SYNTHESIS AND BIOASSAY OF NOVEL HYBRID ANTIMICROBIAL PEPTIDES (AMPS), DESIGNED TO INCREASE THERAPEUTIC ACTIVITY AND DECREASE HEMOLYSIS

Abstract

There is currently a critical health issue of global concern; there are many types of bacterial pathogens that have evolved a resistance to many common, conventional antibiotics. In silico structural tools can be used to identify antimicrobial peptide (AMP) sequence modifications that result in better antibacterial therapeutics without increasing human erythrocyte hemolysis. Out of the five AMPs designed for this Thesis project, two were minor modifications of an existing Pleurocidin-Dermaseptin (P-DER) hybrid sequence. The in silico structural design strategies were used to determine on a biophysical level what made P-DER so effective. The tools included the Chou-Fasman secondary structure algorithm, the Edmondson helical wheel model, and the Hopp-Woods hydrophilicity scale. These design insights were used to model the remaining three AMPs. These novel, hybrid AMPs were created by combining the N-terminal region from CEME with the entire sequences of three other AMPs: Ranatuerin 6, VesCP-M and Temporin. Although all five hybrid peptides were synthesized, only the modified P-DER sequences were purified and characterized. The AMPs were tested by varying the concentration of each peptide in contact with the bacteria or human red blood cells (RBC). The dose-dependent cytotoxic and hemolytic effect of those peptides on cells was determined. Screening for bacterial growth inhibition was performed in vitro against two species each of representative gram positive and gram negative bacteria. These tests on the AMPs were to see if it was active against a broad spectrum of microbial pathogens that have diverse types of cell surfaces, with the caveat that these AMPs are not suitable for therapeutic use if they cause significant human RBC hemolysis.