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Cracking The Metabolism Cocaine

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Title: Cracking The Metabolism Cocaine Author: Gladys DeLeon Last modified by: Merrill Created Date: 11/20/2002 1:57:36 AM Document presentation format – PowerPoint PPT presentation

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Title: Cracking The Metabolism Cocaine


1
Shannon Hoon
Chris Farley
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 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

9
Word from our sponsors
10
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11
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12
Coke in the 20th Century
13
1 Kg of Cocaine
14
Tools of Trade
15
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16
How does Cocaine affect the brain?
17
The Dopamine Hypothesis
F. I. Carroll et al, Journal of Medical Chemistry
42, 2721-36 (1999)
18
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)
19
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20
What are the effects of cocaine on the mind?
  • Thinking and feeling brain
  • Racing thoughts
  • Reward
  • Felling of well being

21
!! EUPHORIA !!
  • Negative effects
  • Increased heart rate
  • Hyperthermia
  • Dilated pupils
  • Diaphoresis
  • Irritability
  • Decrease appetite
  • Sleeplessness
  • Depression
  • Over dose respiratory arrest
  • Myocardial infarction
  • Arrhythmia
  • Seizures

22
Cocaine Dependency Three classical clinical
characteristics that define addiction
  1. Psychological dependence
  2. Tolerance
  3. Physical withdrawal

23
1. Psychological dependence
  • Compulsive drug seeking behavior despite negative
    consequences
  • Drug becomes the highest priority in the
    individuals life

24
2. Tolerance
  • As time goes on, more and more of the drug is
    needed to produce the same high

25
3. Physical withdrawal
  • Constellation signs and symptoms that occur
    following cessation of drug use

26
Emergency Situation
  • COCAINE OVERDOSE

27
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

28
Cocaine Esterase (cocE) is an attractive
candidate for rapid cocaine detoxification in an
emergency setting.
29
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

30
Hydrolysis of cocaine by cocE
D. W. Landry et al, Science, 259, 1899-1901
31
Structural and Biochemical Characterization of
Cocaine Esterase (cocE)
32
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

33
What is the structure of cocE?
N. A. Larsen et al., Nature Structural Biology
9, 17-21 (2002)
34
Domain Structure and Association
35
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)
36
REVIEW
N. A. Larsen et al., Nature Structural Biology
9, 17-21 (2002)
37
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)
38
REVIEW
N. A. Larsen et al., Nature Structural Biology
9, 17-21 (2002)
39
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)
40
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)
41
Proposed Mechanism for acyl intermediate
hydrolysis
42
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

43
J.M. Turner et al. Biochemistry, 41, 12297-12307
( 2002)
44
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.

45
http//cocaine.org/cokehell.html
46
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
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