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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