Work Description

Date:
12 March, 2025

Dataset Title:
Azole Library Description and Binding Parameters for CYP2A6, CYP2D6, and CYP8B1

Dataset Contact:
Emily Scott scottee@umich.edu

Dataset Creators:

Name: Elyse K. Frydendall
Institution: University of Michigan, Ann Arbor
Email: ekfry@umich.edu
ORCID: https://orcid.org/0000-0002-1862-0529

Name: Emily E. Scott
Institution: University of Michigan, Ann Arbor
Email: scottee@umich.edu
ORCID: https://orcid.org/0000-0002-5538-5716

Funding:
Pharmacological Sciences Training Program (PSTP) T32GM007767, R37 GM076343 (NIH)

Key Points:
- The dataset presented herein estimates the binding affinity for 108 imidazole-containing compounds to cytochromes P450 2A6, 2D6, and 8B1
- Vendor information and chemical properties for each compound are listed.
- Compounds were scored for stability based on replicates of assays with CYP2D6.
- Four compounds were deemed unsuitable for replication over time.

Research Overview:
Human cytochrome P450 enzymes are membrane-embedded monooxygenases responsible for xenobiotic metabolism, steroidogenesis, fatty acid metabolism, and vitamin metabolism. Their active sites can accommodate diverse small molecules and understanding these interactions is key to decoding enzymatic functionality and designing drugs. The most common method for characterizing small molecule binding is quantifying absorbance changes that typically occur when substrates enter the active site near the heme iron. Traditionally such titrations are monitored by a spectrophotometer, requiring significant manual time, protein, and increasing solvents. This assay was adapted for semi-automated high throughput screening, increasing throughput 50-fold while requiring less protein and keeping solvent concentrations constant. This 384-well assay was validated for both type I and II shifts typically observed for substrates and heme-coordinating inhibitors, respectively. This assay was used to screen a library of approximately 100 diverse imidazole-containing compounds which can coordinate with the heme iron if compatible with the overall active site. Three human cytochrome P450 enzymes were screened: drug-metabolizing CYP2A6 and CYP2D6 and sterol-metabolizing CYP8B1. Each bound different sets of imidazole compounds with varying K(d) values, providing a unique binding fingerprint. As a final validation, the K(d) values were used to generate pharmacophores to compare to experimental structures. Applications for the high-throughput assay include 1) facilitating generation of pharmacophores for enzymes where structures are not available, 2) screening to identify ligands for P450 orphans, 3) screening for inhibitors of P450s drug targets, 4) screening potential new drugs to avoid and/or control P450 metabolism, and 5) efficient validation of computational predictions.

Methodology:
The methods for data collection are detailed in the publication cited below. The assay was performed using a plate reader to determine changes in absorbance of the P450 spectra in the UV-visible range. Changes in absorbance are quantified and plotted against concentration of compound in each well. The nonlinear regression one site, specific binding in GraphPad Prism was used to determined dissociation constants and maximum changes in absorbance.
Instrument and/or Software specifications: Perkin Elmer EnVision plate reader, GraphPad Prism

Files contained here:
Supporting_Table_1_HTS_Azole_Library.xlsx
-The file deposited here contains each compound included in the azole library along with its vendor information and chemical properties of interest. The compounds are scored for their ability to repeat over the course of three months. Dissociation constants and maximum changes in absorbance are reported for compounds that cause a spectral shift with cytochromes P450 2A6, 2D6, and 8B1.

Supporting_Table_1_HTS_Azole_Library.csv
-.CSV version of the same data

Related publication(s):
Frydendall, E. K. and E. E. Scott (2024). "Development of a high throughput cytochrome P450-ligand binding assay." J Biol Chem: 107799.

Use and Access:
This data set is made available under an Attribution-NonCommercial 4.0 International license (CC BY-NC 4.0).

To Cite Data:
Frydendall, E. K., Scott, E. E. Azole Library Description and Binding Parameters for CYP2A6, CYP2D6, and CYP8B1 [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/8dt0-0d16