Validation of a Phage Display Method for Protease Inhibitor Selection Using SFTI and HiTI Synthetic Hybrid Peptides

Autor(es)

de Marco, Renato ^[1] ; Azzolini, Simone S. ^[1] ; Lovato, Diogo V. ^[1] ; Torquato, Ricardo J. S. ^[1] ; Amino, Rogerio ^[1] ; de Miranda, Antonio ^[2] ; Tanaka, Aparecida S. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo UNIFESP, Dept Biochem, BR-04044020 Sao Paulo - Brazil

^[2] Univ Fed Sao Paulo UNIFESP, Dept Biophys, BR-04044020 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING; v. 13, n. 9, p. 829-835, NOV 2010.
• Citações Web of Science: 2

Resumo

A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1' - P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI - Mw 1533 Da), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH(2); Ki = 14 mu M) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases similar to 20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH(2); Ki = 259 mu M). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction. (AU)

• Processo FAPESP: 02/13960-8 - Estudos de estrutura e funcao de anti-hemostaticos presentes em artropodes hematofagos.
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 05/03514-9 - Estudos da função fisiológica e potencial biotecnológico de inibidores de proteases e anti-hemostáticos presentes em artrópodes hematófagos
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 05/03339-2 - Estudos bioquimicos e estruturais de inibidores de serinoproteases purificados de ovos do inseto triatoma infestans.
• Beneficiário: Renato de Marco
• Modalidade de apoio: Bolsas no Brasil - Mestrado