Inhibition, Cytochrome P450 enzyme (CYP17A1) activity

This KE describes the inhibition of the enzyme CYP17A1, an enzyme involved in making precursors for the synthesis of mineralocorticoids, glucocorticoids and sex steroids. 

CYP17A1 is a membrane bound enzyme that catalyses two reactions, 17α-hydroxylation and 17,20-lyase transformation. Both reactions are dependent on cofactor P450 oxidoreductase, which transfers two electrons to CYP17A1 from NADPH. The lyase reaction is also dependent on the cofactor 

cytochrome B5. The conversion of pregnenolone and progesterone to 17-OH-pregnenolone and 17-OH-progesterone, respectively, takes place through the 17α-hydroxylase reaction at C17 and the conversion of these hydroxylated steroids into dehydroepiandrosterone (DHEA) and androstenedione, respectively, occurs through the 17,20-lyase reaction, which breaks the bond between the C17 and the C20. CYP17A1 is also involved in the backdoor pathway to dihydrotestosterone synthesis (Burris-Hiday & Scott, 2021; Miller & Auchus, 2011, 2019; Peng et al., 2019; Wróbel et al., 2023). CYP17A1 is expressed in traditional steroidogenic tissues (testes, ovary, adrenal, and to a minor degree in the placenta). Studies also detected Cyp17A1 activities in heart, adipose, liver, skin, brain, and kidney tissue. CYP17A1 is expressed in the zona reticularis and zona fasciculate of the adrenal gland, in the Leydig cells of testicles, and in theca cells of the ovaries (Chatuphonprasert et al., 2018; Odermatt et al., 2016; Peng et al., 2019; Petrunak et al., 2014; Storbeck et al., 2011; Wróbel et al., 2023). Cyp17a1 inhibitors bind in the active site of the enzyme by mimicking endogenous substrate, leading to a reduction in the activity of the enzyme. Cyp17A1 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20-lyase activities, the distinction between the two being functional and not genetic or structural. Cyp17a1 is found in all the steroidogenic tissues such as the Leydig cells of the testes, the thecal cells of the ovaries and the adrenal cortex. Studies also detected Cyp17a1 activities in heart, adipose, liver and kidney tissue. CYP17a1 has a decisive function in steroidogenesis by constituting the initial step in a series of biochemical reactions that culminate in synthesis of steroid end-products (testosterone, estradiol, cortisol, and DHEA). Thus, any variation in Cyp17a1’s activity directly or indirectly affect steroidogenesis.

There is currently (2023) no OECD test guideline for the measurement of CYP17A1 inhibition. 

Inhibition of CYP17A1 can be assessed in vitro by modulating mammalian or yeast cells to express the enzyme as well as P450-oxidoreductase with/without B5. Subsequently, enzyme activity can be studied in isolated microsomes that contain the enzyme system. Alternatively, the isolated enzyme can be used for activity measurement in concert with purified P450-oxidoreductase with/without B5. NADPH should be added as the source of electrons for the reaction to take place. The reaction substrate is added and either the substrate removal or product formation is measured (Yoshimoto & Auchus, 2015). 

In the case of examining the ability of a chemical to inhibit the enzyme activity, the chemical is added in increasing concentrations and a constant level of substrate is added – a decreased conversion of the substrate to product, would suggest enzyme inhibition. 

After running inhibition assay, the steroid substrate or product is extracted. There are many ways that these can be measured. They can be resolved by use of HPLC and the levels detected with UV-detection or scintillation counter, if for instance the substrate is radiolabeled. They can be quantified by use of LC-MS, as well as thin-layer chromatography, which requires the use of radiolabeled substrate (Yoshimoto & Auchus, 2015).

Taxonomic applicability. 

The KE is applicable to mammals. The CYP17 gene is present in mammals and show relatively high homology of amino acid sequence; however, the enzyme exhibit differences in activity and requirement of cytochrome B5 (Gilep et al., 2011). For example, in humans and primates the lyase reaction leading to the conversion of 17-OH-pregnenolone to DHEA is dominant compared to the conversion of 17-OH-progestrone to androstenedione, which is predominant in rodents (Lawrence et al., 2022; Miller & Auchus, 2011). 

