This document provides an overview of butyrylcholinesterase (BChE), including its substrates, inhibitors, structure, mechanism of action, and therapeutic indications. It discusses the biochemistry and genetic variability of BChE, as well as its role in protecting against toxicities and disease. BChE preferentially hydrolyzes butyrylcholine and can also hydrolyze acetylcholine and various drugs like cocaine. Inherited BChE deficiencies have implications for responses to drugs like succinylcholine. Enhancing BChE activity may help treat cocaine abuse and toxicity.

1. Butyrylcholinesterase Overview:
Substrates, Inhibitors, Structure,
Mechanism, Therapeutic Indications
(BChE)
Luke Lightning

2. Outline
• Introduction/History
• Biochemistry
• Genetic Variability
• Mechanism and Structure
• Protection from Toxicities and Disease
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3. BChE Substrates
3

4. BChE Introduction
• Preferentially hydrolyzes butyrylcholine, but also hydrolyzes acetylcholine
– Function thought to be a scavenger of toxic molecules
• Serum BChE is synthesized in the liver and then secreted
– But also synthesized in the lungs, heart, and brain
• > 11 different isoforms
– > 60 isoforms of human P450
• Many different names
– Pseudo, plasma, serum, benzoyl, false, non-specific, or type II cholinesterase
– Acyl hydrolase or Acylcholine acylhydrolase
• Member of the type-b carboxylesterase/lipase family
– Inhibited by organophosphates
• type a’s hydrolyze OPs, type c’s do not interact)
4

5. History
• 1920’s
– Loewi in Austria
• Awarded Nobel Prize for work on cholinesterase, etc.
• 1940’s
– Mendel in Toronto, Canada
• “True cholinesterase”: present in red blood cells
• “pseudo-cholinesterase”: present in plasma
5

6. More History
• 1950’s:
– Patients with schizophrenia treated with electroshock
– Good therapeutic success, but also overstimulated some
patients’ skeletal muscles  broken bones
– Succinylcholine would be injected to avoid contractions
• Most times, paralyzing effect is over in a few minutes
– BChE rapidly hydrolyzes succinylcholine
• In some patients, the effect can last > 1 hour
• 1957:
– BChE activity of plasma from patients and their parents was
analyzed
– Genetic difference in BChE activity in humans was described
6

7. • 2 classes
Animal Cholinesterases
– Based on their substrate specificity and susceptibility to inhibitors
• Acetylcholinesterase (AChE)
– Hydrolyzes ACh faster than other choline esters
– Much less active on BCh
– Inhibited by excess substrate
• Butyrylcholinesterase (BChE)
– Preferentially hydrolyzes BCh
– Also hydrolyzes Ach (4X slower)
– Activated by excess substrate
– Hydrolyzes a large number of ester-containing compounds
• Species with higher BChE activity in plasma
– Human, monkeys, guinea pig, mice
• Species with higher AChE activity in plasma
– Rat, bovine, sheep
7

