Clobetasol Propionate Is a Heme-Mediated Selective Inhibitor of Human Cytochrome P450 3A5

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CY...

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Published inJournal of medicinal chemistry Vol. 63; no. 3; pp. 1415 - 1433
Main Authors Wright, William C, Chenge, Jude, Wang, Jingheng, Girvan, Hazel M, Yang, Lei, Chai, Sergio C, Huber, Andrew D, Wu, Jing, Oladimeji, Peter O, Munro, Andrew W, Chen, Taosheng
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 13.02.2020
Subjects
Cell Line, Tumor
Clobetasol - chemistry
Clobetasol - metabolism
Clobetasol - pharmacology
Cytochrome P-450 CYP3A - chemistry
Cytochrome P-450 CYP3A - metabolism
Cytochrome P-450 CYP3A Inhibitors - chemistry
Cytochrome P-450 CYP3A Inhibitors - metabolism
Cytochrome P-450 CYP3A Inhibitors - pharmacology
Enzyme Assays
Heme - chemistry
Heme - metabolism
High-Throughput Screening Assays
Humans
Molecular Docking Simulation
Molecular Dynamics Simulation
Protein Binding
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Summary:The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV–visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme–clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.
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Author Contributions
W.C.W. performed all biochemical high-throughput screening and dose-responsive CYP3A4 and CYP3A5 inhibition assays and analyzed the data, and he also performed CYP panel inhibition data analysis, public data mining and in-house RNA-seq data analysis, CYP cell-based inhibition experiments, LC-MS/MS post-collection data analysis, cell-based imaging and analysis, docking and molecular dynamics simulations, Western blot quantification, and downstream analysis, as well as drafting the corresponding methods sections and the manuscript; J.C., S.C.C., and J. Wang constructed expression vectors, expressed and purified proteins, performed UV-visible spectroscopy, analyzed the resulting data, and drafted the corresponding methods, results, and discussion sections; L.Y. performed all collections of LC-MS/MS samples and LC-MS/MS initial data processing and quality control and drafted the corresponding methods section; A.D.H. designed and optimized all parameters of cell confluence imaging experiments, performed those experiments and analyzed the resulting data, and drafted the corresponding methods section; J. Wu generated inducible CYP3A4 and CYP3A5 constructs and corresponding stable inducible cell lines, extracted their RNA, performed all Western blots, and drafted the corresponding methods sections; P.O.O. grew cultures of all parental PDAC cell lines and extracted the RNA used for RNA sequencing; H.M.G. and A.W.M. performed all the EPR spectroscopic experiments and analyzed the resulting data; W.C.W., J.C., L.Y., J. Wang., S.C.C., A.D.H., J. Wu, P.O.O., H.M.G., A.W.M., and T.C. designed studies and discussed the results. T.C. assembled and finalized the manuscript.
ISSN:0022-2623
1520-4804
DOI:10.1021/acs.jmedchem.9b02067