4.11.Drug Metabolism Cytochrome P450

7/31/2019 4.11.Drug Metabolism Cytochrome P450

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Subjects to be covered

(Professor Kozikowski)

Cytochrome P450, metabolism

mechanisms

HDACs and Inhibitors cancerapplications

Kinases phosphate transfer, inhibitordevelopment for cancer some SARstudies


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Metabolism

Sum of processes by which particular

substances are handled by the body. From the Greek, metabole -- change


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Drug Metabolism

Cytochrome P450Substrates can undergo a broad range of

reactions during metabolism.

These reactions include, for example,oxidation, reduction, hydrolysis, hydration,

conjugation and condensation.

Drug metabolism is divided into 2 Phases --

Phase I which are the functionalizationreactions;

And Phase II, which are the conjugationreactions.


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Drug Metabolism

Foreign organism elicits antibody

response Low molecular weight xenobiotics

nonspecific enzymes convert them intopolar molecules for excretion

Enzymatic biotransformations of drugs

drug metabolism


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Principal site of drug metabolism is the liver; also

kidneys, lungs, GI tract

take via

mouth

absorbed through small

intestine or stomach

bloodstream

liver

(first metabolized)

Drug metabolism by liver enzymes first-pass effect

Pathway of Oral Drugs


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Example of Phase I and II

OH

O-SO3H

Phase I Phase II


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Drug Metabolism

Drug metabolism is desirable once drug

has reached site of action may produceits effect longer than desired or become

toxic.

Drug metabolism studies are essential for

the safety of drugs. Metabolites must be

isolated and shown to be nontoxic.


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Metabolism Studies Can Be a Useful Lead

Modification ApproachApproach:

The antihistamine terfenadine (7.4, R = CH3) was

removed from the drug market because of

arrhythmias. Its metabolite fexofenadine (7.4, R =COOH) is as active, but does not produce

arrhythmias.

7.4

NHO

OH

H3 CR

CH3

terfenadine HCl (R = CH3)fexofenadine HCl (R = COOH)

HCl


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Pathways for Drug Deactivation and Elimination

Rate and pathway of drug metabolism are affected by

species, strain, sex, age, hormones, pregnancy, and liver

diseases.

Drug metabolism is stereoselective, if not stereospecific.

Generally, enantiomers act as two different xenobiotics

different metabolites and pharmacokinetics.

Sometimes the inactive enantiomer produces toxic

metabolites or may inhibit metabolism of active isomer.

Metabolism of enantiomers may depend on the route ofadministration.

For example, the antiarrhythmia drug verapamil is 16 timesmore potent when administered i.v. than orally.


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One enantiomer can be metabolized

to the other.

(Advil)

Inactive (R)-isomer is metabolized to

active (S)-isomerNo need to use a single enantiomer

ibuprofen7.10

* COOH

CH3


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Drug Metabolism

The Phase I reactions create a reactive

functional group on the molecule so that it canbe attacked by Phase II enzymes. Phase II

reactions are the true detoxification pathways

and give rise to products that account for thebulk of the inactive, excreted products of a drug.

Many of the enzymes involved in drug

metabolism are principally involved in themetabolism of, or are capable of metabolizing,

endogenous compounds.


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Phase I/II

Drug metabolism reactions two

categories Phase I transformations introduce or

unmask a functional group, e.g., byoxygenation or hydrolysis

Phase II transformations generatehighly polar derivatives (called conjugates)

for excretion


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Phase I Transformations

Oxidative Reactions Late 1940s, early 1950s

Metabolism of 4-dimethylaminoazobenzeneshown to require O2 and a reducing system

(NADPH). Called a mixed function oxidase.

One atom of O from O2 is incorporated intoproduct; a heme protein is involved.

Cytochrome P450 family of heme enzymesthat catalyzes the same reaction on different

substrates (isozymes)


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Phase I

A diverse array of reactions are performed by themicrosomal mixed-function oxidase system (cytochrome

P-450 dependent). The mixed-function oxidase is found in microsomes

(endoplasmic reticulum) of many cells (liver, kidney,lung, and intestine) and is able to carry out different

functionalization reactions. This is called a mixed function oxidase as both oxygen

and a reducing system (NADPH) is requiredone atomof oxygen is transferred to the substrate, and the other

undergoes a two electron reduction and is converted towater.

