RESUMO
Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2-4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Animais , Calorimetria , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Dicroísmo Circular , Túnica Conjuntiva/citologia , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Bicamadas Lipídicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Coelhos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: To describe mutations in the transforming growth factor-beta induced (TGFBI) gene in Asian patients with Bowman's membrane as well as stromal corneal dystrophies, and to elucidate their structural implications, using model peptides. METHODS: Twenty-two unrelated Asian families were examined clinically including visual acuity testing and ocular examination with slit lamp biomicroscopy. Genomic DNA was extracted and the 17 exons of the TGFBI gene were amplified by PCR and sequenced bi-directionally. Biophysical techniques were used to characterise the wild type and mutant model peptides. RESULTS: Molecular genetic analysis identified a variety of mutations in our patient series including a novel heterozygous C to A transversion mutation in exon 14 (c.1859CâA), resulting in a substitution of a highly conserved alanine residue by aspartic acid (p.A620D). Clinical presentation in the patient with the p.A620D included subepithelial scarring in addition to the linear branching opacities usually seen with lattice dystrophy. Using model peptides, we showed that A620D mutant peptide alters the secondary structure and conformational stability, and increased amyloid formation. CONCLUSION: A novel mutation (A620D) in transforming growth factor-beta induced protein (TGFßIp) is described, expanding the repertoire of mutations in this protein. Using model peptides, we demonstrated that AâD substitution leads to perturbation of the secondary structure that may be responsible for the amyloid formation in lattice corneal dystrophy.