Cytochromes P450 Lecture 1 PDF

Cytochromes P450 BIO-5002A Biochemistry, Professor Julea Butt ([email protected]) Lecture Outline Lecture 1:  Introduction to Cytochrome P450 enzymes  Metabolism of Endogenous and Exogenous Molecules  Induction and Inhibition of Cytochromes P450 Lecture 2:  Th...

Cytochromes P450 BIO-5002A Biochemistry, Professor Julea Butt ([email protected]) Lecture Outline Lecture 1:  Introduction to Cytochrome P450 enzymes  Metabolism of Endogenous and Exogenous Molecules  Induction and Inhibition of Cytochromes P450 Lecture 2:  The catalytic mechanism of Cytochromes P450  Cytochromes P450 Learning Outcomes: Lecture 1 After reviewing the lecture material and reading additional resources relating to P450 biochemistry and pharmocogenetics in your private study time you will be able to:  recognise the general form of the mono-oxygenation reactions catalysed by P450s.  distinguish Class I and II P450s.  demonstrate understanding the classification of the P450 superfamily.  define the major categories of P450 function in humans.  show understanding of spectrophotometric assays for quantifying P450 activity.  describe and identify the nature of drug-drug interactions.  demonstrate understanding of the significance of polymorphisms in Cytochromes P450  a unique family of heme proteins encoded by a gene superfamily with hundreds of members  enzymes that catalyse monooxygenation of structurally diverse compounds endogenous substrates include cholesterol, steroid hormones and fatty acids exogenous (xenobiotic) substrates include drugs, food additives, pesticides and chemicals that enter the body by ingestion, inhalation and absorption through the skin  make significant contributions to medicine through inactivation or activation of therapeutic agents drug-drug interactions Cys- Ligated Heme in the Active Site Cys flurbiprofen heme PDB file 1R9O  Enzymes Named for a Unique Spectroscopic Property oxidised enzyme 0.6 Absorbance 0.4 0.2 0.0 400 500 600 700 Wavelength (nm)  Enzymes Named for a Unique Spectroscopic Property reduced enzyme 0.6 e- Absorbance 0.4 sodium dithionite a chemical reductant 0.2 0.0 400 500 600 700 Wavelength (nm)  Enzymes Named for a Unique Spectroscopic Property reduced enzyme + CO maximum absorbance at 450 nm 0.6 e- CO Absorbance 0.4 P450 = Pigment 0.2 with maximum absorbance at 450 nm 0.0 400 500 600 700 Wavelength (nm) Cytochromes P450 Catalyse Monooxygenation Monooxygenation is the incorporation of one O atom from O2 into an organic molecule R3CH R3CH + O2 + NADPH + H+  R3COH + NADP+ + H2O where R3CH is a hydrophobic / lipophilic compound, and, R3COH is less hydrophobic and more hydrophilic than R3CH. R-CH2-CH3  R-CH2-CH2-OH aliphatic Common reactions R-CH -CH  R-CH(OH)-CH3 hydroxylati 2 3 catalysed by on cytochromes P450 aromatic R-  R- -OH hydroxylati on R3CH may be a steroid, fatty acid, drug or other chemical that has an alkane, alkene, aromatic ring or heterocyclic ring substituent.  Cytochrome P450 Electron Transport Systems 1 NADP+ +2e- + H+ NADPH NADPH/NADP+ a 2e- Redox Co-Substrate Cytochrome P450: Heme Fe3+ + 1e- Fe2+ a 1e- Redox Cofactor  Cytochrome P450 Electron Transport Systems 1 NADP+ +2e- + H+ NADPH NADPH/NADP+ a 2e- Redox Co-Substrate Flavin e - + H+ e - + H+ 2 x 1e- Redox Cofactor Cytochrome P450: Heme Fe3+ + 1e- Fe2+ a 1e- Redox Cofactor Cytochrome P450 Electron Transport Systems 2 Most mammalian cytochromes P450 are found in the endoplasmic reticulum (ER) within hepatocytes, in renal cells and cells of the respiratory tract. These are the Class II cytochrome P450 enzymes. NADPH NADP+ NADPH Cytochrome FAD P450 Oxidoreductase FMN cytosol heme ER membrane R3CH R3COH O2 H2O Cytochrome P450 Cytochrome P450 Electron Transport Systems 3 Class I cytochrome P450 enzymes are found in mitochondria. NADPH NADP+ Ferredoxin Reductase FAD e- Ferredoxin FeS matrix e- inner heme mitochondrial membrane R3CH