Cytochrome P450 represents a family of enzymes thatcatalyze the same reaction on different substrates. The

related enzymes are called isozymes.


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P-450

Cytochrome P450 catalyzes either hydroxylation or

epoxidation of various substrates, and is believed to

involve radical intermediates.

It is closely related with another enzyme NADPH-

cytochrome P-450 reductase, a flavoenzyme that

contains one molecule of flavin adenine dinucleotide

(FAD) and flavin mononucleotide (FMN).


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P-450

Heme, or protoporphyrin IX, is an iron(III) containingporphyrin cofactor for a large number of mixed function

oxygenases, particularly those belonging to the P-450family.

Molecular oxygen binds to the heme cofactor after

reduction of Fe3+ to Fe2+ and is converted to a reactiveform which is used in a number of oxygenation reactions.

The mechanism is still under debate. NADPH is requiredin the heme dependent enzymes to reduce the flavin

coenzymes used to transfer electrons to the heme andheme-oxygen complex.


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Heme

N

N

N

N

CO 2H

HO 2C

Fe3+

Protoporphyrin IX

Reactions Catalyzed by Cytochrome P450


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Reactions Catalyzed by Cytochrome P450Table 7.1

( X = NR, S )

R-X-R'

( X = N, O, S, halogen )RCHO + R'XH

RCH-XR'RCH2 -X-R'

RCHR'RCH2 R'

ArCHRArCH2R

Functional Group

R R OH

R R'

R'

R'R

R' O

OH

R

R

CH2R'

R

CHR'R

OH

O

R CHR'

O

CH2R'R

OH

OH

OH

R X R '

O

Product


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Site of Reactions Catalyzed by

P450Site determined by:

topography of the active site of the isozyme

degree of steric hindrance of the heme iron-oxo

species to the site of reaction

ease of H atom abstraction or electron transfer from

the compound


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Iron-Oxo Species

N

NN

NFeIII

Mechanism for formation of high energy iron-oxo species in heme dependent oxygenases.

S

OH H

N

NN

N

FeIII

S

R-HN

NN

N

FeII

S

R-H

NAD(P)H NAD(P)+

FAD FADH-

FMNH- FMN

FMN

O2

N

N

N

N

FeIII

S

OO

R-HN

N

N

N

FeIII

S

OO-

R-HR-H FMN FMN

H-B+

N

N

N

N

FeIII

S

OOH

R-H

B-H

N

N

N

NFeIII

S

O+N

N

N

NFeIV

S

ON

N

N

NFeV

S

ON

N

N

N

FeIV

S

O

ROH

-H2O


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Heme Dependent Oxidations

N

N

N

N

FeIV

O

HR'

R

R''

N

N

N

N

FeIV

OH

R'R

R''

N

N

N

N

FeIII

R'R

R''OH

oxygen

rebound

N

N

N

N

FeIV

O

R' R

R''

R' R

O

S S

S

S

N

N

N

N

FeIV

O

S

R R'

N

N

N

N

FeIII

S


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Tertiary Amine -Hydroxylation When more readily oxidizable groups are used, heme-dependent oxygenases can

also function as oxidases and reaction take place by electron transfer mechanisms.

N

N

N

N

Fe4+

O

+

N

N

N

N

Fe3+

O-

+

N

N

N

N

Fe3+

N

NN

N

Fe3+

Possible mechanism for alpha-hydroxylation of a tertiary amine by heme dependent cytochrome

ON

Ph

Ph

Me

MeO

NPh

Ph

Me

CH2

ON

Ph

Ph

Me

CH2

HO

ON

Ph

Ph

Me

C

H2

ONH

Ph

Ph

Me

OH-HCHO

H

+


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Aromatic Hydroxylation Reaction

A common reaction for drugs or xenobiotics containing a benzene ring is to

undergo ring hydroxylation. One example of this is the local anesthetic

lignocaine that is converted to its hydroxyl derivative.

CH3

HN

CH3

N(Et)2

O

CH3

HN

CH3

N(Et)2

O

OH


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Aromatic Hydroxylation

Boyland hypothesized in 1950 that aromatic compounds were metabolized first to the

corresponding epoxides. This was confirmed by a group at NIH that isolatednaphthalene 1,2-oxide from microsomal oxidation of naphthalene.

