UDP-Glucuronosyltransferases (UGTs): Overview

Transcription

1 UDP-Glucuronosyltransferases (UGTs): Overview Anna Radominska-Pandya Department of Biochemistry and Molecular Biology University of Arkansas for Medical Sciences Little Rock, Arkansas, US October 2010; Gdansk University of Technology

2 General Concept of Detoxification Endobiotics Xenobiotics Metabolism Inactive Excretion Products Urine Bile Phase I Oxidation (P450s) OH Phase II Conjugation (UGTs) HO HO COOH O OH H O Lipid Soluble Water Soluble

3 Biotransformation of Drugs and Endobiotics via Oxidation and Conjugation Pathways Phase 0: Absorption of Drugs and Endobiotics Phase I: Oxidation CYP450s Warfarin HO Phase II: Conjugation UGTs SULTs GSTs Phase III: Excretion to Bile and Urine Glucuronic Acid O 7-OH-warfarin glucuronide Abbreviations: CYP450 (Cytochrome P450); UGT (UDP-Glucuronosyltransferase); SULT (Sulfotransferase); GST (Glutathione Sulfatransferase)

4 Biotransformation via Glucuronidation Lipophilic Substrates UGT Therapeutic Drugs Carcinogens Environmental Toxins Dietary Constituents Bilirubin Steroids Bile Acids Retinoic Acids Fatty Acids Prostaglandins UDP-GlcUA Co-Substrate Cytoplasm _ COO - Lumen + H3 N _ ER Various Glucuronides Electophilic Glucuronides Acyl glucuronides (NSAIDs) N-O-glucuronides (Hydroxamic Acids) Hydrophilic β-d-glucuronides Bioactivated Glucuronides Retinoic Acid-gluc 6-O-Morphine-gluc 3-O-Lithocholic Acid-gluc D-ring glucuronides of estradiol, testosterone, DHEA

5 Substrate Specificity of UGTs Substrate specificity is broad and overlapping (promiscuous) Wide range of substrates: Phenols Endogenous substrates Xenobiotic substrates Drugs Dietary plant constituents Billirubin Carcinogens Bile acids Amines Types of glucuronides: Coumarins O-glucuronides Opioids Including acyl glucuronides Steroids N-glucuronides S-glucuronides C-glucuronides Simple Complex Aliphatic alcohols Anthraquinones/flavones Carboxylic acids Sapogenins Primary Secondary Tertiary Heterocyclic Estrogens Androgens Progestin Bile acids Tukey R.H

6 Glucuronidation Reactions Catalyzed by UGTs O-Glucuronidation N-Glucuronidation S-Glucuronidation C-Glucuronidation

7 UDP-Glucuronosyltransferases (UGTs) Large family of membrane bound glycosylated proteins located in the ER as well as inner and outer nuclear membranes Conjugate a wide range of endobiotics and xenobiotics with glucuronic acid Glucuronic acid moiety can attach via a hydroxyl, carboxyl, amino, thiol or carbonyl group on the substrates Generate more polar, water soluble metabolites which can be excreted in urine and bile

8 Biological Significance of Glucuronidation Detoxification increases hydrophobic properties soluble in blood/urine structure different from parent compound no favorable interaction with pharmacological target Detoxification leads to: Excretion of catabolic products Elimination of nucleophilic metabolites of carcinogens Inactivation of biologically active components AZT

9 Biological Significance of Glucuronidation Bioactivation of the parent compound Increases toxicity Increases pharmacological properties Metabolic activation leads to: Cholestatic glucuronides Lithocholic acid glucuronide Estradiol glucuronide Chemically reactive glucuronides: Acyl-glucuronides NSAID glucuronides Ketoprofen Zomepirax Metabolically-active glucuronides Morphine-6-O-glucuronide Irinotecan glucuronide Retinoid glucuronides

10 Biological Significance of Glucuronidation Control of homeostasis of the body and cells Control of steady state concentrations of ligands for nuclear receptors and signaling molecules Downregulation of UGTs results in pathological conditions such as: Cancer Alzheimer s Disease Hyperbiliruminemia Crigler-Najjar syndrome Obesity Other Alteration in Glucuronidation can be caused by: Genetic defects Polymorphisms Tissue-specific regulation

11 Elimination More undesired effects Elimination Elimination Increased toxicity and/or immunological reactions Detoxification Increased potency Detoxification Detoxification Increased potency Activation Toxins and Pollutants Olfactory Substances Drugs XENOBIOTICS UGTs ENDOBIOTICS Ligands for nuclear receptors Bilirubin, Steroids, etc Regulation of gene expression Detoxification Up/down regulation of Protein expression Elimination