8. Cholinesterases
• Acetylcholinesterase
– Function is to hydrolyze acetylcholine released at the synaptic cleft and
neuromuscular junction in response to nerve action potential
– Loss of AChE activity  muscle paralysis, seizures, death
– Extremely efficient – rate approaches diffusion
– Membrane bound
• Butyrylcholinesterase
– Physiological role is unclear – no endogenous substrate
• Lipoprotein metabolism
• Myelin maintenance
• Cellular adhesion and neurogenesis
• Processing of amyloid precursor protein (implications for Alzheimer’s)
– Individuals with no BChE have no physiological abnormalities
– Plays an important role in pharmacology and toxicology
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9. Localization Differences
AChE BChE
Brain Plasma (relatively abundant, ~ 2-3 mg/L)
Muscle Liver
Erythrocyte membrane Smooth muscle
Nerve endings Intestinal mucosa
Spleen Pancreas
Lung Heart
Kidney
Lung
White matter of the brain
No carboxylesterases in human blood
Are present in high amounts in mice, rat, rabbit, horse, cat, and tiger blood
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10. Selective Inhibitors
AChE BChE
H
H N O
N O N
N O
N
O
N
Phenserine
Phenethyl-norcymserine
Huperzine A
Ethopromazine
BW284C51
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11. •
Inherited BChE Deficiency
Not clinically significant until plasma activity is reduced to 75% of normal
• No physical characteristics correlate with deficiency
• Most often recognized when respiratory paralysis unexpectedly persists for a
prolonged period after a dose of succinylcholine
• One of the oldest (50’s) and best-studied examples of a pharmacogenetic
condition
– Normally,
• 90-95% of an IV dose of succinylcholine is hydrolyzed before it reaches the neuromuscular
junction
• 5-10% of the dose  flaccid paralysis in 1 min
• Skeletal muscle returns to normal after 5 min
– If BChE deficient,
• Duration of paralytic effect can last 8 hours
• Most common in Europeans and rare in Asians
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12. Genetic Variants
• 96% of population is homozygous for normal genotype
• 4% of the population:
– Atypical (Dibucaine) resistant (most of the 4%) and F- resistant
• Measure % inhibition of enzyme activity in presence of dibucaine or F-
• WT is inhibited 80% and 60%, respectively
• Homozygous variants are inhibited only 20% and 36%, respectively
• Succinylcholine paralysis for > 1hr
– ~ 20 different “silent” genotypes identified  0-2% WT activity
• 1 in 100,000
• No functional BChE synthesized
• Succinylcholine paralysis for > 8 hours
– Cynthiana variant  increased amount of BCh (3X)
• Resistant to succinylcholine treatment
– Johannesburg variant  same amount of BChE, but increased activity
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13. •
Genetic Variability
Deficiencies are due to one or more inherited abnormal alleles
– Failure to produce normal amounts of the enzyme
– Production of BChE with altered structure and activity
• > 11 different variants – all have reduced activity compared to WT
mutation homozygous
– U “usual” WT
– A “atypical” Asp70Gly 1:3,000
“dibucaine resistant”
– K Kalow form Ala539Thr
– J Glu497Val 1:150,000
– F1 F- resistant Thr247Met
– F2 F- resistant Gly390Val
– H Val142Met
– S silent 129STOP 1:100,000
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14. Biochemical Features
• MW ~ 68,000 Da (602 AA’s)
– Human AChE is ~ 60,000 Da, human CE-1 is ~ 63,000 Da and P450s are ~ 50,000 Da
• 9 different glycosylation sites
• 3 internal disulfide bonds
– Cys65-Cys92, Cys252-Cys263, Cys400-Cys519
• Homotetramer
• Made up of 2 dimers linked by a disulfide bond (Cys571-Cys571)
• Catalytic Triad
– Ser198, Glu325, His438 (akin to hCEs)
• “Atypical” variant is identical in every way, except for one AA
– Reduced binding affinity (2X)  reduced activity
14

15. Interspecies Similarities
• Protein Sequence Identity (and Homology) with
Human BChE (~ 50 mg costs $350)
– Rabbit 91% (93%)
– Horse 90% (94%)
– Cat 87% (91%)
– Dog 86% (91%)
– Mouse 80% (87%)
– Rat 79% (87%)
– Chicken 71% (83%)
– Human AChE 53% (65%)
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16. Crystal Structure of BChE
• Comparison to AChE
– Catalytic triads of both are at the bottom of a 20 Å-deep
gorge
• Gorge of BChE is lined with hydrophobic residues instead of
aromatic ones
– Acyl binding pockets are different
• 2 Phe’s  Val, Leu  bulkier substrates can be accommodated
– Peripheral site
• At the outer rim of the gorges
• Proposed to be the initial binding site – attraction center for
substrates
– Anionic site
• Found half-way down the gorges
• In between the peripheral and acylation sites
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17. General Mechanism
BChE:
Ser198
Glu325
His438 oxyanion hole
ESTER
hydrolysis of acyl
enzyme complex
by water
ACID
Confidential 17

18. BChE Mechanism
ES1: substrate binds to PAS (Asp70)
ES2: substrate slides down the active
site gorge (Trp 82)
ES3: substrate rotates to horizontal
position for hydrolysis
(Ser-198)
18

