Butyrylcholinesterase Overview: Substrates Inhibitors Structure Mechanism Therapeutic Indications

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 2

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. 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

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 8

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 9 No carboxylesterases in human blood Are present in high amounts in mice, rat, rabbit, horse, cat, and tiger blood

10. Selective Inhibitors 10 BW284C51 BChE AChE Huperzine A Ethopromazine Phenserine Phenethyl-norcymserine

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 11

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 12

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 13

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%) 15

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 16

17. General Mechanism Confidential 17 oxyanion hole ESTER ACID BChE: Ser198 Glu325 His438 hydrolysis of acyl enzyme complex by water

18. 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)

19. Choline Substrates 19 butyrylthiocholine (optimal substrate) acetylcholine butyrylcholine succinylcholine (powerful muscle relaxant)

20. Prodrugs 20 Heroin (Silent variants Cannot hydrolyze) CPT-11 Bambuterol

21. Drugs 21 Aspirin Benzactyzine Tetracaine

22. Inhibitors 22 Amitryptiline Phosphonothiolate Cocaine Analog

23. Kinetic Parameters K i (µM) k cat (min -1 ) plasma t 1/2 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 K M ’s for (+) and (-) cocaine are 10 and 14 µM, respectively 23

24. Cocaine Structure 24 (-) (+)

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

26. 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

27. 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

28. Cocaine Hydrolysis 28 (-) Cocaine cocaine hydrolysis  95% of metabolites Ecgonine Methyl Ester (EME) BChE hCE-2 ~45% Benzoyl ecgonine (BE) hCE-1 ~45%

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 29

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 30

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] 31

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 t 1/2 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

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 (k cat /K M ): ~ 1 X 10 6 M min-1 Mutant: (k cat /K M ): ~ 1.4 X 10 8 M min-1 ~ 140X increase Half-life in plasma decreases from 45-90 min to 18-36 s 33

34. Organophosphorous Compounds (OPs) 34 VX AChE inhibitor – developed as a pesticide (1952) most deadly nerve agent in existence 3X more deadly than sarin 300  g is fatal Sarin 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

35. OP Poisoning Mechanism – “Aging” 35 - BChE is inactivated by these organophosphates - point mutations in the active site of BChE  efficient organophosphate hydrolase paraoxon phosphonylated enzyme (inactivated) H 2 O

36. OP Poisoning Extrapolate rhesus monkey data to humans ~ 150 mg human BChE in a 70 kg human can protect against 2X LD 50 of soman 1.5X LD 50 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 Å 3 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 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

42. 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