Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display

Autor(es)

Soares, Tatiane Sanches ^[1] ; Soares Torquato, Ricardo Jose ^[1] ; Alves Lemos, Francisco Jose ^[2] ; Tanaka, Aparecida Sadae ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 Sao Paulo - Brazil

^[2] Univ Estadual Norte Fluminense, Lab Biotecnol, Rio De Janeiro, RJ - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Insect Biochemistry and Molecular Biology; v. 43, n. 1, p. 9-16, JAN 2013.
• Citações Web of Science: 5

Resumo

Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1 P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC50 of 1.1 nM) and chymotrypsin-like enzymes (IC50 of 11.6 nM), respectively. The variants 123 (LLGGL) and 1149 (GGVWR) inhibited both larval chymotrypsin-like (IC50 of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC50 of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The Hill variants T6 and T23 are good candidates for the development as a larvicide to control the vector. (C) 2012 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 09/05405-3 - Atualização do sequenciador automático de DNA da Unidade Multi-usuário do INFAR, UNIFESP-EPM
• 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