Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose

Autor(es)

de Carvalho, Daniella Romano ^[1] ; Ximenes, Valdecir Farias ^[2] ; Groppo, Milton ^[3] ; Cardoso, Carmen Lucia ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Grp Cromatog Bioafinidade & Prod Nat, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] Univ Estadual Paulista, Fac Ciencias, Dept Quim, BR-17033360 Bauru, SP - Brazil

^[3] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, BR-14040901 Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Pharmaceutical and Biomedical Analysis; v. 199, MAY 30 2021.
• Citações Web of Science: 0

Resumo

Human tissue kallikreins (KLKs) are serine proteases involved in various physiological and pathological conditions, including cancer and neurological disorders. These enzymes constitute attractive drug targets, which has stimulated the search for new KLK inhibitors. In this study, we have covalently immobilized porcine pancreas KLK on an NHS-activated Sepharose matrix, to obtain KLK-Sepharose-NHS. The immobilized enzyme showed high recovered activity and maintained the ability of free KLK to recognize the synthetic substrate Z-Phe-Arg-AMC (K-Mapp = 10.3 +/- 0.9 mu M). As proof of concept, we used leupeptin as a reference inhibitor to perform inhibition studies for KLK-Sepharose-NHS and to determine the halfmaximal inhibitory concentration (IC50 = 0.13 +/- 0.01 mu M), the inhibition constant (K-i = 0.06 mu M), and the leupeptin inhibition mechanism. We evaluated several complex matrixes (plant crude extract) by the same bioassay, to demonstrate their applicability. The species Solanum lycocarpum, Stryphnodendron adstringens, and Psychotria carthagenensis gave the best results. KLK-Sepharose-NHS was fully active after six consecutive reaction cycles and retained about 60 % of its initial activity after being used for at least five months, so the bioassay developed herein is a promising strategy to screen and to identify KLK ligands. (C) 2021 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 16/06260-2 - Estudos sistemáticos integrados em famílias neotropicais, com ênfase em Rutaceae, Asteraceae e Rubiaceae
• Beneficiário: Milton Groppo Júnior
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/50249-8 - Green chemistry: sustainable synthetic methods employing benign solvents, safer reagents, and bio-renewable feedstock
• Beneficiário: Arlene Gonçalves Corrêa
• Modalidade de apoio: Auxílio à Pesquisa - Programa Centros de Pesquisa em Engenharia
• Processo FAPESP: 19/05363-0 - Triagem de ligantes enzimáticos: uma abordagem on-flow via LC-MS/MS
• Beneficiário: Carmen Lúcia Cardoso
• Modalidade de apoio: Auxílio à Pesquisa - Regular