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Shannon Hoon

Chris Farley. Shannon Hoon. River Phoenix. What did they have in common?. They were all cocaine addicts and they died of cocaine overdose!. COCAINE ESTERASE (cocE) The story of the ‘dope fiend’ Rhodococcus bacteria. By: Gladys de Leon Department of Chemistry and Biochemistry

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Shannon Hoon

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  1. Chris Farley Shannon Hoon River Phoenix

  2. What did they have in common? They were all cocaine addicts and they died of cocaine overdose!

  3. COCAINE ESTERASE (cocE)The story of the ‘dope fiend’ Rhodococcus bacteria. By: Gladys de Leon Department of Chemistry and Biochemistry University of Guelph

  4. Overview • general description of Cocaine • Brain mechanism under influence of cocaine • Effects and dependency • CocE overview • Structural and biochemical analysis of cocE • Concluding remarks

  5. Structure of cocaine

  6. What is cocaine? • C, coke, snow, nose, candy, crack • powerful central nervous system (CNS) stimulant • provides intense feelings of pleasure • Derived from leaves of South American coca bush (erythroxylon coca and erythroxylon novogranatense) • Addictive and can cause death

  7. Erythroxylon coca • cultivated in Bolivia, Peru and Ecuador • grows to a height of up to eight feet • rich in vitamins, protein, calcium, iron and fibre • cocaine content of the leaves ranges from O.1% to 0.9% http://cocaine.org/cokleaf.html

  8. History of cocaine • Used by man since 800 A.D. • Cultivated by the Incan • 1500s - Coca chewing of the South American Indians • 1860s - Isolated from coca leaf by Albert Nieman • 1880s - Sigmund Freud’s Uber Coca • 1886 - Coca Cola a non-alcoholic medicinal tonic from Atlanta Georgia • 1890s – Cocaine containing medicine • 1922 – Narcotic Drug Import and Export Act restricted coca imports

  9. Word from our sponsors:

  10. Coke in the 20th Century

  11. 1 Kg of Cocaine

  12. Tools of Trade

  13. How does Cocaine affect the brain?

  14. The Dopamine Hypothesis F. I. Carroll et al, Journal of Medical Chemistry42, 2721-36 (1999)

  15. The brain NA – nucleus accumbens VT – ventral tegmental FC – frontal cortex CB – cerebeller PT – putamen CN- caudate nucleus F. I. Carroll et al, Journal of Medical Chemistry42, 2721-36 (1999)

  16. What are the effects of cocaine on the mind? • Thinking and feeling brain • Racing thoughts • Reward • Felling of well being

  17. !! EUPHORIA !! Negative effects: Increased heart rate Hyperthermia Dilated pupils Diaphoresis Irritability Decrease appetite Sleeplessness Depression Over dose: respiratory arrest Myocardial infarction Arrhythmia Seizures

  18. Cocaine Dependency : Three classical clinical characteristics that define addiction • Psychological dependence • Tolerance • Physical withdrawal

  19. 1. Psychological dependence • Compulsive drug seeking behavior despite negative consequences • Drug becomes the highest priority in the individual’s life

  20. 2. Tolerance • As time goes on, more and more of the drug is needed to produce the same high

  21. 3. Physical withdrawal • Constellation signs and symptoms that occur following cessation of drug use

  22. Emergency Situation COCAINE OVERDOSE

  23. So what is an ideal cocaine antidote in an emergency setting? • High catalytic proficiency • Lack of observable product inhibition • Ability to hydrolyze both cocaine and cocaethylene

  24. Cocaine Esterase (cocE) is an attractive candidate for rapid cocaine detoxification in an emergency setting.

  25. Overview of cocE: • Identified in the bacteria Rhodococcus sp. Strain MB1 (Rhodococcus thrives in the rhizosphere soil of the cocaine-producing plant Erythroxylum coca) • Cocaine degrading enzyme • First enzyme in the metabolic pathway leading to cocaine catabolism • Inducible and specific for cocaine

