Date of Award

6-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Taosheng Chen, Ph.D.

Committee

Marcus Fischer, Ph.D; Kirk E. Hevener, Ph.D; Leonard Lothstein, Ph.D; David R. Nelson, Ph.D; Erin Schuetz, Ph.D

Abstract

Cytochrome P450 enzymes function to catalyze a wide range of reactions important for various biological processes. In humans, the CYP3A subfamily is particularly critical for drug response. Within this family are CYP3A4 and CYP3A5, which collectively metabolize greater than half of all currently prescribed drugs. These promiscuous enzymes can bind a broad and structurally diverse array of compounds, in turn leading to an increased risk of their modulation via small molecules. In the case of CYP3A5, which is over-expressed in some cancers, this leads to chemoresistance. Such aberrant expression and corresponding drug resistance merit a need to selectively target CYP3A5. However, the significant overlap in sequence and structural identity with CYP3A4 as well as flexible and dynamic binding modes make development of a selective inhibitor challenging, and no progress has been made thus far. Moreover, the cancer-specific regulation of CYP3A5 remains unknown, removing the possibility of targeting a factor upstream of its transcription. While CYP3A4 regulation in liver is well-documented, these regulators don’t control CYP3A5 in extra-hepatic contexts. This warrants further investigation in order to understand the biological basis of CYP3A5 over-expression in disease models. Here we present discovery of the first isoform-selective CYP3A5 inhibitor. We used high-throughput technology to identify clobetasol propionate as capable of selectively inhibiting CYP3A5 enzymatic activity without conferring CYP3A4 inhibition. We further demonstrate the in vitro ability of the compound using a clinically relevant cell model with CYP3A5 overexpression and CRISPR/Cas9-mediated full genetic deletion. Additionally, we explore the mechanism of selectivity, employing computational and biophysical techniques to illustrate how subtle active site differences allow the compound to adopt a tight heme-ligand coordination exclusively in CYP3A5 and serving as the basis of its selective inhibition.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0002-2274-9966

DOI

10.21007/etd.cghs.2020.0509

2020-018-Wright-DOA.pdf (394 kB)
Declaration of Authorship

Available for download on Sunday, June 26, 2022

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