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Profiling the Tox21 Library Compounds for Cytochrome P450 Inhibitory Activity
Citation:
Sakamuru, S., J. Travers, C. Klumpp-Thomas, R. Huang, K. Witt, S. Ferguson, S. Simmons, A. Simeonov, AND M. Xia. Profiling the Tox21 Library Compounds for Cytochrome P450 Inhibitory Activity. SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25556127
Impact/Purpose:
Presentation to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity
Description:
Background and Purpose: The Cytochrome P450 (CYP) enzymes are membrane-bound hemeproteins that play a key role in drug and xenobiotic metabolism. Individual CYPs are classified into families and sub-families based on their sequence similarities. More than 50 CYPs have been identified in humans, but mainly CYP1, 2, and 3 families contribute to the metabolism of about 80% of clinical drugs. We screened the Tox21 10K compound library to identify chemicals that inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 enzymes. The data obtained from these five screenings were analyzed to reveal structural motifs that drive inhibition of multiple CYP enzymes. Methods: P450-Glo™ assays were used to identify the inhibitory activity of test compounds against CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a cell-free system. This assay technology utilizes a luminescent method in which signal is dependent upon the conversion of a pro-luciferin substrate to luciferin by a specific CYP enzyme. The luciferin metabolite generates a signal proportional to CYP activity when treated with the detection reagent. Screening data were analyzed at multiple treatment concentrations ranging from 0.6 nM to 46 µM, producing concentration-response curves and potencies for each test compound. The Tox21 10K compounds were clustered based on structural similarity. Structural clusters were evaluated for enrichment of active compounds (inhibitors) in each corresponding CYP assay. Results: The five CYP screenings performed in three independent runs exhibited high reproducibility, with an active match rate exceeding 30% and minimal mismatch rates. Of the 10K compounds tested, 80% were active in at least one CYP assay and 15% demonstrated activity against all five CYP enzymes. Hierarchical clustering, based on the active compounds from each CYP assay, revealed that CYP2C9, CYP2C19, and CYP1A2 exhibited the most similar activity patterns. Notably, common structural clusters enriched with active compounds including proton-pump inhibitors, which are known weak CYP inhibitors, tricyclic antidepressants known for their inhibitory effects on several CYPs in vitro, and conazole fungicides, recognized as potent CYP inhibitors, were all confirmed from the study. CYP1A2, CYP3A4, and CYP2D6 also showed strong activity with specific classes of compounds, namely anthraquinones, isoflavones, and phenothiazines, respectively, of which over 90% of the class members were active. Conclusions: In the current study, many known inhibitors of CYPs were confirmed, and several novel structural groups with potential inhibitory activity against CYPs were also identified. The data derived from the quantitative high-throughput screenings of five CYP assays can be utilized as training sets for building predictive models for CYP inhibitors. It also offers valuable insights into the chemical structural groups responsible for CYP inhibition during the early phases of drug development. Disclaimer: this abstract does not necessarily represent US EPA and NIH policy.
URLs/Downloads:
DOI: Profiling the Tox21 Library Compounds for Cytochrome P450 Inhibitory Activity
CYP450_POSTER_SOT2024.PDF (PDF, NA pp, 711.902 KB, about PDF)