Lesch-Nyhan Syndrome: Curing the incurable disease

1. Lesch-Nyhan Syndrome: Curing the incurable disease Allison Striano

2. Lesch-Nyhan Syndrome: The Basics What is it? Deficiency in enzyme hypoxanthine-guanine phosphoribosyltransferase(HGPRT) Error in purine metabolism Inheritable chromosomal mutation on X-chromosome How is it transmitted? Familial transmission is possible; X-linked recessive Mutation can arise spontaneously How common is LNS? Very rare in males; virtually absent in females Is LNS curable? NO Treatments do exist Short life expectancy kidney failure muscle loss

3. Lesch-Nyhan Syndrome: The Basics What are the Symptoms of LNS? Uric Acid Overproduction -Gouty Arthritis -Kidney Stones/Renal Failure Neurological Disability -Motor Disability -Involuntary Writhing Behavioral Abnormality -Self Mutilation -Lack of Speech How is it diagnosed? Physical and behavioral abnormalities present from infancy Biochemical confirmation- Azathioprine testing Drug interacts with HGPRT to lower levels of uric acid in urine LNS patients will not demonstrate this change Pre-natal testing available How is it recognized? Characteristic Behavior Self-mutilation Biochemical Distinction HGPRT Deficiency

4. GOUT: The Disease of Kings What is gout? Accumulation of uric acid crystals- especially in joints Who first documented instances of gout? Egyptians (2640BCE) -> Hippocrates (400sBCE) -> van Leeuwenhoek (1600sCE) What lifestyles exaggerate instances of gout? Diets rich in purines (i.e. meats, fish) What is the relation between gout and LNS? Gout is a symptom of LNS resulting from the absence of HGPRT ~1960 LNS was ‘discovered’ as a specific enzyme deficiency associated with gout What are the proposed treatments for gout? Change in diet – reduction of purine intake drug therapy – e.g. allopurinol

5. Lesch-Nyhan Syndrome: The Patients "Diego does not have his teeth anymore. He has his arms in splints. Everything around him is padded. He underwent plastic surgery on his nose. He wears a glove to defend one hand from the other. Nevertheless I can say that he has never lost his will to live and his hope to recover. Almost everyday he says: - If I could walk...I would do...I would go...I would study …” http://www.lesch-nyhan.eu/en/experiences.html

6. Hypoxanthine-Guanine PhosphoribosylTransferase (HGPRT) • Biochemical Role: • Purine Salvage Pathway • -recycle hypoxanthine and guanine nucleotides back to DNA synthesis • -relief from de novo synthesis • * failure of this system results in unused nucleotides • purines are broken down into uric acid • Deficiency in LNS • Two categories of severity: • <1% normal function • 1-50% normal function • *Note: this group of patients does not exhibit the • characteristic LNS nervous system abnormalities • Genetic Cause • -mutation ‘hot spots’ where Arg -> STOP • --> unstable, truncated HGPRT proteins

7. Hypoxanthine-Guanine PhosphoribosylTransferase (HGPRT) Structure Homotetrameric Catalytic Activity -Hypoxanthine/Guanine + PRPP --> IMP/GMP Catalytic Loop Blocks active site from solvent Prevents hydrolysis of PRPP once bound Order of Substrate Binding PRPP Base

8. Hypoxanthine-Guanine PhosphoribosylTransferase (HGPRT) PRPP Binding H-bonding with Serine 103 Displacement of catalytic loop from active site H-bonds with Aspartate 137, Threonine 138, Glycine 138, Serine 141 Disruption of interactions of free enzyme

9. Hypoxanthine-Guanine PhosphoribosylTransferase (HGPRT) Purine Binding -Binds to Lysine 165 Disturbs H-bond between K165 & K185 of free HGPRT -Specificity of K165 for hypoxanthine and guanine -Pi bonds to Phe 186 Catalysis of Transfer -Propogatedwhen H+ removed from the purine ring

10. Lesch-Nyhan Syndrome Treatments Syndrome Hallmarks -Uric Acid Overproduction -Neurological Disability -Behavior Problems Gouty Arthritis Kidney Stones Renal Failure Generous hydration Allopurinol treatments XanthineOxidoreductase (XOR) Motor Disability Involuntary Writhing DOPA Decarboxylase (DDC) Self Mutilation Lack of Speech Impulsive Aggressiveness Physical Restraints Carbidopa treatments

11. Lesch-Nyhan Syndrome Treatments: ALLOPURINOL Target Enzyme -XanthineOxidoreductase (XOR) Desired Effect -Decrease production of uric acid -Reduce gouty arthritis induced pain *effective, common, but must be taken frequently *no effect on neurology or behavior ** Method of Inhibition -inhibit XOR from catalyzing catabolism of hypoxanthine and guanine to uric acid -Allopurinol is a hypoxanthine analog ** Note: both xanthineoxidoreductaseand xanthinedehydrogenasewill be examined in order to illustrate the binding of desired substrates. The two are overwhelmingly similar in binding, catalysis, and inhibition by drugs that they are considered highly identical. (Dietzel, et al.)

