title: Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display.
abstract: 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 (IC(50) of 1.1 nM) and chymotrypsin-like enzymes (IC(50) of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC(50) of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC(50) 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 HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil. tatianesanches@yahoo.com.br
authors.forename: Tatiane Sanches
authors.initials: TS
authors.lastname: Soares
authors.forename: Ricardo Jose
authors.initials: RJ
authors.lastname: Soares Torquato
authors.forename: Francisco Jose
authors.initials: FJ
authors.lastname: Alves Lemos
authors.forename: Aparecida Sadae
authors.initials: AS
authors.lastname: Tanaka
mesh_terms: D000330:Aedes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003715:Dengue / Q000517:prevention & control; D019476:Insect Proteins / Q000037:antagonists & inhibitors*; D007303:Insect Vectors / Q000201:enzymology*; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D009117:Muscidae / Q000235:genetics; D009154:Mutation; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D019476:Insect Proteins; D019151:Peptide Library; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases