Human esterases: Chemical and Biochemical Considerations

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Human esterases: Chemical and Biochemical Considerations

  1. 1. Human Esterases:Chemical and Biochemical Considerations Luke Lightning, PhD 1
  2. 2. Outline: Esterases and Carboxylesterases• Introduction – Esters – Different esterases involved in drug metabolism – Mechanism – Biochemical Characteristics• Human Carboxylesterases – Molecular Structure  • Overall Function • Localization • Substrate Specificity • Evolutionary Relationships 2
  3. 3. Introduction: Esterases• PubMed Search: at least 73 “different” human esterase genes – complicated by duplicate entries• / hydrolase-fold family (as of 9/28/04: 5440 sequences) – Carboxylesterases (329 nucleotide sequences) • Carboxylesterases (hCE-1, 2, 3) – broad substrate specificity – Cholinesterases (114 nucleotide sequences) • Acetylcholine esterase (AChE) – specific for acetylcholine (101 sequences) • Butyrylcholine esterase (BChE) – broad substrate specificity (13 sequences) – Juvenile Hormone Esterase – specific for hormone (12 sequences) – Esterase D – Lipases• Others: – Paraoxonases (Arylesterases) 3
  4. 4. Esterases vs Lipases• water soluble substrates • insoluble or heavily aggregated - short chain fatty acid esters - longer chain fatty acids esters• activity correlates with [substrate] • activity correlates with substrate area • more non-polar residues at the surface • lid openingBoth: • found in all kingdoms • display broad substrate specificity • overlapping protein sequence motifs 4
  5. 5. Clinical Consequences• Activation (prodrugs)• Inactivation (ester drugs)• Inhibition  increased potential for drug-drug interactions• Exposure to environmental pollutants or drugs  induction• Induction  enhanced hydrolysis 5
  6. 6. Famous Esters O OH O CH3 O aspirinO O O Esther Rolle O n polyester 6
  7. 7. Other Substrates for Esterases O CH3 H P N H3C O O NO2 N O O CH3 H3C paraoxon lidocaine CH3 N heroin O O O CH3 H3C O O 7
  8. 8. Esterase Activity O O R2 esterase + R1 O R1 O OH R2OH H2O acid alcohol acid alcohol part part also can metabolize thiols, amides, and carbamates 8
  9. 9. Ester Hydrolysis 4-methylumbelliferone acetate CH3 CH3 O O +H3C O O O H3C OH HO O O acetate 4-methyl-7- acid alcohol hydroxycoumarin part part hCE-1 2,000 catalytic efficiency (kcat/KM): hCE-2 60,000 9
  10. 10. Prodrugs and Esterases• Prodrugs containing esters, amides, lactones  – Increased solubility – Increased bioavailability – Less toxicity• Various esterases activate prodrugs in humans – CPT-11 (irinotecan) • Used in the treatment of colon cancer • Approved by the FDA in 3 days (1996) • Acid form (SN-38) is a topoisomerase I inhibitor • High interpatient variability in SN-38 pharmacokinetics • some patients respond very poorly • Tumor tissue from colon has lower level of esterase activity • only 2% of SN-38 makes it to the tumor • Gene therapy  enhance local production of SN-38 in tumors 10
  11. 11. CPT-11 (Irinotecan) inactive active Km = 5 M SN-38 CPT-11 topoisomerase I poisonno glucuronidationpathway in tumors - can give orally inactive, can be inactive recycled 11
  12. 12. General Mechanism oxyanion hole ESTER ACID 12
  13. 13. P450 Activity H H O Active site: H2O ROH FeIII RH H2O heme ferric, low-spin RH ROH FeIII FeIII Requires: ferric, high-spin e- NADPH lipid HO R . H2O . O2 - RH FeIV FeII O2 ferrous : : : : H2O2 . . O2 = O-OP450 reductase O RH O RH O RHCytochrome b5 FeIII FeIV FeV O-O RH - O-O RH. .+ compound I ferryl oxene FeII FeIII “iron oxo” 2 H+ ferrous dioxy ferric superoxide “oxy-P450” e- (rate determining step) H2O H+ HO-O RH -O-O RH . - O-O RH FeIII FeIII FeII ferric H+ ferric ferrous hydroperoxide peroxy superoxide 13
  14. 14. Crystal Structures: hCE-1 + tacrinecatalytic gorge + tacrine 14
  15. 15. Lovastatin - cholesterol-lowering drug - lipid soluble prodrug - considerable inter-patient variability in therapeutic effect - long term adverse effects include liver damage and myopathy HO O HO COOH O OH O O hCEsH3C O H3C O H H CH3 CH3 CH3 CH3 H3C H3C lactone (inactive) -hydroxy acid form (active) HMG-CoA reductase inhibitor 15
  16. 16. Lovastatin• Approximate % of hydroxy acid formed by esterases in: – Plasma 18% – Liver microsomes 15% – Liver cytosol 67%• Genetic variation in esterase activity is suggested: – 3 of 17 livers showed little or no capacity for lovastatin hydrolysis – inter-individual variation in lovastatin hydrolysis by plasma esterase 16
  17. 17. Interspecies Variability: Esterase Activity carboxylesterase activity in liver microsomes 17
  18. 18. Interindividual VariabilityProtein Levels - Carboxylesterases anti-rat carboxylesterase antibody used to determine protein content 10-fold variation 18
  19. 19. Genetic Polymorphisms: Esterases48 Japanese individuals were screened for single-nucleotide polymorphisms in 9 esterase genes - J. Hum. Genet. 48, 249 (2003) SNPs PolymorphismsArylacetamide deacetylase 23 1Cholesterol esterase 117 15Carboxylesterase 1 and 2 53 8Esterase D 28 1Granzyme A and B 22 1Interleukin 17 11 0Ubiquitin carboxyl terminal esterase 48 12• 302 SNPs were identified in esterases  38 polymorphisms• No variations in the catalytic triad• Is there a correlation between genotype and phenotype?• Do polymorphisms regulate induction?• No analysis of BChE, paraoxonases, etc. 19
  20. 20. Esters Used to Test Human Variability 20
  21. 21. Interindividual Variability esters amides thioester 21
  22. 22. Organophosphorus Pesticides 40 billion pounds of insecticides per year is used CH3 O OH S O H3CO O O esterase P S O S O CH3H3CO H3CO acid P S O CH3 H3CO malathion CH3 P450 O O esterase O O esterase inactivation H3CO P S O CH3 H3CO malaoxon 22
  23. 23. Fatty Acyl Ethyl Ester Synthase/Esterase esterase (H2C)7 (CH2)7 (H2C)7 (CH2)7 OH OCH2CH3 ethanolH3C H3C cis-oleic acid ethyloleate O O • fatty acid + ethanol  fatty acyl ethyl esters – esterases play a role in cholesterol trafficking – build-up in tissues of alcoholics  necrosis of organs 23
  24. 24. Transesterification• cocaine + ethanol  cocaethylene (more lethal in mice)• hCE-1: Km for cocaine = 116 M; Km for ethanol = 43 mM• BChE: Km for cocaine = 12 M• [cocaine] after 100 mg dose IV = 3 M• [ethanol] in blood of people that have OD’d on cocaine = 7-110 mM 24
  25. 25. Sarin, Tabun, and VX gas: Biological Weapons O CH3 O P P CH3F O CH3 H3C O N H3C CH3 H 3C O CH3 Sarin N P N CH3 Tabun H3C S O H3C VX CH3 AChE inhibitor – developed as a pesticide (1952) most deadly nerve agent in existence 3X more deadly than sarin 300 g is fatal "Its one of those things we wish we could disinvent." - Stanley Goodspeed, on VX nerve agent 25
  26. 26. Serine Esterase Inactivation Ser Ser OH O O O P H3C O PH3C O O O NO2 O paraoxon H3C H3C HO NO2- hCE-1 is inactivated by these organophosphates- point mutations in the active site of hCE-1  efficient organophosphate hydrolase- US government is developing variant forms of hCE-1 to treat personnel at risk of exposure to biological weapons 26
  27. 27. Mammalian Carboxylesterases (CEs)• located in the ER and cytosol of many tissues• involved in detoxification or activation of: – Drugs – Environmental toxicants – Carcinogens – Fatty acid esters• multiple isoforms exist in various animal species• activate carcinogens  hepatocarcinogenesis 27
  28. 28. hCEs• Human Carboxylesterases (hCEs) – Originally classified on the basis of substrate specificity and pI – However, they: • are glycoproteins  different pI’s • have overlapping substrate specificities – Now classified based on sequence alignments: • 3 groups for humans with 80% sequence identity within a group • hCE-1 – “liver hCE” • hCE-2 – “intestinal hCE” • hCE-3 – “brain hCE” – large interindividual variation (66-150X) in colon tumors 28
  29. 29. hCEs• serine hydrolases• can metabolize: – esters, thioesters, amide-ester linkages – carbamates• localized in the ER and cytosol of many tissues• glycosylation is essential for maximal catalytic activity – probably assists in folding, solubility, circulatory t1/2 – unknown: if there is a tissue dependence on amount of hydrolytic activity • hCE-1 activity in liver >> hCE-1 activity in heart• importance in industry: – prodrugs  active compound by hCEs – major determinants of pharmacokinetic behavior 29
  30. 30. hCE substrates• Xenobiotics: – Anesthetics: cocaine, lidocaine – Narcotics: heroin, meperidine – Cholesterol lowering: lovastatin – Angiotensin-converting enzyme (ACE) inhibitors: delapril, imidapril, temocapril – Anti-cancer: CPT-11• Endogenous compounds: – Fatty acid esters: • short, and long chain acyl-glycerols, long-chain acyl-carnitine, long-chain acyl-CoA esters 30
  31. 31. hCE Structure• Hydrophobic N-terminus – targets the protein to the ER• HXEL-COOH at C-terminus – retains the protein on the luminal side of the ER• 3 amino acid “catalytic triad” (very similar to serine proteases) – Ser, His, and Asp or Glu• 4 cysteines – Disulfide bonds• N-linked glycosylation sites 31
  32. 32. Subcellular Organization of Membrane Bound hCEs Lumen S-S Glu hCEs His Ser N-linked glycosylation S-S sitesPhospholipid bilayer Cytoplasm 32
  33. 33. Carboxylesterases catalytic oxyanion triad hole 33
  34. 34. Other names:• hCE-1 – Acyl coenzyme A cholesterol acyltransferase – Monocyte/macrophage serine esterase 1 – Monocyte/macrophage serine esterase – Alpha naphthylacetate esterase – Brain carboxylesterase (hBr1) – Cholesteryl ester hydrolase – Liver carboxylesterase – Carboxylesterase, liver – Alveolar esterase – Serine esterase 1 – Acid esterase – Egasyn – HMSE1, HMSE, ACAT, ANAE, SES1, CEH, HU1• hCE-2 – Intestinal carboxylesterase – Liver carboxylesterase 2 – iCE, CE-2 34
  35. 35. hCE-1 and hCE-2• hCE-1 • hCE-2 – 568 Amino Acids – 623 Amino Acids – 62,596 Da – 68,903 Da• sequence identity • sequence identity – AChE 30% – rabbit CE-2 73% – rabbit CE-1 80% • can activate CPT-11 – hCE-2 48% • high-affinity, high velocity • can activate CPT-11 enzyme w/respect to CPT-11• does not activate CPT-11• deficiency may play a role in: – rheumatoid arthritis – non-Hodgkins lymphoma 35
  36. 36. hCE1• is also present in monocytes and macrophages• biological roles: – chemoprotection of proteins in tissues - drug and xenobiotic metabolism – cholesterol trafficking within cells and between tissues • fatty acyl ethyl ester synthase activity • acyl-coenzyme A:cholesterol acyl transferase (ACAT) activity  cholesterol esters • one of 3 cellular binding targets of tamoxifen  cholesterol lowering effects (????) – protein retention and release from the ER • complexes with UGTs and C-reactive protein to retain them in the ER lumen 36
