Effects of N-terminal modifications on the stability of antimicrobial peptide SAMP-A4 analogues against protease degradation.

Infections with multidrug-resistant (MDR) pathogens are increasingly concerning for public health. Synthesized antimicrobial peptide A4 (SAMP-A4), a peptide computationally designed by our research team, is a potential drug candidate. However, the antimicrobial peptide SAMP-A4 is easily degraded in serum. To obtain SAMP-A4 analogues with high biostability, chemical modifications at its N-terminus, including fatty acid conjugation, glycosylation and PEGylation, were carried out. The results showed that the introduction of hydrophobic fatty acids at the N-terminus of SAMP-A4 is better than hydrophilic glycosylation and PEGylation. With increasing fatty acid chain length, the stability of SAMP-A4 analogues in serum and trypsin solutions is significantly improved, and the activities against MDR bacteria and Candida are significantly enhanced. There is no obvious change in haemolysis even when hexanoic acid is coupled with SAMP-A4, so the resulting analogue SAMP-A4-C6, SAMP-A4 conjugated with hexanoic acid, is the most likely of the analogues to become a drug.

Effects of CGA-N12 on the membrane structure of Candida tropicalis cells.

The antimicrobial peptide CGA-N12 (NH2-ALQGAKERAHQQ-COOH) is an active peptide derived from chromogranin A (CGA) and consists of the 65th to 76th amino acids of the N-terminus. The results of our previous studies showed that CGA-N12 exerts anti-Candida activity by inducing apoptosis without destroying the integrity of cell membranes. In this study, the effect of CGA-N12 on the cell membrane structure of Candida tropicalis was investigated. CGA-N12 resulted in the dissipation of the membrane potential, the increase in membrane fluidity, and the outflow of potassium ions in C. tropicalis without significantly changing the ergosterol level. Fluorescence quenching was applied to evaluate the membrane channel characteristics induced by CGA-N12 through detection of the following: membrane permeability of hydrated Cl- (ϕ ≈ 0.66 nm) using the membrane-impermeable halogen anion-selective fluorescent dye lucigenin, passage of the membrane-impermeable dye carboxyfluorescein (CF) (ϕ ≈ 1 nm) through the membrane, and membrane permeation of H3O+ based on the membrane non-permeable pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS). In conclusion, CGA-N12 can induce the formation of non-selective ion channels <1 nm in diameter in the membranes of C. tropicalis, resulting in the leakage of potassium ions, chloride ions, and protons, among others, leading to dissipation of the membrane potential. As a result, the fluidity of membranes is increased without destroying the synthesis of ergosterol is not affected.