Cathepsin D immobilized capillary reactors for on-flow screening assays

Autor(es)

Cornelio, Vivian Estevam ^[1] ; de Moraes, Marcela Cristina ^[2] ; Domingues, Vanessa de Cassia ^[1] ; Fernandes, Joao Batista ^[1] ; das Gracas Fernandes da Silva, Maria Fatima ^[1] ; Cass, Quezia Bezerra ^[1] ; Vieira, Paulo Cezar ^{[1, 3]}

Afiliação do(s) autor(es)

^[1] Fed Univ Sao Carlos UFSCar, Dept Chem, Sao Carlos, SP - Brazil

^[2] Fluminense Fed Univ UFF, Inst Chem, Dept Organ Chem, Niteroi, RJ - Brazil

^[3] Univ Sao Paulo, Dept Chem & Phys, Sch Pharmaceut Sci Ribeirao Preto, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Pharmaceutical and Biomedical Analysis; v. 151, p. 252-259, MAR 20 2018.
• Citações Web of Science: 5

Resumo

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K-M = 81.9 +/- 7.49 mu mol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. (C) 2018 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 13/01710-1 - Ligantes enzimáticos: novos modelos de triagem
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 09/17538-8 - Estudo de novas metodologias para realização de ensaios com a catepsina D na busca de inibidores
• Beneficiário: Vivian Estevam Cornélio
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto