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The Steroid and Xenobiotic Receptor SXR: A Key Regulator of Drug and Xenobiotic Metabolism

The Steroid and Xenobiotic Receptor SXR: A Key Regulator of Drug and Xenobiotic Metabolism. Bruce Blumberg, Ph.D. Department of Developmental and Cell Biology Center for Biomedical Engineering Institute for Genomics and Bioinformatics University of California, Irvine. DNA-binding AGGTCA n

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The Steroid and Xenobiotic Receptor SXR: A Key Regulator of Drug and Xenobiotic Metabolism

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  1. The Steroid and Xenobiotic Receptor SXR: A Key Regulator of Drug and Xenobiotic Metabolism Bruce Blumberg, Ph.D. • Department of Developmental and Cell Biology • Center for Biomedical Engineering • Institute for Genomics and Bioinformatics • University of California, Irvine

  2. DNA-binding AGGTCA n n n D I E Nuclear Receptors - A Large Family of Ligand Modulated Transcription Factors ligand-bindingtranscriptional activationdimerization half-site recognitionhalf-site spacingdimerization

  3. Nuclear Receptors • Bind to specific DNA targets - hormone response elements • Most activate transcription upon ligand binding • Some are constitutive • A few are deactivated by ligand binding • Ligands are small lipophilic molecules that freely enter cells • Diffuse from source • Penetrate to a target • Typically respond to low levels of hormone ~3 ppb (10-8 M) • Regulation of levels • Environmental agents

  4. Mithridates VI Eupator The Royal Toxicologist (120-63 BC) King of Pontus aka Mithridates the Great

  5. Long Standing Questions • Mithridatum - generalized tolerance to poison • Adaptive hepatic response (Hans Selye) • Exposure to certain “catatoxic” chemicals elicits protection against later exposure to others • Apparently mediated via CYP upregulation • What is the mechanism?

  6. SXR and Close Relatives

  7. GAL-SXR Responds to Many Steroids

  8. The Steroid Sensor Hypothesis • Removal of bioactive steroids and xenobiotics is required for physiologic homeostasis • Steroid production is regulated, why not catabolism? • Hundreds of steroid metabolites make it unreasonable to have individual regulation • Hypothesize a broad specificity sensor that monitors steroid levels and regulates the expression of degradative enzymes, e.G. P450s • Broad specificity probably necessitates low-affinity

  9. Predictions and Requirements of the Model • Sensor should be expressed in tissues that catabolize steroids and xenobiotics • Catabolic enzymes should be targets for the sensor • Compounds known to induce catabolic enzymes should activate the sensor • Partially metabolized (reduced) steroids should activate sensor

  10. Expression of SXR mRNA a l l x e u e n d t i r t e o s m c e s t a h l a l n t s c a e a d e i y n l u a i n r n l c s e e t o l n c r i e e m m s r g r a i c t n a n r r y e s a u y n a o d d m d e a r l h v i t e h u m a i b a k s s t h p p t t l l 9 . 5 9 . 0 7 . 5 6 . 5 5 . 2 4 . 4 3 . 5 2 . 4 1 . 4

  11. SXR DNA-binding Specificity βD βD R - 2 βD R - 3 R - 4 βD R - 5 M M T V T R E p S X R a D R - 1 0 1 2 3 4 5 6 7 1 0 1 5 h R X R

  12. Candidate SXR Response Elements in Genes Encoding Steroid Degradative Enzymes

  13. SXR Activates Transcription Through bDR Elements 1 6 s o l v e n t c o r t i c o s t e r o n e 1 4 p r e g n e n o l o n e D H E A 1 2 t e s t o s t e r o n e n o p r o g e s t e r o n e i 1 0 t c P C N u d e s t r a d i o l n 8 I c o r t i s o l d l c o r t i s o n e o 6 F 4 2 0 βD R - 4 βD R - 5 βD D R - 1 D R - 2 R - 3 D R - 4 T R E p t k - l u c R e p o r t e r C o n s t r u c t

