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Human Esterases: Chemical and Biochemical Considerations
Luke Lightning, PhD
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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
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Introduction: Esterases• PubMed Search: at least 73 “different” human esterase genes
– complicated by duplicate entries
• a/b 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)
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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 opening
Both:
• found in all kingdoms
• display broad substrate specificity
• overlapping protein sequence motifs
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Clinical Consequences
• Activation (prodrugs)
• Inactivation (ester drugs)
• Inhibition increased potential for drug-drug interactions
• Exposure to environmental pollutants or drugs induction
• Induction enhanced hydrolysis
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Famous Esters
Esther Rolle
O O
OO
n
polyester
OHO
O CH3
O
aspirin
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Other Substrates for Esterases
lidocaine
NO2O
P
O
O
O
H3C
H3C
paraoxon
CH3
CH3
HN
N
O
OO
O
CH3
O
H3C
O
N
CH3
heroin
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Esterase Activity
R1 O
O
R2R1 OH
O
R2OH+esterase
acid alcohol
acidpart
alcoholpart
H2OO
also can metabolize thiols, amides, and carbamates
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Ester Hydrolysis
O
CH3
OO
O
H3C O
CH3
OHOH3C OH
O
+
acetate
4-methylumbelliferoneacetate
4-methyl-7-hydroxycoumarinalcohol
partacidpart
hCE-1 2,000hCE-2 60,000
catalytic efficiency (kcat/KM):
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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
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CPT-11 (Irinotecan)
inactive
inactive
inactive,can be
recycled
CPT-11
active
SN-38
topoisomerase I poison
no glucuronidation pathway in tumors
- can give orally
Km = 5 M
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General Mechanism
oxyanion hole
ESTER
ACID
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P450 Activity
FeIII
H HO
RH
FeIII
FeIII
RH
H2O
ferric,low-spin
ferric, high-spin
ROH
ROHH2O
e-
FeIIRH
FeIIRH
O2 = O-O
O-O
FeIIIRH-O-O
e- (rate determining step)
FeVRHO
FeIVR.HO
H2O
H2O2
O2 .-
FeIVRHO
FeIIIRHO
.+
H2O
compound I ferryl oxene“iron oxo”
ferric hydroperoxide
ferrous dioxy
ferrous
ferricperoxy
FeIIIRHHO-O
H+
FeIIIRHO-O
ferric superoxide“oxy-P450”
-.
FeIIRHO-O
ferroussuperoxide
-.
2 H+
H+
.
:::
:
.
Active site:heme
Requires:NADPH
lipidO2
P450 reductaseCytochrome b5
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Crystal Structures: hCE-1 + tacrine
catalytic gorge+ tacrine
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Lovastatin- cholesterol-lowering drug- lipid soluble prodrug- considerable inter-patient variability in therapeutic effect- long term adverse effects include liver damage and myopathy
hCEs
H
CH3
H3C
OH3C
CH3
O
COOH
HO
OH
b-hydroxy acid form (active)HMG-CoA reductase inhibitor
H
CH3
O
HO O
H3C
OH3C
CH3
O
lactone (inactive)
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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
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Interspecies Variability: Esterase Activitycarboxylesterase activity in liver microsomes
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Interindividual Variability
Protein Levels - Carboxylesterases
anti-rat carboxylesterase antibody used to determine protein content10-fold variation
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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 538Esterase 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.
