Cathepsin D immobilized capillary reactors for on-flow screening assays.

Autor: Cornelio VE; Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil., de Moraes MC; Department of Organic Chemistry, Chemistry Institute, Fluminense Federal University (UFF), Niterói, RJ, Brazil., Domingues VC; Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil., Fernandes JB; Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil., da Silva MFDGF; Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil., Cass QB; Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil. Electronic address: quezia@pq.cnpq.br., Vieira PC; Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil; Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP, Brazil. Electronic address: dpcv@ufscar.br.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2018 Mar 20; Vol. 151, pp. 252-259. Date of Electronic Publication: 2018 Jan 06.
DOI: 10.1016/j.jpba.2018.01.001
Abstrakt: 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 μmol/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.
(Copyright © 2018 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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