Roles of cytochrome P450 enzymes in pharmacology and toxicology: Past, present, and future

Abstract

The development of the cytochrome P450 (P450) field has been remarkable in the areas of pharmacology and toxicology, particularly in drug development. Today it is possible to use the knowledge base and relatively straightforward assays to make intelligent predictions about drug disposition prior to human dosing. Much is known about the structures, regulation, chemistry of catalysis, and the substrate and inhibitor specificity of human P450s. Many aspects of drug-drug interactions and side effects can be understood in terms of P450s. This knowledge has also been useful in pharmacy practice, as well as in the pharmaceutical industry and medical practice. However, there are still basic and practical questions to address regarding P450s and their roles in pharmacology and toxicology. Another aspect is the discovery of drugs that inhibit P450 to treat diseases.

Keywords: CYP3A4; Cytochrome P450; Drug metabolism; Drug toxicity; Drug-drug interactions; Enzyme inhibition; P450 drug targets.

Figure 1.

Percentages of total P450 in human liver samples accounted for by each P450. The data points were compiled (Guengerich, 2021a) from four sets with multiple liver samples (Achour, Russell, Barber, & Rostami-Hodjegan, 2014; Kawakami et al., 2011; Shimada et al., 1994) and one with a single liver sample high in P450 1A1 (Lang, Radtke, & Bairlein, 2019). The estimates were made immunochemically in one case (Shimada et al., 1994) and by LC-MS proteomic methods in the others (Achour et al., 2014; Kawakami et al., 2011; Lang et al., 2019). The value for P450 1A1 is a mean of measurements of 30 samples (Lang et al., 2019). The individual colors have no meaning but are added to facilitate visualization.

Figure 2.

General scheme for transcriptional regulation of P450s. L: ligand, R: receptor, Ŕ-heterodimeric partner, Coactiv: co-activator protein (e.g., hepatic nuclear factor (HNF) α in the case of P450 3A4), RNA pol: RNA polymerase (Guengerich, 2018a, 2021a).

Figure 3.

P450 catalytic cycle. The nine labeled steps show sequential (1) substrate binding, (2) 1-electron reduction, (3) oxygen binding, (4) second 1-electron reduction, (5) protonation of “Compound 0”, (6) loss of water to form “Compound I”, (7) hydrogen atom abstraction by Compound I, (8) oxygen rebound to form product, and (9) product dissociation. As indicated, ferrous P450 can also bind substrate (Yun, Kim, Calcutt, & Guengerich, 2005). In some cases, b₅ can provide the electron in step 2 or 4. In some sequential reactions, step 9 does not occur and a second oxidation of the initial product is observed (E. Gonzalez & Guengerich, 2017; Reddish & Guengerich, 2019).

Figure 4.

A structure of P450 3A4 (Protein Data Bank (PDB) 1TQN), with major helices labeled (Yano et al., 2004). The heme prosthetic group is shown in gray.

Figure 5.

Hypotheses to explain complex substrate recognition data (Gianni et al., 2014; Vogt & Di Cera, 2012).

Figure 6.

Scheme summarizing interaction of P450 3A4 with inhibitors. The times of appearance of individual species are indicated in blue (Guengerich et al., 2020).

Figure 7.

Fractions of small molecule drugs approved by US FDA in 2015–2020 metabolized by individual enzymes (Bhutani et al., 2021). UGT: uridine diphosphate glucuronosyl transferase; FMO, flavin-containing monooxygenase; AO, aldehyde oxidase. Reprinted from J. Med. Chem., Vol. 64, Bhutani, P., Joshi, G., Raja, N., Bachhav, N., Rajanna, P. K., Bhutani, H., Paul, A. T. and Kumar, R. US FDA approved drugs from 2015-June 2020: A perspective, pages 2339–2381, Copyright (2021), with permission from the American Chemical Society.

Figure 8.

Frequency of new molecular entities (NMEs, i.e. new drug candidates) in inhibition-based drug-drug interactions (DDIs) with drugs approved by the Food and Drug Administration (FDA) in the United States between 2013 and 2016 (Yu et al., 2018). A, Grouping by therapeutic class. B, Grouping by enzymes involved. Pgp and OAT1B1 are transporters. COMT: catechol O-methyl transferase.

Figure 9.