+ O2 + NADPH + H+

O

+ NADP+ + H2OP450

A id F ti


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Arene oxide Formation

Kinetic isotope studies indicate that direct areneepoxidation is unlikely, but rather an activated hemeiron-oxo species may add to the aromatic ring, similar tothe reaction with alkenes.

A tetrahedral intermediate is formed that can rearrangevia the epoxide or ketone pathway to finally give an

arenol.

The arene oxide can also undergo hydration by epoxide

hydrolase to give a trans-diol, react with glutathionecatalyzed by glutathione S-transferase to form a -hydroxy sulfide, as well as react with macromolecularnucleophiles.


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Addition-Rearrangement Mechanism

OO OH

N

N

N

N

FeIV

S

O

R

N

N

N

N

FeIV

S

O

HR

RRR

N

N

N

N

FeIII

S

O

HR

electron

transfer

+

N

N

N

N

FeIII

S


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Reactions of Arene Oxides

O

R

H H

HO

OH

R

GS

OH

X

OH

macromolecular

nucleophile

glathione S-transferase

GSH

epoxide hydrolase

H2O

R

R


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Glutathione

The tripeptide glutathione is found in virtually all mammalian tissues.It contains a potent nucleophilic thiol group, and its function appearsto be to scavenge harmful electrophiles that are ingested orproduced by metabolism.

Drug toxicity can result from the reaction of cellular nucleophiles withelectrophilic metabolites if GSH does not intercept them first.

Electrophilic species include any group capable of undergoing SN2,SNAr-like reactions, acylations, Michael additions, reductions(disulfides and radicals). All of the reactions catalyzed by glutathioneS-transferase occur nonenzymatically as well, but at a slower rate.

-O2C

HN

NH

NH3+

O

HS

O

CO2-

NIH Shift


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NIH Shift

Rearrangement of an arene oxide to arenol is known as the NIHshift. Ring opening occurs in the direction that gives the most stable

carbocation. Because of an isotope effect on cleavage of the C-D

bond, the proton is preferentially removed.

R

D

R R

OD

H

P-450

O2, NADPHD-O

+

R

D

O

H:B

H+

R

HO

D

The NIH Shift.


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Direct Loss of H+ or D+

Competing pathway to NIH shift is simple lost of a proton or

deuterium from the cation intermediate.

This depends upon R; the more stabilizing the R group is the moredeprotonation that occurs (when R is NH2, OH, NHCOCF3 or

NHCOCH3 only 0-30% of the product retains deuterium; when R is

Br, CONH2, F, CN, or Cl, 40-54% retention of D is found)

R R

OD

H

D-O

+

:B

R

HO

H

Deprotonation Pathway.

Migration of Other Groups in NIH


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Migration of Other Groups in NIH

Shift The NIH shifts also works with substituents other than H,

like chlorine. Rat liver metabolizes p-chloroamphetamine

to 3-chloro-4-hydroxyamphetamine.

Cl

CH 3

NH 2Cl

CH 3

NH 2

Cl

CH3

NH 2

O

+-O

CH 3

NH 2

CH 3

NH 2

Cl

OHO

Cl


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Selectivity in Hydroxylations

The more electron rich the aromatic ring, the faster the

aromatic hydroxylation takes places. Aniline undergoes

ortho and para hydroxylation.

Electron deficient drug like probenecid undergoes no

detectable hydroxylation. For drugs with two or more

aromatic rings, generally the more electron rich one ishydroxylated. The antipsychotic chlorpromazine

undergoes hydroxylation at the 7-position.

HO 2C SO2N(n-Pr)2

Probenecid

N

SR

Cl

NM e2


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This reaction is electrophilic aromatic substitutionFavors electron-donating substituents

No aromatic hydroxylation

probenecid7.24

HOOC SO2N(CH2CH2CH3)2

e- withdrawing

uricosuric

agent

A common approach to slow down or block


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A common approach to slow down or block

aromatic hydroxylation is to substitute the phenyl

ring with a para-fluorine or para-chlorine

(deactivates the ring).

The half-life for the anti-inflammatory drug

diclofenac (7.21) is 1 h; for fenclofenac (7.22) is

>20 h.

diclofenac7.21

COOH

NH

ClCl

fenclofenac7.22

COOH

O

Cl

Cl


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Species Specificity Aromatic hydroxylation is species specific and the site of the

reaction can change in one species to the next. In the case of the

antiepilepsy drug phenytoin, this is para-hydroxylated in the pro-(S)

phenyl ring (R1 = OH) 10 times more often than the pro-(R) ring (R3

= OH). In dogs meta-hydroxylation of the pro-(R) ring (R2 = OH)

takes place.