19. Choline Substrates
O
+ O N
+ O
N O + N
O O
acetylcholine
succinylcholine
(powerful muscle relaxant)
O + S
N N
O O
butyrylcholine butyrylthiocholine
(optimal substrate)
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20. Prodrugs
N
H
CPT-11
O O
O
O O N O O N
O O
Heroin
H
(Silent variants N
Cannot hydrolyze) HO
Bambuterol
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21. Drugs
O
O
N
O HO
O
H3C N
N
H
Tetracaine
Benzactyzine
O OH
O
O
Aspirin
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22. Inhibitors
H3C N O
O
O
P
N S
O
Amitryptiline
Phosphonothiolate
Cocaine Analog
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23. Kinetic Parameters
Ki (µM) kcat (min-1) plasma t1/2
Butyrylthiocholine ~ 20 33,900
Benzoylcholine ~ 8000
Succinylcholine ~ 1500
Aspirin 5,000-12,000
(+) Cocaine (synthetic) ~5 7500 seconds
(-) Cocaine (natural) ~ 10 3.9 45-90 min
Butyryl and propionyl choline are hydrolyzed ~ 2X faster than acetyl choline
KM’s for (+) and (-) cocaine are 10 and 14 µM, respectively
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24. Cocaine Structure
(-) (+)
24

25. BChE-Cocaine Crystal Structure
(-) (+)
25

26. Cocaine Structure
BCh (-) cocaine • Carbonyl C-N distance
– BCh
• 4.92 Å
– Cocaine
• 5.23 Å (benzoyl)
• 2.95 Å (methyl)
– Explains hydrolysis at
benzoyl
By BChE
• Non-enzymatic
hydrolysis
methyl > benzoyl
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27. BChE Mechanism
ES1: substrate binds to PAS (Asp70)
ES2: substrate slides down the active
site gorge (Trp 82)
ES3: substrate rotates to horizontal
position for hydrolysis
(Ser-198)
MD simulations: cocaine goes
through same pathway
Difference in (+) vs. (-) cocaine
is in the rotation step
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28. Cocaine Hydrolysis
H C N 3 O
O
OH
H3C N O
BChE
Ecgonine Methyl Ester (EME)
O hCE-2 ~45%
O
O hCE-1 H3C N O
(-) Cocaine OH
O
O
cocaine hydrolysis  95% of metabolites Benzoyl ecgonine (BE)
~45%
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29. Cocaine Metabolism
• EME
– vasodilative effects
• BE
– potent vasoconstriction effects
• Norcocaine
– local anesthetic and hepato- and cardiotoxic properties
• Plasma BChE accounts for all the cocaine hydrolysis in
blood
• Deficiency in BChE shifts metabolism to norcocaine and BE
• Enhancing BChE may mediate cocaine-induced
complications
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30. Cocaine Toxicity Rats
• Tetraisopropylpyrophosphoramide (iso-OMPA)
– Selective BChE inhibitor
– Increases cocaine lethality in mice and rats
• Exogenous BChE in rats
– 400-800X (5000 IU IV-7.8 mg/kg IV) increase in plasma
levels 
• decrease in cocaine-induced: locomotor activity, hypertension,
and cardiac arrhythmias
• saline-induced rats exhibited no change
– 3200-6400X increase  protection against seizures and
death
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31. Cocaine Toxicity Monkeys
• Monkeys have different basal BChE activities than
rats
– Squirrel monkeys used
– + saline, + plasma, + plasma + BChE
– Cocaine 3 mg/kg IV
– BChE half-life = 72 h (rhesus monkeys)
– 3X decrease in [cocaine], 3X increase in peak [EME], no
change in [BE]
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32. Cocaine Abuse and Toxicity in Humans
• Cocaine abuse is major medical and public health problem
– Affected > 40 million in US since 1980
• ~ 400,000 daily users in US
• ~ 5,000 new users each day
– Overdose  respiratory depression, cardiac arrhythmia, acute hypertension
• Serum [cocaine] on overdose ~ 20 mg/L
– Requires > 100 mg BChE for “timely” detoxification
• Increase BChE levels to treat cocaine abuse and toxicity
– ~ 12X increase in BChE (3-37 µg/mL) decreases t1/2 of cocaine (2 µg/mL) in plasma
from 116 to 10 min (~ 12X)
– Higher turnover than catalytic antibodies for cocaine
• Patients with lower BChE activity  more severe problems
– Acceleration of benzoylester hydrolysis
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33. BChE Variants for Cocaine Toxicity
• Used molecular dynamic simulations to
– Optimize hydrogen bonding energies between oxyanion
hole and carbonyl oxygen on benzoyl group of (-) cocaine
– Simulated the transition state
•  A199S/F227A/A328W/Y332G BChE Mutant
– Engineered BChE mutant that hydrolyzes cocaine very
efficiently
• WT (kcat/KM): ~ 1 X 106 M min-1
• Mutant: (kcat/KM): ~ 1.4 X 108 M min-1
• ~ 140X increase
• Half-life in plasma decreases from 45-90 min to 18-36 s
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34. Organophosphorous Compounds (OPs)
O CH3 O H3C CH3
O
P P CH3
F O CH3 H3C O N P N CH3
H 3C H3C S
CH3 O
H3C
Sarin N
VX
CH3
Tabun
AChE inhibitor – developed as a pesticide (1952)
most deadly nerve agent in existence
3X more deadly than sarin
300 g is fatal
Widely used as: pesticides, plasticizers, pharmaceuticals, chemical warfare agents
"It's one of those things we wish we could disinvent."
- Stanley Goodspeed, on VX nerve agent
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35. OP Poisoning Mechanism – “Aging”
Ser
Ser
OH O
O
O
P
H3C O
P
H3C O O
O NO2
O
paraoxon
H3C
H3C HO NO2
H2O
- BChE is inactivated by these organophosphates phosphonylated enzyme
- point mutations in the active site of BChE (inactivated)
 efficient organophosphate hydrolase
35

