title: Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae.
abstract: BACKGROUND
  Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4' selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1).
  
  
  METHODS
  The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4.
  
  
  RESULTS
  The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the μM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested.
  
  
  CONCLUSIONS
  The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue 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.
authors.forename: Tatiane Sanches
authors.initials: TS
authors.lastname: Soares
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.
authors.forename: Ricardo José Soares
authors.initials: RJ
authors.lastname: Torquato
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.
authors.forename: Yamile Gonzalez
authors.initials: YG
authors.lastname: Gonzalez
authors.affiliation: Laboratório de Biotecnologia, Universidade Estadual do Norte Fluminense, Rio de Janeiro, RJ, Brazil.
authors.forename: Francisco Jose Alves
authors.initials: FJ
authors.lastname: Lemos
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. tanaka.bioq@epm.br.
authors.forename: Aparecida Sadae
authors.initials: AS
authors.lastname: Tanaka
mesh_terms: D000330:Aedes / Q000187:drug effects* / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002782:Cholestenes / Q000378:metabolism / Q000494:pharmacology; D003715:Dengue / Q000517:prevention & control*; D006801:Humans; D007306:Insecticides / Q000378:metabolism / Q000494:pharmacology; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000201:enzymology; D016296:Mutagenesis; D009154:Mutation; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002782:Cholestenes; D007306:Insecticides; C584785:N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine; D015842:Serine Proteinase Inhibitors; D007532:Isoleucine