R3COH O2 H2O Cytochrome P450 Cytochromes P450: A Superfamily with Hundreds of Members Isoform 1 Gene Isoform 2 Cytochrome P450 (CYP) Nomenclature Sequence Identity Sequence Identity Order > 40% > 55% Identified CYP1 CYP1A CYP1A1 CYP1B CYP1A2 CYP1C CYP1A3 etc etc CYP2 CYP2A CYP1B1 CYP2B CYP1B2 CYP2C CYP1B3 etc etc CYP2A1 CYP3 CYP3A CYP2A2 etc etc etc FAMILY SUB-FAMILY SPECIFIC ISOFORM Human Cytochromes P450 In humans there are 57 cytochromes P450. The most significant are: CYP1 CYP2 CYP3 CYP4 CYP11 CYP17 CYP19 CYP21 CYP26 1A1 2A6 3A4 4A11 11A1 17A1 19A1 21A2 26A1 1A2 2A7 3A5 4B1 11B1 26B1 1B1 2A13 3A7 4F2 11B2 2B6 4F3 2C8 4F8 2C9 2C18 2C19 2D6 2E1 2J2 7 cytochromes P450 are mitochondrial. Cytochromes P450 for Processing Endogenous Molecules Cytochromes P450 with high substrate specificity process cholesterol, steroids, prostaglandins and fatty acids. A series of reactions catalysed by CYP11A1 in the adrenal mitochondria are shown here. 21 20 22 NADPH + O2 Cholesterol 22-Hydroxycholesterol NADPH + O2 20,22-Dihydroxycholesterol Pregnenolone Steroid Hormone Synthesis in the Adrenal Gland Cholesterol CYPs 11A1, 11B1 and 11B2 mitochondrial CYPs 17A1, 19A1 and 21A2 in the ER HSD = hydroxysteroid dehydrogenases CYP17A1 CYP17A1 CYP17A1 CYP17A1 CYP19A1 CYP21A2 CYP21A2 Testosterone Estradiol CYP19A1 Cortisol: Governs protein, carbohydrate and lipid metabolism Fig. 11.9 Textbook of Biochemistry with Clinical Correlations, 7e edited by Aldosterone: regulates salt and water balance Thomas M. Devlin © 2011 John Wiley & Sons, Inc. All of the following are correct about every cytochrome P450 EXCEPT: A. contains heme as a cofactor B. is associated with the endoplasmic reticulum C. catalyses monooxygenation D. receives electrons from NADPH via FAD containing redox partners Conversion of cholesterol to steroid hormones in the adrenal gland involves: A. a cytochrome P450 that catalyses more than one transformation B. only mitochondrial cytochromes P450 C. only cytochromes P450 of the endoplasmic reticulum D. transformation of a xenobiotic Which of the following best describes cholesterol? A. amino acid B. lipid C. sugar D. nucleotide Cytochromes P450 are Membrane Associated Their substrates are mainly hydrophobic and soluble in lipid bilayers. matrix flurbiprofen membrane heme PDB file 1R9O Cytochromes P450 Processing Xenobiotics 1 Cytochromes P450 that process exogenous molecules, xenobiotics, are much less substrate specific than those for processing endogenous molecules. They probably evolved to allow lipophilic toxins acquired from the environment to be made more water soluble for excretion via kidneys or in bile. Three families are involved in processing many environmental contaminants, food additives, drugs etc : CYP1, CYP2 and CYP3 (with 23 isoforms). Many different substrates can bind adjacent to the catalytic Cys- ligated heme because of the large and cavernous active site pocket within the structure of these proteins. P.A. Williams et al Nature 424, 464-468 (2003) Cytochromes P450 Processing Xenobiotics 2 Many xenobiotics are highly lipophilic and they will accumulate within cells potentially interfering with cellular function unless they are metabolized to more hydrophilic products and then excreted from the body. HO OH OH OH NADPH NADPH NADPH O2 O2 OH O2 OH water soluble lipid soluble retained excreted in lipid from the bilayers body Cytochromes P450 Processing Xenobiotics 3 Types of Reactions Catalysed by Cytochromes P450 Example Reaction Type Hydroxylation of ibuprofen at its alkyl Aliphatic sidechain hydroxylation Hydroxylation of S-warfarin Aromatic hydroxylation O-demethylation of codeine to Oxygen morphine, dealkylation its active analgesic agent N-demethylation of diazepam (Valium) Nitrogen  Cytochromes P450 and Metabolism of Therapeutic Drugs Broad substrate specificity of CYPs for exogenous substrates can also result in compounds being processed by