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Esters Used to Test Human Variability
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Interindividual Variability
esters amides thioester
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Organophosphorus Pesticides
40 billion pounds of insecticides per year is used
malathion
esterase
H3CO
P
SH3CO
S
OO
O
O CH3
CH3
acidH3CO
P
SH3CO
S
OHO
O
O CH3
malaoxon
P450
H3CO
P
OH3CO
S
OO
O
O CH3
CH3
esteraseinactivation
esterase
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Fatty Acyl Ethyl Ester Synthase/Esterase
• fatty acid + ethanol fatty acyl ethyl esters– esterases play a role in cholesterol trafficking– build-up in tissues of alcoholics necrosis of organs
cis-oleic acid
ethanol
esterase(H2C)7 (CH2)7
O
OH
H3C
ethyloleate
(H2C)7 (CH2)7
O
OCH2CH3
H3C
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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
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Sarin, Tabun, and VX gas: Biological Weapons
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 sarin300 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
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Serine Esterase Inactivation
NO2O
P
O
O
O
H3C
H3C
Ser
OH
P
O
O
O
H3C
H3C
Ser
O
NO2HO
- 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
paraoxon
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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
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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
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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
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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
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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
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Lumen
Cytoplasm
Subcellular Organization of Membrane Bound hCEs
hCEs
Phospholipid bilayer
N-linkedglycosylation
sites
GluHis
Ser
S-S
S-S
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Carboxylesterasesoxyanion
hole
catalytictriad
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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
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hCE-1 and hCE-2• hCE-1
– 568 Amino Acids– 62,596 Da
• sequence identity– AChE 30%– rabbit CE-1 80%
• can activate CPT-11– hCE-2 48%
• can activate CPT-11
• does not activate CPT-11• deficiency may play a role in:
– rheumatoid arthritis– non-Hodgkins lymphoma
• hCE-2– 623 Amino Acids– 68,903 Da
• sequence identity– rabbit CE-2 73%
• high-affinity, high velocity enzyme w/respect to CPT-
11
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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
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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
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Crystal Structures: hCE-1
hCE-1:tacrinehCE1:naloxone
hexamer
trimer
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hCE-1 Crystal Structure
oligomer analysis by AFM
monomers
trimers
hexamers
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hCE Tissue Localizationbr
ain
colo
nhe
art
kidn
eyliv
erlu
ngm
uscl
esm
all i
nest
ine
plac
enta
stom
ach
sple
ente
stis
liver > colon > SI > heart
liver >> heart > stomachspleen = testis = kidneyalso present in plasma
liver clearance: bothkidney clearance: hCE-1
SI and colon clearance: hCE-2
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hCE Induction• In rats:
– Phenobarbital– Aroclor 1254– Polycyclic aromatic hydrocarbons– Aminopyrine– Clofibrate– Pregnenolone 16-a-carbonitrile– Di(2-ethylhexyl)phthalate
– Not 3-methycholanthrene
– Testosterone, but not estrogen sex differences?
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hCE-1 Substrates
N
H3C
O
O
O
O
CH3
hCE-1
O
ONH3C CH3
hCE-1
NH
O O
O
N
OH
H3C
CH3
O
hCE-1
cocaine
meperidine
delapril
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hCE-2 Substrates
N
H3C
O
O
O
O
CH3
hCE-2(and BChE)
cocaine
6-acetylmorphine
NN
O
CH3
O
O
O
HO
H3C
O
NN
hCE-2OHO
O
H3C
O
N
CH3
hCE-2
CPT-11
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hCE-1substrates
hCE-2substrates
hCE-1: does not hydrolyze cmpds with bulky alcohol groupshCE-2: does not hydrolyze cmpds with bulky acid groups
IN GENERAL:
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hCE-1 Binding
Compounds with more hydrophobic R groups
(larger log P)bind more tightly
(smaller Ki)
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swap R3 and R4
atropine
hydrolysis products
enantiomer
swap R1 and R2
extend length of R1
remove R1
hCE-1 SubstrateSpecificity
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Cocaine Metabolism (hCE-1 and P450)
hCE-1 hCE-1
P450
transesterification, hCE-1
P450
transesterification, hCE-1
P450
hCE-1 hCE-1
• longer t 1/2
• more toxic• higher brain:plasma ratio
MAJOR
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hCE-1 Substrate Specificity
• 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
in vivot1/2 (min)
330-40
--
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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
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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/
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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