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

Authors :
Cornelio VE
de Moraes MC
Domingues VC
Fernandes JB
da Silva MFDGF
Cass QB
Vieira PC
Source :
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.
Publication Year :
2018

Abstract

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 <subscript>M</subscript>  = 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.<br /> (Copyright © 2018 Elsevier B.V. All rights reserved.)

Details

Language :
English
ISSN :
1873-264X
Volume :
151
Database :
MEDLINE
Journal :
Journal of pharmaceutical and biomedical analysis
Publication Type :
Academic Journal
Accession number :
29367161
Full Text :
https://doi.org/10.1016/j.jpba.2018.01.001