12 Phylogenetic Tree of Mammalian UGTs Divergence of 49 mammalian UGT proteins Known mammalian UGTs have been separated into two families, UGT1 and UGT2. UGT1 family is localized on chromosome 2q37 and is divided into 2 subfamilies, UGT1A and UGT1B. UGT2 is localized on chromosome 4q13 and is divided into 3 subfamilies, UGT2A, UGT2B, and UGT2C (not shown). 20 human UGTs have been identified Guillemette C, DMR 2009

13 Guillemette C, DMR 2009

14 UGT1A Gene Cluster and Putative Protein Structure in Humans Ritter JK, 1992 Signal peptide Variable N-terminal domain amino acids Conserved C-terminal domain amino acids Transmembrane fragment UDP- NH 2 Substrates UDP-GlcUA COO - Substrate binding domain UDP-GlcUA binding domain Retention signal Radominska-Pandya A, 1999

15 Hypothetical Model of UGT Topology UDP-GT N + H 3 lumen Transmembrane fragment UDP- GlcUA binding domain Substrate binding domain ER cytosol COO - UGT UDP Gluc-O Lumen HO Transporter Cytosol UDP-GlcUA?? Gluc-O

16 Tissue-Specific Expression of Human UGTs Isoform UGT1A1 Protein Expression Biliary tissue, colon, intestine, liver, stomach, Tissue mrna expression Kidney, trachea, adrenal gland, lung, prostate, testis, thymus, thyroid UGT1A3 UGT1A4 UGT1A6 UGT1A7 UGT1A8 UGT1A9 UGT1A10 UGT2B4 UGT2B7 Biliary tissue, colon, liver, stomach, brain Biliary tissue, colon, liver, intestine, lung, Biliary tissue, colon, liver, stomach, brain, kidney, larynx, lung Orolaryngeal tissue, esophagus, stomach Colon, esophagus, intestine, kidney, larynx Breast, colon, esophagus, liver, kidney, ovary, prostate, skin, testis Orolaryngeal tissue, colon, biliary tissue, esophagus, intestine, stomach Adipose tissue, adrenals, breast, ovary, liver, lung, placenta, prostate, skin, testis, kidney Breast, brain, colon, esophagus, intestine, kidney, liver, lung, and pancreas Adrenal gland, placenta, prostate, salivary gland, small intestine, testis, thymus, thyroid gland, trachea, uterus. Breast Testis, uterus UGT2B10 Liver Adrenal gland, colon, heart, skeletal muscle, testis, uterus UGT2B11 UGT2B15 UGT2B17 Lung Breast, testis, uterus, prostate, lung, ovary, esophagus, kidney, liver, skin Liver, prostate Colon, pancreas, small intestine, stomach, testis, trachea Adrenal gland, bone marrow, brain, colon, lung, pancreas, peripheral leukocytes, salivary gland, small intestine, spinal cord, spleen, stomach, testis, thymus, trachea UGT2A1 Lung Trachea

17 UGT Polymorphisms and Genetic Deficiencies

18 Examples of UGT Polymorphisms UGT1A1 UGT1A1*28 A common variant [A(TA)7TAA] in the TATA-box region of the UGT1A1 promoter UGT1A1*1 Results in: Decrease level of UGT1A1 gene expression Increased breast cancer risk (due to estrogen metabolism), specifically in African American women UGT1A6 Metabolizes aspirin and other NSAIDs Two missense mutations leading to T181A and R184S amino acid substitution UGT1A6*2 Has a frequency of 30% in Caucasian pop. Positively modified protective effect of aspirin (decreased glucuronidation leads to higher levels of aspirin)

19 Examples of UGT Polymorphisms UGT1A7 Glucuronidates polycyclic aromatic hydrocarbons and dietary heterocyclic aromatic amines Three missense mutations in exon 1 result in four alleles: UGT1A7*1 (N129, R131, W208) UGT1A7*2 (K129, K131, W208) UGT1A7*3 (K129, K131, R208) Increased risk of orolaryngeal, liver, and colon cancer UGT1A7*4 (N129, R131, R208) Increased risk of orolaryngeal, liver, and colon cancer UGT2B7 Single nucleotide polymorphisms in coding and regulatory region of UGT2B7 gene are thought to play a role in morphine glucuronidation Cytosine to thymine polymorphism at 802 bp UGT2B7*1 (Y268) 3 times more likely Asian pop than 2B7*2. UGT2B7*2 (H268)

20 Genetic Deficiencies of UGT1A1 Crigler-Najjar disease: Severe, chronic, non-hemolytic, unconjugated hyperbilirubinemia Defect in the gene encoding bilirubin UGT1A1 Caused by mutations to common exons 2-5 or by a mutation to exon 1 Type I Complete loss of bilirubin-conjugating activity Type II Partial loss of bilirubin-conjugating activity (typically <10% of normal) Responds to phenobarbital treatment Gilbert s syndrome: Mild, unconjugated hyperbilirubinemia Defect in the gene encoding bilirubin UGT1A1 Missense mutation in the coding region Homozygous insertion into promoter

21 Conclusions

22 Practice Questions