  14. 1 1 6 1 4 1 2 n o i t c 1 0 u d n I 8 d l o F 6 4 2 0 e n n l n n e e n n n A e l l i i i o i i n e o o t l n n e c n r c e f r e o e o i u t i z y o i o t t p r c e s x n p t q r s s d i m r t e i i c e o e i e e l r d m t e n a t a o u b o s e m l o m e u l l a r d f i p o o r f t a q h i g u i p s c t n n s t r i o l y o y t o y r l c r c r c h i e o y t p c e s c i d Classes of SXR Activators 8 x e n o b i o t i c s s t e r o i d s p h y t o e s t r o g e n s t n e v l o s

  15. SXR Senses Total Steroid Concentration 4 m 1 0 M i n d i v i d u a l s t e r o i d s m 1 0 0 M t o t a l i n c o c k t a i l 3 n o i t c u d n 2 I d l o F 1 0 e e e e e e l A t l T i n o n n n N n n n E a H s e o o o o o o t C i H l r v r r k s s t D o r i l P e e e c a D t o o n t t t r o h s s s s c o e t c e e o c e n g d g g m l o o e a r a r r p p x p e H d O - 7 1

  16. Predictions and Requirements of the Model • Sensor should be expressed in tissues that catabolize steroids and xenobiotics • Expressed in liver, small and large intestine • Catabolic enzymes should be targets for the sensor • CYP genes are known targets of SXR, in vivo • Compounds known to induce catabolic enzymes should activate the sensor • Majority of known CYP3 inducers activate SXR • Partially metabolized (reduced) steroids should activate sensor • Mixture of steroids, each at concentrations below that required to activate SXR, will collectively activate SXR-mediated gene expression

  17. CYP3A4 and Human Steroid Metabolism • Steroid levels are tightly regulated. Increased catabolism will lead to ACTH release and upregulated adrenal synthesis • Observation is elevated ACTH, slightly increased circulating steroids • Decreased circulating steroid metabolites • Increased catabolism will be reflected by urinary metabolites • Large increases in urinary steroids caused by rifampicin therapy have led to misdiagnosis of Cushing’s syndrome • VP16 SXR transgenic mice have drastically elevated urinary steroid metabolite levels • Induction of CYP3A4 should lead to decreases in administered steroid levels • Steroid crisis in Addison’s patients on rifampicin and oral steroids • Pregnancy in rifampicin-treated patients on oral contraceptives

  18. O C H O H 2 O N a t u r a l a n d S y n t h e t i c S t e r o i d s Many CYP3A4 Inducers Are SXR Activators O s p i r o n o l a c t o n e H O c o r t i c o s t e r o n e O H ( C 2 1 ) O N O O Steroid receptorantagonists S t e r o i d r e c e p t o r t a m o x i f e n a g o n i s t s e s t r a d i o l ( C 1 8 ) H O O H O C N t e s t o s t e r o n e P C N ( C 1 9 ) O H O p h y t o e s t r o g e n s ( i s o f l a v o n e s ) x i o e n o b t i c d r u g s H O O O H O H O C C O H O O c o u m e s t r o l 3 O H O H O O H C H O N H 3 H O O N N N O O H O r i f a m p i c i n e q u o l clotrimazole O H

  19. Pharmacology of Mouse and Human SXR 3 0 3 0 h E R h S X R 2 5 2 5 n o 2 0 2 0 i t c u d 1 5 1 5 n I d l 1 0 1 0 o F 5 5 3 0 3 0 r E R m P X R 2 5 2 5 n o 2 0 2 0 i t c u d 1 5 1 5 n I d l 1 0 1 0 o F 5 5

  20. Model Systems • Central tenet of model system is parallel biochemistry and endocrinology • Toxicology: effects on animals predict effects on humans • Nuclear receptors behave virtually identically across species • Different pharmacology of SXR and PXR suggests that there are important differences in metabolism • These differences may be highly relevant for toxicology, drug interactions and endocrine disruption • Cross-species extrapolation must account for differences in response of xenobiotic sensors • SXR • CAR

  21. Drug Interactions • CYP3A4 is the primary steroid and xenobiotic metabolizing enzyme • Drugs interactions arise from: • Induction or inhibition of CYP3A4 expression • rifampicin and oral steroids • St John’s Wort and many drugs • Modulation of CYP3A4 enzyme activity • macrolide antibiotics (e.g. erythromycin) and many drugs (e.g. Seldane) • Activated SXR mediates induction of CYP3A4 • SXR activation is a direct molecular test for potential drug interactions • Pharmacological differences between inducibility of rodent and human CYP3 genes explained by receptor pharmacology • Differences suggest rodents may not be an appropriate model for human drug interactions • Rabbit CYP3A induction closely parallels human • Mouse now exists that expresses human SXR instead of mouse gene