  26. Hydrolysis of cocaine by cocE D. W. Landry et al, Science, 259, 1899-1901

  27. Structural and Biochemical Characterization of Cocaine Esterase (cocE)

  28. Structural Overview of cocE • Belongs to the / hydrolase superfamily • 574 amino acids • ~65,000 Daltons • 3 domains – (DOM 1, DOM 2, DOM 3) • 30 % -sheet • 23 % -helix

  29. What is the structure of cocE? N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  30. Domain Structure and Association

  31. Domain 1: / sandwich • Residues 1-144 and residues 241-354 • Hydrolase fold (repeating --  motifs) • Central -sheet is predominantly parallel • Contains the active site His-287 N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  32. REVIEW N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  33. Domain 2: The -helical domain • 95 amino acids • Compose of 7 helices • Inserted between 6 and 7 of Domain 1 • Five helix core – helices H2-H6 N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  34. REVIEW N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  35. Domain 3: jelly-roll -barrel • 221 amino acids • Overall fold has a jelly-roll-like topology ( i.e. Hgg-Haemagglutinin) • Mostly -structure • -barrel-like core • Strands connected by 6 cross over loops • Important role in the overall tertiary structure N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  36. Active site of cocE • Located in a cleft formed at the interface of the three domains • Catalytic triad – Ser 117, Asp 259 and His 287 • Oxyanion hole – backbone amide of Y118 and Y44 N. A. Larsen et al.,Nature Structural Biology9, 17-21 (2002)

  37. Proposed Mechanism for acyl intermediate hydrolysis

  38. Biochemical Characterization and Structural Analysis of cocE • cocE hydrolyzes cocaine faster than any other reported cocaine esterase • Follows Michaelis-Menten kinetics with kcat= 7.8 s-1 and Km 640 nm. • Similar rate for cocaethylene • Validated the proposed mechanism • Reveal contributions of active site towards substrate recognition and catalysis

  39. J.M. Turner et al. Biochemistry, 41, 12297-12307 ( 2002)

  40. In conclusion: cocE enzyme itself has therapeutic potential as an enzyme-based treatment for cocaine overdose, furthermore, the crystal structure of the bacterial cocaine esterase provides a basis for further antibody engineering.

  41. http://cocaine.org/cokehell.html

  42. References • W.F. Borson and T.D. Hurley, Nat Struct Biol.9, 4-5 (2002). • N.A. Larsen et al., Nat Struct Biol. 9, 17-21 (2002). • J.M. Turner et al., Biochemistry, 41, 12297-12307 (2002). • Larsen, N. A., Zhou, B., Heine, A., Wirsching, P., Janda, K. D., and Wilson, I. A. J. Mol. Biol. 311, 9-15 (2001) • F.I. Carroll, L.L. Howell, and M.J. Kuhar, J Med Chem. 42, 2721-2736 (1999). • L.L. Howell and K.M. Wilcox, Perspectives in Pharmacology, 298, 1- 6 (2000). • A. M. Washton and M.S. Gold. “Cocaine: A clinician’s handbook”, The Guilford Press, New York. 1987. Pp. 73-79. • D.W. Landry et al., Science, 259, 1899- 1901 (1993). • P.H. Earley. “The Cocaine Recovery Book”, Sage Publications, London. 1991, pp.9-35. • L.M. Kamendulis et al., Jour. Phar. Exp. Ther., 279, 713-717 (1996). • M.R. Brzezinski et al., Drug Metab Dispos., 9, 1089-1096 (1997). • C. E. Mattes et al., Addict Biol., 2, 171-188 (1998). • http://www.cocaine.org/cokeleaf.html • http://www.hc-sc.gc.ca/hppb/alcohol-otherdrugs/pube/straight/stimulants.htm • http://www.a1b2c3.com/drugs/coc03.htm

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