Life stage applicability 

This KE is applicable throughout all life stages as CYP17A1 is expressed throughout life in mammals (Chatuphonprasert et al., 2018; Odermatt et al., 2016; Peng et al., 2019; Petrunak et al., 2014; Storbeck et al., 2011; Wróbel et al., 2023). 

Sex applicability 

CYP17A1 is expressed in the Leydig cells of testicles, and in theca cells of the ovaries (Chatuphonprasert et al., 2018; Odermatt et al., 2016; Peng et al., 2019; Petrunak et al., 2014; Storbeck et al., 2011; Wróbel et al., 2023). This KE is therefore applicable to both sexes.

Burris-Hiday, S. D., & Scott, E. E. (2021). Steroidogenic cytochrome P450 17A1 structure and function. Molecular and Cellular Endocrinology, 528. https://doi.org/10.1016/j.mce.2021.111261 

Chatuphonprasert, W., Jarukamjorn, K., & Ellinger, I. (2018). Physiology and pathophysiology of steroid biosynthesis, transport and metabolism in the human placenta. In Frontiers in Pharmacology (Vol. 9, Issue SEP). Frontiers Media S.A. https://doi.org/10.3389/fphar.2018.01027 

Gilep, A. A., Sushko, T. A., & Usanov, S. A. (2011). At the crossroads of steroid hormone biosynthesis: The role, substrate specificity and evolutionary development of CYP17. In Biochimica et Biophysica Acta - Proteins and Proteomics (Vol. 1814, Issue 1, pp. 200–209). https://doi.org/10.1016/j.bbapap.2010.06.021 

Lawrence, B. M., O’Donnell, L., Smith, L. B., & Rebourcet, D. (2022). New Insights into Testosterone Biosynthesis: Novel Observations from HSD17B3 Deficient Mice. In International Journal of Molecular Sciences (Vol. 23, Issue 24). MDPI. https://doi.org/10.3390/ijms232415555 

Miller, W. L., & Auchus, R. J. (2011). The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine Reviews, 32(1), 81–151. https://doi.org/10.1210/er.2010-0013 

Miller, W. L., & Auchus, R. J. (2019). The “backdoor pathway” of androgen synthesis in human male sexual development. PLoS Biology, 17(4). https://doi.org/10.1371/journal.pbio.3000198 

Odermatt, A., Strajhar, P., & Engeli, R. T. (2016). Disruption of steroidogenesis: Cell models for mechanistic investigations and as screening tools. In Journal of Steroid Biochemistry and Molecular Biology (Vol. 158, pp. 9–21). Elsevier Ltd. https://doi.org/10.1016/j.jsbmb.2016.01.009 

Peng, Z., Xueb, G., Chen, W., & Xia, S. (2019). Environmental inhibitors of the expression of cytochrome P450 17A1 in mammals. In Environmental Toxicology and Pharmacology (Vol. 69, pp. 16–25). Elsevier B.V. https://doi.org/10.1016/j.etap.2019.02.007 

Petrunak, E. M., DeVore, N. M., Porubsky, P. R., & Scott, E. E. (2014). Structures of human steroidogenic cytochrome P450 17A1 with substrates. Journal of Biological Chemistry, 289(47), 32952–32964. https://doi.org/10.1074/jbc.M114.610998 

Storbeck, K. H., Swart, P., Africander, D., Conradie, R., Louw, R., & Swart, A. C. (2011). 16α-Hydroxyprogesterone: Origin, biosynthesis and receptor interaction. In Molecular and Cellular Endocrinology (Vol. 336, Issues 1–2, pp. 92–101). https://doi.org/10.1016/j.mce.2010.11.016 

Wróbel, T. M., Jørgensen, F. S., Pandey, A. V., Grudzińska, A., Sharma, K., Yakubu, J., & Björkling, F. (2023). Non-steroidal CYP17A1 Inhibitors: Discovery and Assessment. In Journal of Medicinal Chemistry (Vol. 66, Issue 10, pp. 6542–6566). American Chemical Society. https://doi.org/10.1021/acs.jmedchem.3c00442 

Yoshimoto, F. K., & Auchus, R. J. (2015). The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1). In Journal of Steroid Biochemistry and Molecular Biology (Vol. 151, pp. 52–65). Elsevier Ltd. https://doi.org/10.1016/j.jsbmb.2014.11.026