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

Advances in pharmacology (San Diego, Calif.)

Published On 2022

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.

Journal

Advances in pharmacology (San Diego, Calif.)

Published On

2022

Volume

95

Page

1

Authors

Fred Guengerich

Fred Guengerich

Vanderbilt University

Position

Professor of Biochemistry

H-Index(all)

175

H-Index(since 2020)

51

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Enzymology

drug metabolism

cytochrome P450

mutagenesis

University Profile Page

Other Articles from authors

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Medicinal Chemistry

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Chemical Research in Toxicology

In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 μM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Formation of potentially toxic metabolites of drugs in reactions catalyzed by human drug-metabolizing enzymes

Data are presented on the formation of potentially toxic metabolites of drugs that are substrates of human drug metabolizing enzymes. The tabular data lists the formation of potentially toxic/reactive products. The data were obtained from in vitro experiments and showed that the oxidative reactions predominate (with 96% of the total potential toxication reactions). Reductive reactions (e.g., reduction of nitro to amino group and reductive dehalogenation) participate to the extent of 4%. Of the enzymes, cytochrome P450 (P450, CYP) enzymes catalyzed 72% of the reactions, myeloperoxidase (MPO) 7%, flavin-containing monooxygenase (FMO) 3%, aldehyde oxidase (AOX) 4%, sulfotransferase (SULT) 5%, and a group of minor participating enzymes to the extent of 9%. Within the P450 Superfamily, P450 Subfamily 3A (P450 3A4 and 3A5) participates to the extent of 27% and the Subfamily 2C (P450 2C9 and P450 2C19 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Proteomics, modeling, and fluorescence assays delineate cytochrome b5 residues involved in binding and stimulation of cytochrome P450 17A1 17, 20-lyase

Cytochrome b5 (b5) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b5 are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b5 variant (Alexa 488-T70C-b5) to characterize human P450 17A1-b5 interactions, but subsequent proteomic analyses indicated that lysines in b5 were also modified with Alexa 488 maleimide in addition to Cys-70, due to disulfide dimerization of the T70C mutant. A series of b5 variants were constructed with Cys replacements for the identified lysine residues and labeled with the dye. Fluorescence attenuation and the function of b5 in the steroid lyase reaction depended on the modified position. Apo-b5 (devoid of heme group) studies revealed the lack of involvement of the b5 heme in …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Angewandte Chemie

Oxygen‐18 Labeling Reveals a Mixed Fe− O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α‐Demethylation

The 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2−) or Compound I (perferryl oxygen, FeO3+) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18O incorporation into formic acid from 18O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18O (one atom) into formic acid, consistent with a major but not exclusive FeO2− mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Ninety-eight semesters of cytochrome P450 enzymes and related topics—What have I taught and learned?

This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have …

Fred Guengerich

Fred Guengerich

Vanderbilt University

ACS catalysis

Oxygen-18 Labeling Defines a Ferric Peroxide (Compound 0) Mechanism in the Oxidative Deformylation of Aldehydes by Cytochrome P450 2B4

Most cytochrome P450 (P450) oxidations are considered to occur with the active oxidant being a perferryl oxygen (FeO3+, Compound I). However, a ferric peroxide (FeO2̅, Compound 0) mechanism has been proposed, as well, particularly for aldehyde substrates. We investigated three of these systems, the oxidative deformylation of the model substrates citronellal, 2-phenylpropionaldehyde, and 2-methyl-2-phenylpropionaldehyde by rabbit P450 2B4, using 18O labeling. The formic acid product contained one 18O derived from 18O2, which is indicative of a dominant Compound 0 mechanism. The formic acid also contained only one 18O derived from H218O, which ruled out a Compound I mechanism. The possibility of a Baeyer–Villiger reaction was examined by using synthesized possible intermediates, but our data do not support its presence. Overall, these findings unambiguously demonstrate the role of the …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Principles of Xenobiotic Metabolism (Biotransformation)

This chapter provides a general overview of metabolic reactions and their significance. Basic concepts and terminology related to biotransformation, activity, and toxicityToxicity are explained and discussed. Major enzymes involved in oxidationOxidation, reductionReduction, hydrolytic, and conjugationConjugation are covered including enzyme nomenclature, localization, catalytic cycle, coenzymes, relevance of individual enzymes, types of reactions, substrates and metabolites, influence of metabolic reactions on the activity/toxicity of xenobiotics, enzyme inhibition, and relevance if applicable.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate …

2023/11/24

Article Details
Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Nucleic Acids Research

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Xenobiotica

The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyse warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C.2. Compared with that seen at 37 °C, the intrinsic clearance rates (Vmax/Km) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Food and Chemical Toxicology

FEMA GRAS assessment of natural flavor complexes: Lemongrass oil, chamomile oils, citronella oil and related flavoring ingredients

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, eleventh in the series, evaluates the safety of NFCs characterized by primary alcohol, aldehyde, carboxylic acid, ester and lactone constituents derived from terpenoid biosynthetic pathways and/or lipid metabolism. The scientific-based evaluation procedure published in 2005 and updated in 2018 that relies on a complete constituent characterization of the NFC and organization of the constituents into congeneric groups. The safety of the NFCs is evaluated using the threshold of toxicological concern (TTC) concept in addition to data on estimated intake, metabolism and toxicology of members of the congeneric groups and for the NFC under evaluation. The scope of the safety evaluation …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Food and Chemical Toxicology

FEMA GRAS assessment of derivatives of basil, nutmeg, parsley, tarragon and related allylalkoxybenzene-containing natural flavor complexes

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural …

Fred Guengerich

Fred Guengerich

Vanderbilt University

International Journal of Molecular Sciences

Identification of Three Human POLH Germline Variants Defective in Complementing the UV-and Cisplatin-Sensitivity of POLH-Deficient Cells

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1—432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3’-T and 5′-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants—substantially reduced in TLS activity—failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of inorganic biochemistry

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1)

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance …

Fred Guengerich

Fred Guengerich

Vanderbilt University

The Importance of Biotransformation

Biotransformation is important in considerations of toxicity of chemicals. What begins as a well-defined compound may lead to a mixture of chemicals after it enters the body. The changes may be beneficial or detrimental. A potentially harmful chemical may be rapidly inactivated, at low doses. Conversely, an innocuous compound may be transformed into a toxic one. There are cases of both detoxication and bioactivation for the same chemical, sometimes even with the same enzyme being involved in both changes (e.g., aflatoxin B1 and cytochrome P450 3A4). A proper understanding of the chemical changes, the enzymes involved, and the kinetics of changes is needed to understand the outcomes regarding safety assessment.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Drug Metabolism and Disposition

Cytochrome P450 enzymes as drug targets in human disease

Although the mention of cytochrome P450 (P450, CYP) inhibition usually brings to mind unwanted variability in pharmacokinetics, in several cases P450s are good targets for inhibition. These P450s are essential but in certain disease states it is desirable to reduce the concentrations of their products. Most of the attention to date has been with human P450s 5A1, 11A1, 11B1, 11B2, 17A1, 19A1, and 51A1. In some of those cases, there are multiple drugs in us, e.g., exemestane, letrozole, and anastrozole with P450 19A1, the steroid aromatase target in breast cancer. There are also several targets that are less developed, e. g. P450s 2A6, 8B1, 4A11, 24A1, 26A1, and 26B1.Significance Statement The selective inhibition of certain P450s that have major physiological functions has been shown to be very efficacious in certain human diseases. In several cases the search for better drugs continues.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Steroid 17α-hydroxylase/17, 20-lyase (cytochrome P450 17A1)

Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b5, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Direct addition of flavors, including taste and flavor modifiers

The addition of flavorings to food and beverages provides practically unlimited opportunities for innovation, for maintaining and enhancing palatability, and is one essential element of a stable supply of nutritious consumer products. A safety evaluation by the Flavor and Extract Manufacturers Association (FEMA) Expert Panel provides a pathway for flavor producers and users to achieve regulatory authority to use for substances under the conditions of intended use as a flavoring. This chapter describes the factors that contribute to the safety assessment process that is conducted by the Expert Panel, and provides examples of specific flavorings and types of flavorings that are considered. The chapter also describes future issues and opportunities likely to be encountered within the context of the FEMA generally recognized as safe assessment of flavorings.

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Fred Guengerich

Vanderbilt University

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

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.

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Atypical kinetics of cytochrome P450 enzymes in pharmacology and toxicology.

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