multiple CYPs at more than one site. % Drugs Metabolised 60 50 40 30 20 10 0 CYP3A4 is present in gastrointestinal tract and liver. It is responsible for the poor bioavailability of many drugs because it hydroxylates them to inactive forms. Fig. 11.12 of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc. Quantifying CYP Catalytic Activity One of the substrates of all cytochrome P450 reactions gives a characteristic colour change when it is converted to the corresponding product. R3CH + O2 + NADPH + H+  R3COH + NADP+ + H2O where R3CH is a hydrophobic / lipophilic compound. NADPH gives solutions NADP+ a yellow colour NADPH NADP+ is colourless Monitoring a reaction at 340 nm quantifies the rate of a P450 catalysed reaction. Spectrophotometric assays of steady-state enzyme activity are powerful tools in quantifying the activities of cytochromes P450 to identify the consequences of polymorphisms and the consequences of drug:drug interactions. Steady-State Assay of Cytochromes P450 Inject catalytic (small amount of) P450 and mix Measure loss of colour with NADPH spectrometer and organic substrate Gradient gives Absorbance reaction rate at 340 nm 0 0 time At 340 nm the  for NADPH is 6220 M-1 cm-1 Absorbance Rate of NADPH consumption = M time 1  t l Substrate Inhibition of Cytochromes P450 Most cytochrome P450 catalysed reactions are well-described by the classical Michaelis-Menten description of an enzyme catalysed reaction. However, in some cases substrate inhibition is observed as illustrated below for two reactions catalysed by CYP 3A4. Substrate Inhibition Kinetics for Cytochrome P450-Catalyzed Reactions Yuh Lin et al Drug Metabolism and Disposition 2001, 29:368-374 Substrate Inhibition: A Special Case of Competitive Inhibition no substrate inhibition E Reaction Velocity (nM/sec) S Kd substrate inhibition ES P S Ki ESS Substrate Concentration (µM) 1/rate rate  Vmax S  where K i  S ES   S   K M  S  1  ESS   Ki  0 1/[substrate] Moderated Drug Efficacy 1: Substrate Inhibition and Drug:Drug Interactions Explained by the cavernous pocket adjacent to the catalytic heme cofactor. S-warfarin flurbiprofen Zharkova et al Journal of Molecular Recognition (2013) 26:86-91 Moderated Drug Efficacy 2: Regulation of CYP Expression Compounds that Anaesthetics Damage Liver (halothane and enflurane) CYP2E1 Proteins hepatitis People with high levels of CYP2E1 are at higher risk for hepatitis as an adverse reaction to anaesthetic. Alcohol and certain anti-depressants trigger increased levels CYP2E1 in the body. As a consequence those with chronic alcohol intake and/or taking those drugs are at increased risk of developing hepatitis after surgery. Moderated Drug Efficacy 2: Regulation of CYP Expression The increased levels of CYP2E1 that occur in response to certain anti- depressants or chronic alcohol are an example of drug induced regulation of cytochrome P450 expression. In other cases expression is decreased. R D L Binding of drug (D) to specific receptor (R) R D Binding of receptor partner (P) to the (DR) P complex R D P Receptor complex binds to response element of specific P450 genes R D P P450 gene Moderated Drug Efficacy 3: CYP Polymorphisms Polymorphism is a difference in DNA sequence found at 1% or higher in a population. It is thought to occur in genes of 40% of drug metabolising cytochromes P450. As a consequence individuals contain unique cytochrome P450 genes or alleles and can exhibit very different rates of metabolising individual drugs. Specific genetic variations are often associated with specific ethnic groups. CYP polymorphisms occur in CYP 2C9 found in human liver where it processes several commonly used drugs such as non-steroidal anti-inflammatory drugs and S-warfarin. Wild-type CYP2C9*1 (shown here) Ile359 and Asp360. Polymorphisms in CYP2C9 include: Caucasian variant CYP2C9*3 Leu359 (0.4% of this population are homozygous carriers and 15% are