VII Simposio Internacional de Ciencias Farmacéuticas 2019 "VII SICF" -VII Simposio "Diseño, Obtención y Desarrollo de Fármacos"

VII Simposio Internacional de Ciencias Farmacéuticas 2019

VII SICF

VII Simposio "Diseño, Obtención y Desarrollo de Fármacos"

Identification of a potent inhibitor of LAPTc, an M17 aminopeptidase of Trypanosoma cruzi

Resumen [ES]

A novel target in Chagas disease is the leucil-aminopeptidase M17 of Trypanosoma cruzi (LAPTc). The objective of this work was to identify a potent inhibitor of LAPTc. The optimized LAPTc gene was synthesized and cloned in the vector pET-19b for its expression in Escherichia coli. The genetic construction pET-19brLAPTc allows an soluble and active production of rLAPTc in E. coli BL21(DE3)pLysS, by induction during 20 h at 25ºC with IPTG 1 mM. A level of expression of 12.53 % of the total cellular protein was obtained. The rLAPTc enzyme was purified in a single step by IMAC, exploiting the N-terminal tag of 10 His encoded by the vector. The recombinant protein was obtained with a 90 % of purity and a yield of 90 mg per liter of culture. The enzyme has an optimal pH of 9.0, and preference for Leup-nitroanilide among nine tested substrates (KMapp = 74 M, kcatap = 4.4 s-1, kcat/KM (ap) = 59,459 L mol-1 s-1). The optimal temperature is 50ºC, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity in a 60 % or more, but Mn2+ inhibited only a 15 % and Co2+ activated in a 40 %. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. The enzymatic activity of rLAPTc on Leu-pNA was inhibited in the micromolar order by bestatin and a synthetic tetrazole-peptidomimetic. The last one has potentialities as an in vitro antichagasic agent.

Resumen [EN]

A novel target in Chagas disease is the leucil-aminopeptidase M17 of Trypanosoma cruzi (LAPTc). The objective of this work was to identify a potent inhibitor of LAPTc. The optimized LAPTc gene was synthesized and cloned in the vector pET-19b for its expression in Escherichia coli. The genetic construction pET-19brLAPTc allows an soluble and active production of rLAPTc in E. coli BL21(DE3)pLysS, by induction during 20 h at 25ºC with IPTG 1 mM. A level of expression of 12.53 % of the total cellular protein was obtained. The rLAPTc enzyme was purified in a single step by IMAC, exploiting the N-terminal tag of 10 His encoded by the vector. The recombinant protein was obtained with a 90 % of purity and a yield of 90 mg per liter of culture. The enzyme has an optimal pH of 9.0, and preference for Leup-nitroanilide among nine tested substrates (KMapp = 74 M, kcatap = 4.4 s-1, kcat/KM (ap) = 59,459 L mol-1 s-1). The optimal temperature is 50ºC, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity in a 60 % or more, but Mn2+ inhibited only a 15 % and Co2+ activated in a 40 %. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. The enzymatic activity of rLAPTc on Leu-pNA was inhibited in the micromolar order by bestatin and a synthetic tetrazole-peptidomimetic. The last one has potentialities as an in vitro antichagasic agent.

Sobre el ponente

Mirtha Elisa Aguado Casas

Lic. Mirtha Elisa Aguado Casas

CEP,Facultad de Biologia,Universidad de la Habana Flag of Cuba
Información Práctica
English (US)
No definido
30 minutos
No definido
Autores
Maikel Izquierdo
Daniel García
Lic. Mirtha Elisa Aguado Casas
Jorge Gozález
Martin Zoltner
Yanira Méndez
Palabras clave
antichagasic agent
chagas disease
inhibitor
leucil-aminopeptidase