Butyrylcholinesterase Overview:Substrates, Inhibitors, Structure,

Mechanism, Therapeutic Indications(BChE)

Luke Lightning

Outline• Introduction/History

• Biochemistry

• Genetic Variability

• Mechanism and Structure

• Protection from Toxicities and Disease

2

BChE Substrates

3

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

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

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

Animal Cholinesterases• 2 classes

– 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

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

8

Localization DifferencesAChE BChEBrain 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

9

No carboxylesterases in human blood

Are present in high amounts in mice, rat, rabbit, horse, cat, and tiger blood

Selective Inhibitors

HN

O

O

N

N

HN

O

O

N

N

10

BW284C51

BChEAChE

Huperzine A

Ethopromazine

PhenserinePhenethyl-norcymserine

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

11

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

12

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

13

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

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%)

15

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

16

General Mechanism

Confidential 17

oxyanion hole

ESTER

ACID

BChE:

Ser198

Glu325

His438

hydrolysis of acyl

enzyme complex

by water

BChE Mechanism

18

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)

Choline Substrates

NO

O+ N

+

O

O

NO+

O

NO

O

NS+

O

19

butyrylthiocholine

(optimal substrate)

acetylcholine

butyrylcholine

succinylcholine

(powerful muscle relaxant)

Prodrugs

OO O

O O

H

N

O N

O

HO

HN

ON

O

20

Heroin

(Silent variants

Cannot hydrolyze)

CPT-11

Bambuterol

Drugs

O OH

O

O

HO

O

O

N

ON

NH

H3C

O

21

Aspirin

Benzactyzine

Tetracaine

Inhibitors

N

NH3C O

O

OP

OS

22

AmitryptilinePhosphonothiolate

Cocaine Analog

Kinetic ParametersKi (µM) kcat (min-1) plasma t1/2

Butyrylthiocholine ~ 20 33,900Benzoylcholine ~ 8000Succinylcholine ~ 1500Aspirin 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 cholineKM’s for (+) and (-) cocaine are 10 and 14 µM, respectively

23

Cocaine Structure

24

(-) (+)

BChE-Cocaine Crystal Structure

25

(-) (+)

Cocaine Structure• 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

26

BCh (-) cocaine

BChE Mechanism

27

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

Cocaine Hydrolysis

NH3C O

O

O

O

28

(-) Cocaine

cocaine hydrolysis 95% of metabolites

NH3C O

O

OH

Ecgonine Methyl Ester (EME)BChE

hCE-2 ~45%

NH3C O

OH

O

O

Benzoyl ecgonine (BE)

hCE-1

~45%

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

29

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

30

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]

31

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

32

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

33

Organophosphorous Compounds (OPs)

34

P

S

N

H3C

CH3

CH3

CH3H3C

OH3C

O

VX

AChE inhibitor – developed as a pesticide (1952)

most deadly nerve agent in existence

3X more deadly than sarin

300 g is fatal

F

P

H3C

O

O

CH3

CH3

Sarin

O

P

O

N

CH3

N

H3C

CH3

Tabun

"It's one of those things we wish we could disinvent."

- Stanley Goodspeed, on VX nerve agent

Widely used as: pesticides, plasticizers, pharmaceuticals, chemical warfare agents

OP Poisoning Mechanism – “Aging”

35

NO2O

P

O

O

O

H3C

H3C

Ser

OH

P

O

O

O

H3C

H3C

Ser

O

NO2HO

- BChE is inactivated by these organophosphates

- point mutations in the active site of BChE

efficient organophosphate hydrolase

paraoxon

phosphonylated enzyme

(inactivated)

H2O

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

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

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

AChE Inhibitors

for AD • Benefits of treatment are not sustained long-term and illness continues to progress

Confidential 39

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

Novel BChE Inhibitors for AD

41

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

Summary• BChE can metabolize a broader spectrum esterase than AChE

• 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