Roles of P450s in the bioactivation and detoxication of chemicals: the complex example of phenacetin (Guengerich, 2019a). Acetaminophen (paracetamol, Tylenol ^®) is widely used as an analgesic, safe at low doses and hepatotoxic at high levels (S. S. T. Lee, Buters, Pineau, Fernandez-Salguero, & Gonzalez, 1996). Phenacetin has been classified as a carcinogen and withdrawn from use. The metabolism of acetaminophen has been investigated in detail (Dahlin, Miwa, Lu, & Nelson, 1984; Dahlin & Nelson, 1982; Guengerich, 2021a). Only in a few cases are the structures of the protein and DNA adducts known. Some of the indicated P450s have been identified in different species, including humans (Distlerath et al., 1985; S. S. T. Lee et al., 1996).

Figure 10.

Metabolism of terfenadine (Guengerich, 2014; D. Thompson & Oster, 1996; Yun et al., 1993). All steps are catalyzed primarily by P450 3A4. Oxidations of the antihistamine terfenadine catalyzed by P450 3A4. The oxidation of terfenadine was attenuated in individuals who have inherently low levels of P450 3A4 (Yang et al., 2010) or used P450 3A4 inhibitors (e.g., erthyromycin, ketoconazole) concomitantly with terfenadine (Guengerich, 2014; Yun et al., 1993).

Figure 11.

Boundary line for k_obs for time-dependent inhibition and relation to in vivo drug-drug interactions (DDI) (Eng et al., 2021). A, Fifty drugs were evaluated for P450 3A4 time-dependent inhibition in human liver microsomes (at 30 μM unless noted otherwise) and ranked by k_obs, the first-order rate of inactivation, as judged using midazolam 1´-hydroxylation (○), presented on a log₁₀ scale (right y-axis). The filled bars show the in vivo drug-drug interactions as judged by the AUC_R (AUC with the drug divided by the AUC without the drug, Clinical DDI magnitude). B, The study in Part A was repeated in human hepatocytes. The stippled line indicates a 2-fold in vivo difference. Also indicated are p < 0.05 statistical limits and a k_obs “boundary” of the lowest in vitro value with 2-fold in vivo difference. Reprinted from Drug Metab. Dispos., Vol. 49, Eng, H., Tseng, E., Cerny, M. A., Goosen, T. C. and Obach, R. S., Cytochrome P450 3A time-dependent inhibition assays are too sensitive for identification of drugs causing clinically significant drug-drug interactions: a comparison of human liver microsomes and hepatocytes and definition of boundaries for inactivation rate constants, pages 442–450, Copyright (2021), with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 12.

Some multi-step steroid biosynthetic reactions catalyzed by human P450s that are targets for drugs. A, P450 17A1; B, P450 19A1.

Figure 13.

P450 11B2 oxidation of 11-deoxycorticosterone to aldosterone, a drug target (Hu, Yin, & Hartmann, 2014). Reprinted from J. Med. Chem., Vol.67, Hu, Q., Yin, L., & Hartmann, R. W. (2014). Aldosterone synthase inhibitors as promising treatments for mineralocorticoid dependent cardiovascular and renal diseases, pages 5011–5022, Copyright (2014), with permission from the American Chemical Society.

Figure 14.

Posaconazole bound in the C. albicans CYP51 active site (Hargrove et al., 2017). A, An early lead compound in the program (SCH 51048) and posaconazole. B, X-ray crystal structure of C. albicans P450 51A1 bound to posaconazole. The arrow in Part B is pointed to the extra hydroxyl group in posconazole.

Figure 15.

P450 21A2 variants. Amino acid changes which give rise to the (A) salt-wasting (SW), (B) simple virile (SV), and (C) non-classical (NC) congenital adrenal hyperplasia phenotypes are mapped in the crystal structure of P450 21A2 (Pallan et al., 2015). Carbon atoms of wild type (*1) residues are highlighted in blue (SW), green (SV), and purple (NC). Reprinted from Molecular Endocrinology, Vol. 29, Pallan, P. S., Lei, L., Wang, C., Waterman, M. R., Guengerich, F. P., & Egli, M. (2015). Research Resource: Correlating human cytochrome P450 21A2 crystal structure and phenotypes of mutations in congenital adrenal hyperplasia, pages 1375–1384, Copyright (2015), with permission from The Endocrine Society.