Bioreactors based on cathepsin D: A new analytical approach for inhibitors screening of natural products

Cathepsin D (CatD) inhibitors could form the basis of novel anti-malarial and schistosomiasis chemotherapies. In this work, we report the preparation of bioreactors based on CatD (CatD-IMER) for rapid on-line screening of natural and combinatorial libraries. To monitoring the CatD-IMER activity, a multidimensional chromatography (Figure 1) method was developed and validated. The CatD-IMER retained the catalytic activity (K _M=78.7±6.85µM) and remained stable for over one month, an important finding in proteases since they are susceptible to autoproteolysis. The adopted strategies for the method development, enzyme immobilization and kinetic studies will be presented. So far, the developed procedure has demonstrated to be a useful strategy in natural products analytical technologies.

Figure 1. Scheme of the multidimensional chromatographic system. CatD-IMER inserted in the first dimension (50 mM phosphate buffer, pH 4.0, 50µL/min) and in the second dimension an octadecyl column Luna-Phenomenex (100 Å, 5µm, 10×0.46cm) which separates the substrate and product obtained with CatD catalyzed reaction (H₂O with 0.1% TFA/ACN (61:39 v/v), 0.5 mL/min).