(154e) Antifungal Peptide Variants with Reduced Degradation By Fungal Proteases and Improved Antifungal Activity Against Planktonic and Biofilm Cells

Histatin 5 is a highly cationic peptide found in human saliva that has specific and potent antifungal activity against the fungal pathogen Candida albicans. Although histatin 5 has potential as a therapeutic to combat C. albicans infections, C. albicans cells secrete proteases called secreted aspartic proteases (Saps) that can degrade and inactivate histatin 5. Two of the most important Saps are Sap2 and Sap9, due to their high levels of expression, and these enzymes cleave histatin 5 at its lysine residues. We designed analogs of histatin 5 with substitutions at its four lysine residues to study the effect on resistance to proteolysis by Sap2 and Sap9 and on antifungal activity. The single amino-acid substitutions of K17R and K17L were the most resistant to proteolysis, with 100% and 77% intact peptide remaining after incubation with purified Sap2 and Sap9, respectively, under conditions that led to only 61% (Sap2) and 47% (Sap9) of Hst-5 remaining. The decrease in proteolysis resulted mainly from reduced cleavage of the peptides on the C-terminal side of the K17 residue, and the decrease in proteolysis was also observed following incubation with C. albicans cells and culture supernatant. The protease-resistant variants of histatin 5 maintained the antifungal activity of the parent peptide, and the K17R, K17L, and K11R peptides showed significant increases in residual antifungal activity compared to histatin 5 following incubation with Sap2 and Sap9. By combining the K11R and K17R mutations, we were able to further improve the protease resistance and the antifungal activity of the peptides. The K11R-K17R variant had stronger antifungal activity than the parent peptide and maintained full antifungal activity following incubation with the proteases. The modified peptides also have improved activity against C. albicans biofilms and lack toxicity towards mammalian cells, further illustrating their potential application in designing improved therapeutics. We are currently exploring the incorporation of the histatin 5 variants into surface coatings to determine their potential in preventing biofilm-associated infections. Our results provide a unique perspective on antimicrobial peptide design by focusing on proteolytic resistance, in addition to antifungal activity, which could be particularly relevant in applications involving sustained contact with pathogen and host proteases.