12. XanthineDehydrogenase (XOR) Biochemical Role: Purine Catabolism -breakdown of un-recycled hypoxanthine & guanine for excretion -exacerbated by absence of HGPRT Structure -heterotetrameric -each of the 4 monomers has 2 subunits -each monomer has one α chain and one β chain *Note: XOR bound to Hypoxanthine

13. XanthineDehydrogenase (XDH) Reaction Specifics -Glu 730 deprotonation of Molybdenum -Molybdenum nucleophilicattack on hypoxanthine -Glu 232 binding & stability H-bonds hypoxanthine -Arg310 lowers Ea through stabilization via electrostatic interactions H-bonds hypoxanthine -Thr460 positioning H-bonds hypoxanthine -Phe 459 & 344 Van derWaals interactions with hypoxanthine form opposing walls of active site

14. ALLOPURIONL Inhibition of XanthineOxidoreductase (XOR) Inhibtion Specifics -upon binding, allopurinol is hydroxylated to form oxypurinol -oxypurinol: competitive inhibition -covalently bound to molybdenum -H-bonding with residues Glu 802 Arg 880 Glu 1261 -time dependence Spontaneous oxidation of molybdenum cofactor -->inhibited XOR reactivated t1/2 = 300 minutes Promising New Inhibitors -longer-lasting inhibitory complexes

15. Biochemical Explanations for Behavioral Abnormalities in LNS How does HGPRT deficiency affect nervous system? Link: Dopaminergic Dysfunction 70-90% redution of dopamine in LNS patients Dopamine -regulate movement -inhibit tendency to make unwanted decisions

16. Biochemical Explanations for Behavioral Abnormalities in LNS What is the said dopaminergic dysfunction? Testing with fluorodopa (F-18) revealed: -F-18 activity low in dopaminergic regions of LN brain -Deficit of dopamine is stable, not degenerative Testing of TH enzyme revealed: -decreased TH activity -no loss of dopaminergic fibers, but reduction of projections into dopaminergic regions -dopaminergic consequences proposed to be secondary effect of HGPRT deficit Testing of Norepinephrine levels revealed: -low levels in LNS patients -low activity of DβH (due to substrate limitation)

17. Lesch-Nyhan Syndrome Treatments: CARBIDOPA Target Enzyme -Dopamine Decarboxylase (DDC) Desired Effect -Decrease PNS [dopamine] -Increase CNS [L-dopa] --> increase CNS [dopamine] -Bypass PNS production/storage of dopamine dopaminergic vesicles in which to store the NT are limited in LNS Method of Inhibition -Inhibit DDC in PNS -Increased levels of L-DOPA cross BBB -Increased L-DOPA in brain --> Dopamine in brain -carbiDOPA is an analog of L-DOPA

18. CarbiDOPA inhibition of Dopamine Decarboxylase (DDC) Biochemical Role: Dopamine Formation -decarboxylation of L-DOPA Structure -dimeric -each of the 2 monomers has 3 domains PLP binding site C-terminus N-terminus Function of PLP -co-enzymatic prosthetic group -assists catalytic function

19. CarbiDOPA inhibition of Dopamine Decarboxylase (DDC) PLP Binding -H-bonding with Lys 303 - Salt bridge formation with Asp 271 Active Site -Ile101 & Phe103 Van derWaals with carbiDOPA

20. CarbiDOPA inhibition of Dopamine Decarboxylase (DDC) Active Site cont’d.. -Thr 82 bottom of binding pocket H-bonding with carbiDOPA -His 192 H-bonding with carbiDOPA -carbiDOPA sits deep within site aids inhibitory activity Flexible Loop -residues 328-339 -at dimer interface -conserved residues Tyr 332 & 334(Lys or Arg)

21. Avenues of Possible Future Research LNS is incurable as we know it -Further investigation of potential drugs to ameliorate neurological and behavioral symptoms -Closer examination for links between HGPRT shortage and dopaminergic deficiency -> is HGPRT stored in dopamine containing fibers? Wherever the search for a cure leads, may the scientists driving the research not let the science of Lesch-Nyhan Syndrome overshadow the faces Of the disease.

22. Resources Breese, George R., et al. “Behavioral Differences Between Neonatal and Adult 6-hydroxydopamine-treated Rates to Dopamine Agonists: Relevance to Neurological Symptoms in Clinical Syndromes with Reduced Brain Dopamine.” Journal of Pharmacology and Experimental Therapeutics. 231.2 (1984):343-354. J Pharmacol Exp Ther. 31 March 2010. http://jpet.aspetjournals.org/content/231/2/343.long Burkhard, P., et al. “Structural Insight into Parkinson’s Disease Treatment from Drug-inhibited DOPA Decarboxylase.” Natural Structural Biology. 8:11 (2001): 963-967. Nature Structural and Molecular Biology. 22 March 2010. http://www.nature.com/nsmb/journal/v8/n11/abs/nsb1101-963.html Dietzel, Uwe, et al. “Mechanism of Substrate and Inhibitor Binding of RhodobactercapsulatusXanthineDehydrogenase.” J BiolChem. 284.13 (2009): 8768-8776. PubmedCentral. 1 April 2010. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659235/ Ernst, Monique, et al. “PresynapticDopaminergic Deficits in Lesch-Nyhan Syndrome.” New England Journal of Medicine. 334.24 (1996): 1568-1572. NEJM. 31 March 2010. http://content.nejm.org/cgi/content/full/334/24/1568 Harris, James C. “Social Neuroscience, Empathy, Brian Integration, and Neurodevelopment Disorders.” Physiology and Behavior. 79.3 (2003): 525-531. ScienceDirect. 1 April 2010. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0P-49569GX1&_user=10&_coverDate=08%2F31%2F2003&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1280985662&_rerunOrigin=scholar.google&_acct=C000050221&_version=1&_urlVersion=0&_user id=10&md5=0a3343f80e3ed9b126da86772dfb32b4 Jinnah, H. A. "Lesch-NyhanSyndrome.“ www.lesch-nyhan.org. Lesch-Nyhan Disease International Study Group, 2 Oct. 2010. Web. 22 Mar. 2010. <http://www.lesch-nyhan.org/index.php?section=&topic=Welcome&subtopic=>. Jinnah, H.A., et al. “Dopamine Deficiency in a Genetic Mouse Model of Lesch-Nyhan Disease.” The Journal of Neuroscience. 14.3 (1994): 1164-1175. The Journal of Neuroscience. 31 March 2010. http://www.jneurosci.org/cgi/reprint/14/3/1164

23. Resources cont’d.. Keough, Dianne T., et al. “The Crystal Structure of Free Human Hypoxanthine-Guanine Phosphoribosyltransferase Reveals Extensive Conformational Plasticity Throughout the Catalytic Cycle.” Journal of Molecular Biology. 351.1 (2005): 170-181. ScienceDirect. 1 April 2010. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-4GD4PS9- 2&_user=10&_coverDate=08%2F05%2F2005&_rdoc=1&_fmt= high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221 &_version=1&_urlVersion=0&_userid=10&md5=f79ab6ff953f71b5d945d dcfb4edff93  Lake, Raymond C. and Ziegler, Michael G. “Lesch-Nyhan Syndrome: Low Dopamine-B-Hydroxylase Activity and Diminished Sympathetic Response to Stress and Posture.” Science. 196.4292 (1977): 905-906. Jstor. 31 March 2010. http://www.jstor.org/pss/1744110 Nuki, George and Simkin, Peter A. “A Consise History of Gout and Hyperuricemia and Their Treatment.” Arthritis Research and Therapy. 8.1 (2006). Biomedcentral. 1 April 2010. http://www.biomedcentral.com/content/pdf/ar1906.pdf Nyhan, William L. “Lesch-Nyhan Disease.” Journal of the History of the Neurosciences. 14.1(2005): 1-10. Informaworld. 31 March 2010. http://www.informaworld.com/smpp/content~content=a713734366&db=all Nyhan, William L., et al. “Lesch-Nyhan.” www.Lesch-nyhan.eu. 2 June 2009. Web. 4 April 2010. http://www.lesch- nyhan.eu/en/experiences.html Okamoto, Ken, et al. “Mechanism of Inhibition of XanthineOxidoreductase by Allopurinol: Crystal Structure of Reduced Bovine Milk XanthineOxidoreductase Bound with Oxipurinol.” Nucleosides, Nucleotides, and Nucleic Acids. 27.6 (2008): 888-893. http://pdfserve.informaworld.com/584840_731209523_794699692.pdf Shi, Wuxian, et al. “The 2.0 A Structure of Human Hypoxanthine-guanine phosphoribosyltransferase in complex with a transition-state analog inhibitor.” Natural Structural Biology. 6.6 (1999): 588-593. Nature Structural and Molecular Biology. 22 March 2010. http://www.nature.com/nsmb/journal/v6/n6/abs/nsb0699_588.html