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 General Concept of Detoxification

Endobiotics Inactive Excretion Metabolism Xenobiotics Products

Urine Bile

OH COOH O Phase I HO Phase II O HO OH H Oxidation Conjugation (P450s) (UGTs) Lipid Soluble Water Soluble Biotransformation of Drugs and Endobiotics via Oxidation and Conjugation Pathways

Phase 0: Absorption of Drugs and Endobiotics

Warfarin Phase I: Oxidation CYP450s

HO

Phase II: Conjugation UGTs SULTs GSTs Glucuronic Acid O 7-OH-warfarin glucuronide Phase III: Excretion to Bile and Urine

Abbreviations: CYP450 (Cytochrome P450); UGT (UDP-Glucuronosyltransferase); SULT (Sulfotransferase); GST (Glutathione Sulfatransferase) Biotransformation via Glucuronidation

Lipophilic Substrates

Bilirubin Steroids Therapeutic Drugs Bile Acids Carcinogens Retinoic Acids Environmental Toxins Fatty Acids Dietary Constituents Prostaglandins UDP-GlcUA UGT Co-Substrate

Cytoplasm _COO- ER + _ Lumen H3N

Various Glucuronides

Electophilic Glucuronides Hydrophilic β-D-glucuronides Bioactivated Glucuronides Acyl glucuronides (NSAIDs) Retinoic Acid-gluc N-O-glucuronides (Hydroxamic Acids) 6-O-Morphine-gluc 3-O-Lithocholic Acid-gluc D-ring glucuronides of estradiol, testosterone, DHEA Substrate Specificity of UGTs

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

Tukey R.H. 2000. Glucuronidation Reactions Catalyzed by UGTs

O-Glucuronidation N-Glucuronidation

S-Glucuronidation C-Glucuronidation 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 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 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 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 More undesired effects Elimination Increased toxicity and/or Elimination Elimination immunological reactions

Increased Detoxification Increased Detoxification potency Detoxification potency Activation

Toxins and Olfactory Drugs Pollutants Substances

XENOBIOTICS UGTs ENDOBIOTICS

Ligands for nuclear receptors Bilirubin, Steroids, etc

Regulation of gene expression Detoxification

Up/down regulation of Protein Elimination expression 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 Guillemette C, DMR 2009 UGT1A Gene Cluster and Putative Protein Structure in Humans

Ritter JK, 1992

Signal Transmembrane Variable N-terminal domain Conserved C-terminal domain peptide fragment amino acids 25 - 286 amino acids 287 - 530

UDP- Substrates - NH2 UDPGlcUA-GlcUA COO

Substrate binding domain UDP-GlcUA binding domain Retention signal

Radominska-Pandya A, 1999 UDP-GT Hypothetical Model of UGT Topology

+ N H3 Substrate UDP- binding GlcUA domain lumen binding domain Transmembrane fragment ER UDP cytosol UGT - COO Gluc-O

HO Lumen Transporter

Cytosol ? ? UDP-GlcUA Gluc-O Tissue-Specific Expression of Human UGTs Tissue Isoform Protein Expression mRNA expression Kidney, trachea, adrenal gland, lung, prostate, testis, thymus, UGT1A1 Biliary tissue, colon, intestine, liver, stomach, thyroid

UGT1A3 Biliary tissue, colon, liver, stomach, brain

UGT1A4 Biliary tissue, colon, liver, intestine, lung,

Adrenal gland, placenta, prostate, salivary gland, small intestine, UGT1A6 Biliary tissue, colon, liver, stomach, brain, kidney, larynx, lung testis, thymus, thyroid gland, trachea, uterus.

UGT1A7 Orolaryngeal tissue, esophagus, stomach

UGT1A8 Colon, esophagus, intestine, kidney, larynx

UGT1A9 Breast, colon, esophagus, liver, kidney, ovary, prostate, skin, testis

Orolaryngeal tissue, colon, biliary tissue, esophagus, intestine, UGT1A10 Breast stomach Adipose tissue, adrenals, breast, ovary, liver, lung, placenta, UGT2B4 prostate, skin, testis, kidney Breast, brain, colon, esophagus, intestine, kidney, liver, lung, and UGT2B7 Testis, uterus pancreas UGT2B10 Liver Adrenal gland, colon, heart, skeletal muscle, testis, uterus

UGT2B11 Lung

Breast, testis, uterus, prostate, lung, ovary, esophagus, kidney, liver, UGT2B15 Colon, pancreas, small intestine, stomach, testis, trachea skin Adrenal gland, bone marrow, brain, colon, lung, pancreas, peripheral UGT2B17 Liver, prostate leukocytes, salivary gland, small intestine, spinal cord, spleen, stomach, testis, thymus, trachea UGT2A1 Lung Trachea UGT Polymorphisms and Genetic Deficiencies 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) 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) 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 Conclusions Practice Questions