  37. 37. Crystal Structures: hCE-1• Philip Potter’s group (St. Jude’s, Memphis, TN, April 2003): – in complex: • with naloxone methiodide (heroin analog) • with homatropine (cocaine analog) • with tacrine (human AChE inhibitor (Ki = 38 nM), Alzheimer’s)• large substrate binding gorge with rigid and flexible pockets• binding gorge is lined with hydrophobic residues• catalytic triad = Ser-221, His-468, Glu-354 37
  38. 38. Crystal Structures: hCE-1hCE-1:tacrinehCE1:naloxone hexamer trimer 38
  39. 39. hCE-1 Crystal Structure oligomer analysis by AFMmonomers hexamers trimers 39
  40. 40. hCE Tissue Localization liver >> heart > stomach spleen = testis = kidney also present in plasma liver > colon > SI > heart liver clearance: both kidney clearance: hCE-1 SI and colon clearance: hCE-2 40
  41. 41. hCE Induction• In rats: – Phenobarbital – Aroclor 1254 – Polycyclic aromatic hydrocarbons – Aminopyrine – Clofibrate – Pregnenolone 16- -carbonitrile – Di(2-ethylhexyl)phthalate – Not 3-methycholanthrene – Testosterone, but not estrogen  sex differences? 41
  42. 42. hCE-1 Substrates hCE-1 O meperidineH3C CH3 N O H3C N O CH3 O O O hCE-1 cocaine H3C hCE-1 O O CH3 N N H delapril O O OH 42
  43. 43. hCE-2 Substrates OH3C CH3 N N hCE-2 N O O O O CH3 O cocaine hCE-2 (and BChE) CPT-11 N N O CH3 N HO 6-acetylmorphine O H3C O O hCE-2 HO O H3C O 43
  44. 44. hCE-1 substrates hCE-2 substratesIN GENERAL: hCE-1: does not hydrolyze cmpds with bulky alcohol groups hCE-2: does not hydrolyze cmpds with bulky acid groups 44
  45. 45. hCE-1 Binding Compounds with more hydrophobic R groups (larger log P) bind more tightly (smaller Ki) 45
  46. 46. hCE-1 Substrate Specificity swap R3 and R4 swap R1 and R2 enantiomerextend length of R1hydrolysis products remove R1 atropine 46
  47. 47. Cocaine Metabolism (hCE-1 and P450) transesterification, hCE-1 • longer t 1/2 hCE-1 hCE-1 • more toxic • higher MAJOR brain:plasma ratioP450 P450 P450 hCE-1 hCE-1 transesterification, hCE-1 47
  48. 48. hCE-1 Substrate Specificity in vivo t1/2 (min) 3 30-40 -- • hCE-2 was the 1st human enzyme reported to hydrolyze 6-AM • Km’s ( 6.8 mM) are > than in vivo [heroin] • < 270 M in abusers; 3 M in patients treated for pain • 1st order kinetics in vivo • cocaine and heroin are metabolized by same enzymes • “speedballing”  enhanced drug levels 48
  49. 49. Uses of hCEs• Regulating hCE activity to treat narcotic abuse or overdose• Regulating hCE activity to treat soldiers affected by sarin or other biological weapons• Directed Evolution –  regio- and enantio-selective reactions in organic synthesis • improved activity in organic solvent, high temperatures, acidic pH 49
  50. 50. Web and Meeting Information• ESTHER database – http://bioweb.ensam.inra.fr/ESTHER/general?what=index• International Paraoxonase Meeting (1st, 2004) – http://sitemaker.umich.edu/pons-conference• International Cholinesterase Meeting (8th, 2004) – http://www-b.unipg.it/~cholinpg/ 50
  51. 51. Conclusions• Wide variety of esterases present in humans – different substrate specificity, localization, catalytic mechanism• Esterases can act as hydrolases and synthases – gaining prominence in the field of drug metabolism• Interspecies and inter-individual variability in esterase activity exists – does this affect drug metabolism? – more studies needed: • genetic polymorphisms • Induction• hCEs play important roles in the metabolism of drugs and endogenous cmpds• Crystal structures are now possible 51

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