  22. SXR and Endocrine Disruption • SXR regulates the P450-mediated breakdown of ingested steroids and xenobiotics • Activation of SXR may predict effects of suspected EDC • SXR activators may be detoxified by CYP3A action and not a human risk • But activators may also be toxified by CYP3A action, increasing the risk. • EDC may have no effect on SXR and therefore more likely to act on other receptors, e.g. ER • SXR is a molecular assay for potential activity of EDCs • Different pharmacology of SXR and PXR suggests that differences in metabolism may exist and be relevant for risk assessment

  23. Approach to Studying EDC Metabolism • Test potential for metabolism by investigating SXR activation by a panel of known and candidate EDCs • Pesticides: DDT, DDE, methoxychlor, endosulfan, dieldrin, alachlor, chlordane, transnonachlor, chlorpyrifos, kepone • Plasticizers: bisphenol A, phthalates • PCBs: e.g. 184, 196 • Alkylphenols: 4-nonylphenol • xenobiotics: thalidomide, dichlorophenol, triclosan, BHA, BHT • Extend analysis to SXRs from other model organisms of interest • fish - e.g., zebrafish, medaka, fathead minnow • reptiles - alligator, sea turtle • birds - Japanese quail, zebra finch • amphibians - Xenopus, Rana • mammals - monkey, canine • Investigate actual metabolism in animal models • model organisms including humanized mouse • wild populations

  24. C l C l C l C l C l C l C l C l C l C l C l O H H O C l C l C l C l b i s p h e n o l A P C B 1 8 4 P C B 1 9 6 C l C l C l C l C l C l S C H O 2 5 P O C l C H O C l C l 2 5 C l C l D D E O D D T C h l o r p y r i f o s C l O H O O n o n y l p h e n o l O b i s - p h t h a l a t e EDCs can activate SXR

  25. SXR and its mammalian homologs 3 5 1 0 7 1 4 1 4 3 4 1 human D N A L I G A N D 1 3 8 1 3 8 1 0 4 4 3 1 mouse 9 5 % 7 3 % 1 3 8 1 3 8 1 0 4 4 3 1 rat 7 6 % 9 5 % 8 4 1 1 8 4 1 1 1 1 8 rabbit 8 4 % 9 4 %

  26. EDC activation of SXR Ligand human mouse rat rabbit 4-nonylphenol + + + - bisphthalate + + + + alachlor + + + - chlordane + + + + transnonachlor + + + + chlorpyrifos + + + + DDT + + + + DDE + + + - dieldrin + + + + endosulfan + + + + kepone + - + + methoxychlor + + + + PCB184 - + + - PCB196 - + + - genistein + - - + bisphenol A + - - + rifampicin + - - + PCN - + + +

  27. Conclusions and prospects SXR and EDC • Many compounds activate SXR from all four species, suggesting metabolism is the same • SXR is a molecular assay for interspecies variations in metabolism • There are significant differences that suggest metabolism of certain compounds of great interest is different • bisphenol A • phytoestrogens • PCBs • Animal models used for extrapolation of toxicology and drug interaction testing to humans must be validated for each compound. • Is the activation profile of SXR, and by implication metabolism, the same or different? • Are the compounds in fact metabolized? • What is the nature and fate of the metabolites?

  28. O H H O b i s p h e n o l A SXR – A steroid and Xenobiotic Sensor CYP3A mdr1 SXR Target genes mrp1

  29. SXR - A Steroid and Xenobiotic Sensor • SXR has properties predicted for a steroid and xenobiotic sensor • Expression • Targets • Activators Endogenous and dietary steroids Xenobiotic drugs Environmental toxicants • Expected responses from induction Increased ACTH Increased urinary metabolites Increased circulating steroids but decreased metabolites • SXR is an important molecular test for potential species-specific metabolism of drugs and xenobiotics • Understanding SXR regulation and identifying target genes is an important goal to aid in understanding the xenobiotic response • SXR must be considered when working with model organisms

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