heterozygous). African American variant CYP 2C9*5 PDB entry 1OG2 Glu360 (approximately 3% of this population carries the CYP2C9*5 allele). Moderated Drug Efficacy 3: CYP2C9 and Warfarin CYP2C9 is solely responsible for the metabolism of S-warfarin and its elimination from the body. This drug [warfarin] is orally administered over weeks to harmful in blood inhibit blood coagulation in patients who have suffered heart attack or stroke in order to prevent beneficial reoccurrence of clots that may cause another life-threatening episode. Too much drug will cause no effect uncontrolled bleeding and too little has no effect so the level in blood must be tightly maintained in a time specific range. For the majority of the population, a 4 to 5 mg dose of warfarin per day is beneficial. However, substitution of Leu359 in CYP2C9 causes a substantial loss of enzymatic activity and this is found in the CYP2C9*3 allelic variant. If a 5 mg dose of warfarin was taken by patient with CYP2C9*3 uncontrollable bleeding could result from a simple cut. In such individuals a warfarin dose of 0.5 to 1 mg per week may be sufficient to maintain blood levels of warfarin at the therapeutically appropriate level. Pharmacogenetics – how genes influence an individual’s response to drugs. drug toxic but drug toxic but beneficial not beneficial patient group drug not toxic but drug not toxic not beneficial and beneficial one person – one drug – one dose Pharmacogenetics and Personalised Prescriptions Detailed knowledge of the cytochromes P450 within an individual could lead to personalised prescriptions. The Roche AmpliChip CYP450 Test aims to ‘aid to clinicians in determining therapeutic strategy and treatment dose for therapeutics metabolized by the CYP2D6 or CYP2C19 gene product’. Pharmacogenetics informed decision making in adolescent psychiatric treatment: a clinical case report. Smith T, Sharp S, Manzardo AM, Butler MG. Int J Mol Sci. 2015 Feb 20 https://web.archive.org/web/20110906084820/http://molecular.roche.com/assays/Pages/AmpliChipCYP450Test.aspx  Cytochromes P450 Learning Outcomes: Lecture 1 After reviewing the lecture material and reading additional resources relating to P450 biochemistry and pharmocogenetics in your private study time you will be able to:  recognise the general form of the mono-oxygenation reactions catalysed by P450s.  distinguish Class I and II P450s.  demonstrate understanding the classification of the P450 superfamily.  define the major categories of P450 function in humans.  show understanding of spectrophotometric assays for quantifying P450 activity.  describe and identify the nature of drug-drug interactions.  demonstrate understanding of the significance of polymorphisms in human P450s. Do NOT memorise the pathways or molecular structures that are discussed. Focus on understanding key features of the References Principles of Bioinorganic Chemistry SJ Lippard JM Berg , University Science Books (1994) Mill Valley, CA, USA ISBN 978-0-935702-72-9 Textbook of Biochemistry with Clinical Correlations, 7th Edition Thomas M. Devlin, John Wiley and Sons (2010) USA ISBN 978-0-470-28173-4 Chapter 11. Crystal Structure of Human Cytochrome P450 2C9 with Bound Warfarin. P.A. Williams et al Nature 424:464-468 (2003). The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0A Resolution. Wester et al. J. Biol. Chem. 279:35630-35637 (2004) Substrate Inhibition Kinetics for Cytochrome P450-Catalyzed Reactions. Y. Lin et al Drug Metabolism and Disposition, 29:368-374 (2001) Heme Enzyme Structure and Function T. Poulos Chemical Reviews 114:3919 (2014) What makes a P450 tick? A. Munro et al Trends Biochem. Sci. 38:140 (2013) Glimpsing the Critical Intermediate in Cytochrome P450 Oxidations S. Sligar Science 330:924 (2010). Cytochrome P450 Compound I: Capture, Characterization, and C-H Bond Activation Kinetics. J. Rittle, M. T. Green, Science 330:933 (2010).