Profiling the Tox21 Compound Library for Their Inhibitory Effects on Cytochrome P450 Enzymes

Abstract

Cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins crucial for drug and xenobiotic metabolism. While more than 50 CYPs have been identified in humans, the isoforms from CYP1, 2, and 3 families contribute to the metabolism of about 80% of clinically approved drugs. To evaluate the effects of environmental chemicals on the activities of these important CYP enzyme families, we screened the Tox21 10K compound library to identify chemicals that inhibit CYP1A2, 2C9, 2C19, 2D6, and 3A4 enzymes. The data obtained from these five screenings were analyzed to reveal the structural classes responsible for inhibiting multiple and/or selective CYPs. Some known structural compound classes exhibiting pan-CYP inhibition, such as azole fungicides, along with established clinical inhibitors of CYPs, including erythromycin and verapamil inhibiting CYP3A4 and paroxetine and terbinafine inhibiting CYP2D6, were all confirmed in the current study. In addition, some selective CYP inhibitors, previously unknown but with potent activity (IC₅₀ values < 1 µM), were identified. Examples included yohimbine, an indole alkaloid, and loteprednol, a corticosteroid, which showed inhibitory activity in CYP2D6 and 3A4 assays, respectively. These findings suggest that assessment of a candidate compound's impact on CYP function may allow pre-emptive mitigation of potential adverse reactions and toxicity during drug development or toxicological characterization of environmental chemicals.

Keywords: CYP inhibitors; CYP1A2; CYP2C19; CYP2C9; CYP2D6; CYP3A4; cytochrome P450 (CYP); quantitative high-throughput screening (qHTS).

Figure 1

Concentration–response curves of the positive control compounds from five CYP screenings. The positive control compound is plated as 16-point titrations in the control column of every assay plate. The concentration–response curves shown in the figure are from all 408 plates per assay.

Figure 2

Reproducibility from three runs of five CYP assays. For each assay, the reproducibility was calculated for the 10K compound library with compounds plated in different well locations (three copies) by the active match, inactive match, mismatch, and inconclusive classes.

Figure 3

Common structural classes identified from at least three or more CYP assays. The structural classes are enriched with active compounds (inhibitors). The significance of enrichment was determined by p-value from Fisher’s exact test. The heat map is colored by the significance (negative logarithmic p-value) of enrichment, where the darker shades of red and blue indicates a higher degree of enrichment and deficiency of actives, respectively.

Figure 4

Assay outcomes of the Tox21 10K compounds from five CYP screenings. Each compound was assigned an activity outcome such as activator, inconclusive activator, inconclusive, inhibitor, inconclusive inhibitor, and inactive based on its curve class and efficacy.

Figure 5

Concentration–response curves of CYP-selective compounds from each CYP assay. (A) Arylamines from the CYP1A2 assay; (B) indanediones and organophosphates from the CYP2C9 and 2C19 assays, respectively; (C) indole alkaloids from CYP2D6 assay; and (D) corticosteroids from the CYP3A4 assay. The core structures representing each chemical class are shown in the inserts (with R and X representing alkyl/benzyl groups and halogen, respectively) and the IC₅₀ values for each compound are given in parentheses.