in the presence of physiological salts and displayed rapid binding and penetration activity which resulted in membrane depolarization and permeabilization. Moreover, PA-13 showed an anti-inflammatory response via lipopolysaccharide (LpS) neutralization with dose-dependent, inhibiting, LpS-mediated toll-like receptor activation. this study revealed the therapeutic potency of a novel hybrid peptide, and supports the use of rational design in development of new antibacterial agents. The increasing emergence and dissemination of antibiotic resistance among bacterial pathogens has become a global public health challenge 1,2. Therefore, there is an urgent need to develop new antimicrobial agents to overcome this problem. Antimicrobial peptides (AMPs) are an essential component of the innate immune system produced as a first line of defense by all multicellular organisms 3. With such exceptional properties as broad-spectrum antimicrobial activity, rapid action and infrequent development of resistance 4,5 , AMP-based pharmaceuticals provide excellent templates for a wide range of antimicrobial agents and biomedical applications. In general, naturally occurring AMPs are between 12 and 50 amino acids in length, and often contain cationic and hydrophobic residues 6. Previous studies contributing to the understanding of the structure-activity relationship of AMPs show that one important class of membrane-active AMPs infers an amphipathic α-helical conformation 4,7,8. The initial electrostatic interaction between a positively-charged AMP and the negatively-charged
