Hb40-61a: Novel analogues help expanding the knowledge on chemistry, properties and candidacidal action of this bovine α-hemoglobin-derived peptide

• Published in: Biochimica et biophysica acta Vol. 1848; no. 12; pp. 3140 - 3149
• Main Authors: Carvalho, L.A.C., Remuzgo, C., Perez, K.R., Machini, M.T.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2015
• Subjects: Amino Acid Sequence; Animals; Antifungal Agents - chemistry; Antifungal Agents - pharmacology; Antimicrobial peptides; Candida - drug effects; Cattle; Erythrocytes - drug effects; Fluorescent Dyes - chemistry; Fungicidal action; Hemocidins; Hemoglobin-derived peptides; Hemoglobins - chemistry; Humans; Membrane permeabilization; Membrane Potentials - drug effects; Microbial Sensitivity Tests; Molecular Sequence Data; Peptide synthesis at 60 °C; Peptides - chemistry; Peptides - pharmacology; Hemoglobin-derived peptides; Membrane permeabilization; Peptide synthesis at 60°C; Fungicidal action; Hemocidins; Antimicrobial peptides
• ISSN: 0005-2736; 0006-3002; 1879-2642; 1878-2434
• DOI: 10.1016/j.bbamem.2015.09.010

This study expands the knowledge on chemical synthesis and properties of Hb40-61a as well as provides results of the first steps given towards knowing how it kills Candida cells. For the first time, this peptide, its all-D analogue (D-Hb40-61a) and its fluorescently labeled analogue (FAM-Hb40-61a) were successfully assembled on resin at 60°C using conventional heating in all steps. Purified and characterized, these peptides exhibited very low toxicity on human erythrocytes. Hb40-61a and D-Hb40-61a were equally active against Candida strains, ruling out sterically specific interactions on their working mechanism. Cell permeabilization assays confirmed progressive damage of the yeast plasma membrane with increasing concentrations of Hb40-61a. While experiment using the fluorescent probe DiBAC4(5) revealed that this synthetic hemocidin alters the yeast plasma membrane potential, test employing DPH indicated that Hb40-61a might affect its dynamics. Exposure of the yeast cells to FAM-Hb40-61a showed that the peptide accumulates in the cell membrane at the ½ MIC, but stains about 97% of the cells at the MIC. Such effect is salt-dependent and partially energy-dependent. These new findings indicate that the central target of Hb40-61a in Candida cells is the plasma membrane and that this synthetic hemocidin should be considered as a potential candidacidal for topic uses. [Display omitted] •We expanded the knowledge on synthetic hemocidins derived from bovine α-hemoglobin.•We first describe productive, fast and relatively clean syntheses of Hb40-61a and analogues using high temperature.•We first show that, likewise Hb40-61a, its D-isomer and its fluorescently labeled analogue inhibit the growth of Candida strains.•We first show that the low hemolytic Hb40-61a acts through a non-quiral mechanism.•We first show that Hb40-61a damages Candida cell plasma membrane altering its permeability, potential and dynamics.•We first show that Candida plasma membrane is the first and ultimate target of Hb40-61a.