Fragments of cathelicidins PMAP-36 and BMAP-27 and their D-enantiomers: Effects of all D substitutions on structure, protease resistance and antimicrobial properties

Antimicrobial peptides (AMPs) are natural molecules of great interest in the fight against bacteria and in addressing antibiotic resistance. Their use as antimicrobial drugs is still limited due to their cytotoxicity and poor resistance to proteolysis. PMAP-36 and BMAP-27 are cathelicidins that contribute to innate immunity. Two short sequences, PMAP(12-24) and BMAP(1-18), are the most promising analogues identified but despite their broad antibacterial activity, they undergo rapid proteolytic degradation. In this study, the D and L enantiomers of the two peptides were synthesised, and their structures were elucidated and compared by circular dichroism (CD) spectroscopy and 2D-NMR analysis. Conformational studies revealed that, despite their short sequence, all peptides adopted helical structures in membrane-mimetic environments, with subtle differences between the two AMP groups, PMAP(12-24) being more prone to adopt a mixed alpha/310-helical conformation (R([0]222/[0]206 = 0.4-0.5) and BMAP(1-18) being closer to a pure alpha-helix (R ([0]222/[0]208 = 0.7-0.8). These structural differences between the two peptides were found to influence their antimicrobial activity and mode of membrane permeabilization. Moreover, the D enantiomers of both analogues were resistant to proteolysis. All peptides showed a broad spectrum of antibacterial activity (main MIC range: 1-16 mu M). Cytotoxicity studies in fibroblast showed that the peptides were non-cytotoxic at concentrations corresponding to their antibacterial activity. Overall, this study led to the identification of structurally interesting short peptides that could serve as prototypes for the development of effective and practical antibacterial drugs.