Abstract
The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism and metabolizes at least 20% of all clinically relevant drugs. Therefore, it is an important target for drug-drug interaction (DDI) studies. High-throughput screening (HTS) assays are commonly used tools to examine DDI, but show certain drawbacks with regard to their applicability to natural products. We propose an in silico – in vitro workflow for the reliable identification of natural products with CYP2D6 inhibitory potential. In order to identify candidates from natural product-based databases that share similar structural features with established inhibitors, a pharmacophore model was applied. The virtual hits were tested for the inhibition of recombinant human CYP2D6 in a bioluminescence-based assay. By controlling for unspecific interferences of the test compounds with the detection reaction, the number of false positives were reduced. The success rate of the reported workflow was 76%, as most of the candidates identified in the in silico approach were able to inhibit CYP2D6 activity. In summary, the workflow presented here is a suitable and cost-efficient strategy for the discovery of new CYP2D6 inhibitors with natural product libraries.
Highlights
The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism in which xenobiotics are oxidized to increase their excretion from the body[1]
The starting point for the combinatorial approach to identify CYP2D6 inhibitors was the validation of the first two in silico steps of the workflow depicted in Fig. 3 that consisted of the building and virtual screening of a 3D-database, starting from a 2D-database
In search of suitable 2D-databases for the theoretical validation process, the PubChem Assay AID 89115 turned out to be a feasible dataset as it incorporates 1623 CYP2D6 inhibitors, 6338 non-inhibitors, and 1424 compounds with inconclusive behaviour towards CYP2D6. These compounds have been characterised previously with a bioluminescence-based CYP2D6 inhibition assay similar to the assay that we used for the CYP2D6 inhibition studies
Summary
The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism in which xenobiotics are oxidized to increase their excretion from the body[1]. It is of utmost importance to get comprehensive information about the metabolic profile of all ingested xenobiotics, especially of bioactive compounds such as drugs and natural products Both computer-based activity prediction studies[5,6,7] and high-throughput screening (HTS) assays are commonly used tools to examine drug-drug interactions (DDI) and enzymatic activity of CYP-isoforms[8]. The read-out of a CYP reaction is a fluorogenic or luminogenic signal[9], depending on the probe-substrate Such assay systems have been used in investigations with herbal medicinal products[10]. Application of HTS assays in this specific research area, it has become evident that fluorescence-based assays are vulnerable to natural products, as these often exhibit intrinsic fluorescence or quenching These effects can lead to a masking of enzyme inhibition or a simulation thereof, respectively[10]. The polyphenol resveratrol was reported to inhibit firefly-luciferase in the lower micromolar range and to interfere with such bioluminescence-based assays[11]
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