36. OP Poisoning
• Extrapolate rhesus monkey data to humans
– ~ 150 mg human BChE in a 70 kg human can protect against
• 2X LD50 of soman
• 1.5X LD50 of VX
• Want to reduce initial blood levels of OPs by 50% in <10 s
• Protection of at least 30% of red blood cell AChE activity
• Intrinsically limited since its binding is stoichiometric to OPs
– Requires a significant amount of enzyme to detoxify a lethal dose
– To make a more a more efficient OP hydrolyzing enzyme:
• Use crystal structures of human BChE to direct mutations
• Use random mutagenesis of human BCHE to create a library of variants
• Bioscavenger (DVC) and Protexia (Pharmathene) in development for
Army
– Human plasma derived and recombinant (probably mutated) versions of
human BChE
– For pre- and post-exposure to chemical warfare agents 36

37. Exogenous BChE Therapy
• BChE chosen instead of AChE because it:
– Comprises 0.1 % of human plasma protein
• AChE is found only in the erythrocyte membrane
– Can be purified in large amounts from human serum
• AChE from other species could be immunoreactive
– Has a larger active site (200 Å3 larger)
• more substrates will be accommodated
– Has a long half-life in vivo (8-12 days)
• Single injection could increase plasma levels of BChE for several days
• No adverse FX reported with increased BChE plasma activity
– Is thermally stable on prolonged storage
37

38. Alzheimer’s Disease
• Chronic and progressive neurodegenerative disease
– Degeneration of cholinergic neurons  loss of neurotransmission
– Reduced levels of Ach
• Leading cause of dementia among older people – affects:
– 10% of people > 65 years old
– 50% of people > 85 years old
• Aging population  numbers could increase exponentially
• Reversible AChE inhibitors are viable therapies for AD
– Protect residual ACh levels in the brains of patients with AD
• Tacrine (1993) Donepezil (1996)
• Rivastigmine (2000) Galantamine (2001)
– However, associated with ADRs: liver damage, nausea, vomiting
38

39. AChE
Inhibitors
for AD • Benefits of
treatment
are not
sustained
long-term
and illness
continues
to progress
Confidential 39

40. Alzheimer’s Disease
• AChE levels decrease 85-90% at the more severe
stages of AD
• BChE levels increase 2X
– Normal brain: 10-15% of cholinergic neurons possess BChE not AChE
– Brain affected by AD: glial cells express and secrete more BChE
– Also BChE can catalyze:
• Amyloid precursor protein  β-amyloid proteins  plaques  AD
– Maybe increased BChE activity  increased risk of AD
• BChE inhibition may provide therapeutic value at
later stages
• Novel BChE inhibitors were recently described
(2005):
– Tacrine heterobivalent ligands
– Flexible docking procedures
– Molecular modeling studies
40

41. Novel BChE Inhibitors for AD
Tacrine analogs
427X preference for binding BChE (Ki = 110 pM) over AChE
Confirmed extra interaction sites in the mid-gorge and peripheral sites of BChE
41

42. •
Summary than AChE
BChE can metabolize a broader spectrum esterase
• There is an important pharmacogenetic condition that is associated with
BChE activity
• The binding and catalysis of cocaine hydrolysis has been described using a
host of different techniques
• Organophosphorus compounds can act MBIs of BChE
• Administration of exogenous BChE could be a useful therapy for certain
toxic and overdose situations
• Inhibitors of BChE are being developed to treat AD
42