Cracking The Metabolism Cocaine

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Shannon Hoon
Chris Farley
River Phoenix
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What did they have in common?

• They were all cocaine addicts and they died of
  cocaine overdose!

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COCAINE ESTERASE (cocE)The story of the dope
fiend Rhodococcus bacteria.

• By Gladys de Leon
• Department of Chemistry and Biochemistry
• University of Guelph

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Overview

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

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Structure of cocaine
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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

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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
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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 Freuds 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

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Coke in the 20th Century
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1 Kg of Cocaine
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Tools of Trade
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How does Cocaine affect the brain?
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The Dopamine Hypothesis
F. I. Carroll et al, Journal of Medical Chemistry
42, 2721-36 (1999)
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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 Chemistry
42, 2721-36 (1999)
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What are the effects of cocaine on the mind?

• Thinking and feeling brain
• Racing thoughts
• Reward
• Felling of well being

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!! EUPHORIA !!

• Negative effects
• Increased heart rate
• Hyperthermia
• Dilated pupils
• Diaphoresis
• Irritability
• Decrease appetite
• Sleeplessness
• Depression
• Over dose respiratory arrest
• Myocardial infarction
• Arrhythmia
• Seizures

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Cocaine Dependency Three classical clinical
characteristics that define addiction

1. Psychological dependence
2. Tolerance
3. Physical withdrawal

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1. Psychological dependence

• Compulsive drug seeking behavior despite negative
  consequences
• Drug becomes the highest priority in the
  individuals life

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2. Tolerance

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

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3. Physical withdrawal

• Constellation signs and symptoms that occur
  following cessation of drug use

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Emergency Situation

• COCAINE OVERDOSE

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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

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

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Hydrolysis of cocaine by cocE
D. W. Landry et al, Science, 259, 1899-1901
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Structural and Biochemical Characterization of
Cocaine Esterase (cocE)
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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

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What is the structure of cocE?
N. A. Larsen et al., Nature Structural Biology
9, 17-21 (2002)
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Domain Structure and Association
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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 Biology
9, 17-21 (2002)
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REVIEW
N. A. Larsen et al., Nature Structural Biology
9, 17-21 (2002)
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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 Biology
9, 17-21 (2002)
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REVIEW
N. A. Larsen et al., Nature Structural Biology
9, 17-21 (2002)
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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 Biology
9, 17-21 (2002)
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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 Biology
9, 17-21 (2002)
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Proposed Mechanism for acyl intermediate
hydrolysis
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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

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

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http//cocaine.org/cokehell.html
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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
  clinicians 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/pub
  e/straight/stimulants.htm
• http//www.a1b2c3.com/drugs/coc03.htm