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Butyrylcholinesterase Overview: Substrates, Inhibitors, Structure,Mechanism, Therapeutic Indications              (BChE)  ...
Outline• Introduction/History• Biochemistry• Genetic Variability• Mechanism and Structure• Protection from Toxicities and ...
BChE Substrates                  3
BChE Introduction• Preferentially hydrolyzes butyrylcholine, but also hydrolyzes acetylcholine    – Function thought to be...
History• 1920’s  – Loewi in Austria     • Awarded Nobel Prize for work on cholinesterase, etc.• 1940’s  – Mendel in Toront...
More History• 1950’s:   – Patients with schizophrenia treated with electroshock   – Good therapeutic success, but also ove...
•   2 classes              Animal Cholinesterases     – Based on their substrate specificity and susceptibility to inhibit...
Cholinesterases• Acetylcholinesterase    – Function is to hydrolyze acetylcholine released at the synaptic cleft and      ...
Localization Differences  AChE                             BChE  Brain                            Plasma (relatively abund...
Selective Inhibitors        AChE                              BChE                                      HH                ...
•              Inherited BChE Deficiency   Not clinically significant until plasma activity is reduced to 75% of normal• N...
Genetic Variants• 96% of population is homozygous for normal genotype• 4% of the population:    – Atypical (Dibucaine) res...
•                         Genetic Variability    Deficiencies are due to one or more inherited abnormal alleles     – Fail...
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...
Interspecies Similarities• Protein Sequence Identity (and Homology) with  Human BChE (~ 50 mg costs $350)  – Rabbit     91...
Crystal Structure of BChE• Comparison to AChE  – Catalytic triads of both are at the bottom of a 20 Å-deep    gorge     • ...
General MechanismBChE:Ser198Glu325His438                                   oxyanion hole                        ESTERhydro...
BChE Mechanism       ES1: substrate binds to PAS (Asp70)       ES2: substrate slides down the active               site go...
Choline Substrates                          O+         O                       N                                          ...
Prodrugs             N         H                                        CPT-11         O       OO     O       O           ...
Drugs                   O                                            O                            N                       ...
Inhibitors                         H3C N     O                                        O                                   ...
Kinetic Parameters                                       Ki (µM)             kcat (min-1)        plasma t1/2Butyrylthiocho...
Cocaine Structure(-)        (+)                    24
BChE-Cocaine Crystal Structure     (-)             (+)                                 25
Cocaine StructureBCh   (-) cocaine   • Carbonyl C-N distance                      – BCh                         • 4.92 Å  ...
BChE Mechanism       ES1: substrate binds to PAS (Asp70)       ES2: substrate slides down the active               site go...
Cocaine Hydrolysis                                H C N      3           O                                                ...
Cocaine Metabolism• EME   – vasodilative effects• BE   – potent vasoconstriction effects• Norcocaine   – local anesthetic ...
Cocaine Toxicity Rats• Tetraisopropylpyrophosphoramide (iso-OMPA)  – Selective BChE inhibitor  – Increases cocaine lethali...
Cocaine Toxicity Monkeys• Monkeys have different basal BChE activities than  rats  – Squirrel monkeys used  – + saline, + ...
Cocaine Abuse and Toxicity in Humans• Cocaine abuse is major medical and public health problem   – Affected > 40 million i...
BChE Variants for Cocaine Toxicity• Used molecular dynamic simulations to  – Optimize hydrogen bonding energies between ox...
Organophosphorous Compounds (OPs)            O        CH3                       O                                         ...
OP Poisoning Mechanism – “Aging”      Ser                                                        Ser            OH        ...
OP Poisoning• Extrapolate rhesus monkey data to humans   – ~ 150 mg human BChE in a 70 kg human can protect against       ...
Exogenous BChE Therapy• BChE chosen instead of AChE because it:   – Comprises 0.1 % of human plasma protein      • AChE is...
Alzheimer’s Disease• Chronic and progressive neurodegenerative disease   – Degeneration of cholinergic neurons  loss of n...
AChEInhibitors  for AD                    • Benefits of                              treatment                            ...
Alzheimer’s Disease• AChE levels decrease 85-90% at the more severe  stages of AD• BChE levels increase 2X  – Normal brain...
Novel BChE Inhibitors for AD           Tacrine analogs         427X preference for binding BChE (Ki = 110 pM) over AChECon...
•                            Summary than AChE    BChE can metabolize a broader spectrum esterase• There is an important p...
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Butyrylcholinesterase Overview: Substrates Inhibitors Structure Mechanism Therapeutic Indications

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Butyrylcholinesterase Overview: Substrates Inhibitors Structure Mechanism Therapeutic Indications

  1. 1. Butyrylcholinesterase Overview: Substrates, Inhibitors, Structure,Mechanism, Therapeutic Indications (BChE) Luke Lightning
  2. 2. Outline• Introduction/History• Biochemistry• Genetic Variability• Mechanism and Structure• Protection from Toxicities and Disease 2
  3. 3. BChE Substrates 3
  4. 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. 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. 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. 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. 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. 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 bloodAre present in high amounts in mice, rat, rabbit, horse, cat, and tiger blood 9
  10. 10. Selective Inhibitors AChE BChE HH N ON O N N O N O N Phenserine Phenethyl-norcymserine Huperzine A Ethopromazine BW284C51 10
  11. 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. 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. 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. 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. 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. 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. 17. General MechanismBChE:Ser198Glu325His438 oxyanion hole ESTERhydrolysis of acylenzyme complex by water ACID Confidential 17
  18. 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. 19. Choline Substrates O+ O N + ON O + N O O acetylcholine succinylcholine (powerful muscle relaxant) O + S N N O O butyrylcholine butyrylthiocholine (optimal substrate) 19
  20. 20. Prodrugs N H CPT-11 O OO O O N O O N O O Heroin H (Silent variants NCannot hydrolyze) HO Bambuterol 20
  21. 21. Drugs O O N O HO OH3C N N H Tetracaine Benzactyzine O OH O O Aspirin 21
  22. 22. Inhibitors H3C N O O O P N S OAmitryptiline Phosphonothiolate Cocaine Analog 22
  23. 23. Kinetic Parameters Ki (µM) kcat (min-1) plasma t1/2Butyrylthiocholine ~ 20 33,900Benzoylcholine ~ 8000Succinylcholine ~ 1500Aspirin 5,000-12,000(+) Cocaine (synthetic) ~5 7500 seconds(-) Cocaine (natural) ~ 10 3.9 45-90 minButyryl and propionyl choline are hydrolyzed ~ 2X faster than acetyl cholineKM’s for (+) and (-) cocaine are 10 and 14 µM, respectively 23
  24. 24. Cocaine Structure(-) (+) 24
  25. 25. BChE-Cocaine Crystal Structure (-) (+) 25
  26. 26. Cocaine StructureBCh (-) 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 26
  27. 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 27
  28. 28. Cocaine Hydrolysis H C N 3 O O OHH3C 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% 28
  29. 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. 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. 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. 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 32
  33. 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 33
  34. 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 fatalWidely used as: pesticides, plasticizers, pharmaceuticals, chemical warfare agents "Its one of those things we wish we could disinvent." - Stanley Goodspeed, on VX nerve agent 34
  35. 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. 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. 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. 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. 39. AChEInhibitors for AD • Benefits of treatment are not sustained long-term and illness continues to progress Confidential 39
  40. 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. 41. Novel BChE Inhibitors for AD Tacrine analogs 427X preference for binding BChE (Ki = 110 pM) over AChEConfirmed extra interaction sites in the mid-gorge and peripheral sites of BChE 41
  42. 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

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