title: Chemical synthesis, structure-activity relationship, and properties of shepherin I: a fungicidal peptide enriched in glycine-glycine-histidine motifs.
abstract: Although glycine-rich antimicrobial peptides (AMPs) are found in animals and plants, very little has been reported on their chemistry, structure activity-relationship, and properties. We investigated those topics for Shepherin I (Shep I), a glycine-rich AMP with the unique amino acid sequence G(1)YGGHGGHGGHGGHGGHGGHGHGGGGHG(28). Shep I and analogues were synthesized by the solid-phase method at 60 °C using conventional heating. Purification followed by chemical characterization confirmed the products' identities and high purity. Amino acid analysis provided their peptide contents. All peptides were active against the clinically important Candida species, but ineffective against bacteria and mycelia fungi. Truncation of the N- or C-terminal portion reduced Shep I antifungal activity, the latter being more pronounced. Carboxyamidation of Shep I did not affect the activity against C. albicans or C. tropicalis, but increased activity against S. cerevisiae. Carboxyamidated analogues Shep I (3-28)a and Shep I (6-28)a were equipotent to Shep I and Shep Ia against Candida species. As with most cationic AMPs, all peptides had their activity significantly reduced in high-salt concentrations, a disadvantage that is defeated if 10 µM ZnCl2 is present. At 100 µM, the peptides were practically not hemolytic. Shep Ia also killed C. albicans MDM8 and ATCC 90028 cells. Fluo-Shep Ia, an analogue labeled with 5(6)-carboxyfluorescein, was rapidly internalized by C. albicans MDM8 cells, a salt-sensitive process dependent on metabolic energy and temperature. Altogether, such results shed light on the chemistry, structural requirements for activity, and other properties of candidacidal glycine-rich peptides. Furthermore, they show that Shep Ia may have strong potential for use in topical application.
authors.affiliation: Laboratory of Peptide Chemistry, Department of Biochemistry, Institute of Chemistry, University of São Paulo, P.O. Box 26077, 05513-970, São Paulo, SP, Brazil.
authors.forename: César
authors.initials: C
authors.lastname: Remuzgo
authors.forename: Thaís S
authors.initials: TS
authors.lastname: Oewel
authors.forename: Sirlei
authors.initials: S
authors.lastname: Daffre
authors.forename: Thiago R S
authors.initials: TR
authors.lastname: Lopes
authors.forename: Fabio H
authors.initials: FH
authors.lastname: Dyszy
authors.forename: Shirley
authors.initials: S
authors.lastname: Schreier
authors.forename: Gláucia M
authors.initials: GM
authors.lastname: Machado-Santelli
authors.forename: M
authors.initials: M
authors.lastname: Teresa Machini
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000890:Anti-Infective Agents / Q000737:chemistry; D001419:Bacteria / Q000378:metabolism; D002175:Candida / Q000378:metabolism; D002176:Candida albicans; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002942:Circular Dichroism; D005434:Flow Cytometry; D005998:Glycine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D000596:Amino Acids; D000890:Anti-Infective Agents; C417582:GRP protein, Capsella bursa-pastoris; D010455:Peptides; D010940:Plant Proteins; D006639:Histidine; D005998:Glycine