.

HN

NH

OO

R3

R2

R1 humans

dogs

M H d l i


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Meta-Hydroxylation

Meta-hydroxylation may be catalyzed byan isozyme of P-450 that works through adifferent mechanism.

Metabolite of chlorobenzene is 3-

chlorophenol; however, neither 3- nor 4-chlorophenol oxide afford 3-chlorophenolin the presence of rat liver microsomes.

Direct insertion mechanism may occur inthis case.

Further Reactions of Arene Oxides


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The arene oxides are very reactive and react rapidly with

nucleophiles. Toxicity can result from their reaction with cellular nucleophiles.

Epoxide hydrolase catalyzes the hydration of arene oxides to givetrans-dihydrodiols.

The reaction involves general base-catalyzed nucleophilic attack ofwater, with attack occurring from the backside at the less stericallyhindered side.

The trans dihydrodiol product can be oxidized to catechol; thecatechols are further oxidized to ortho-quinones or semiquinones

HOOH

R

HO

OH

R

O

O

R

O

OH

R

Mechanism of Epoxide Hydrolase


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Mechanism of Epoxide Hydrolase

Hydration of Arene Oxide

R R

OH

HO

R

OH

R

OH

O

R

O

O

HO

OH

R

OO O-

R

OH

O O

H

O H

B:

[O]

O O-

Anti-attack

GSH R ti


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GSH Reactions

Glutathione S-transferase is another enzyme that protects cell fromelectrophilic arene oxide metabolites. The adducts can undergo

rearrangement upon dehydration.

GSH

OOH

SG

P450

OSG

OH

HO

SG

SG

OHGSH

only isomernot detected

Carcinogens


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g

If arene oxides escape enzymatic reaction, toxicity may result.

Benzo[a]pyrene is metabolized to a potent carcinogen found in soot.

The resulting arene oxide can react with RNA, DNA and proteins to

generate covalent adducts.

Covalent bond formation of the benzopyrene with DNA leads to malignantcellular transformation. The arene oxide reacts with DNA to form a covalent

adduct with the C-2 amino group of guanosine.

HO

OHO

HO

OH

HO

OH

OHO

NHN

NHN

N

O

R

Alkene Epoxidation


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p

Alkenes also undergo epoxidation by P-450, and are more reactivethan aromatic systems. The anticonvulsant agent carbamazepine isconverted to its epoxide (the metabolite may be responsible foranticonvulsant activity). This is converted in turn to the trans diol byepoxide hydrolase, and then conjugated to the glucuronide by UDP-

glucuronosyltransferase.

N

H2NOC

N

H2NOC

N

H2NOC

HOOG

N

H2NOC

HOOHO

epoxide

hydrolase

UDPGT

Aliphatic Hydroxylation


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Another very common reaction is hydroxylation in the aliphatic side

chain as shown in the case of pentobarbitone. In practice, a non-

activated alkyl group undergoes and -1 oxidation. n-Hexadecaneis oxidized to hexadecanol in the liver, and this then further oxidized

to hexadecanoic acid.

HN

HN

O

O

O

CH3

(CH2)2-CH3

CH3

Pentobarbitone

HN

HN

O

O

O

CH3

CH 3OH



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In the case of the sedative-hypnotic (-)-glutethimide thisis oxidized at the -1 position (ethyl group)butenantiomer difference in metabolism.

glutethimide (R = R' = H)7.40

N

H

O O

R Ph

R'

hydroxylation herefor (+)-isomer

hydroxylation here

for (-)-isomer

sedative/hypnotic

Preferential Oxidation


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In the case of activated sp3 carbon atoms in allylic, propynylic, or

benzylic positions, these activated carbon atoms are preferentiallyhydroxylated.

Carbons adjacent to carbonyl or imine groups can also be oxidized.

(ease of oxidation parallels C-H bond dissociation energiesOH

HOmajor minor

R R

HO

RX

RX

OH

RX-H

O

+

R R

OH

Benzylic Oxidation


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Benzylic Oxidation

Stereochemistry in side chain can influence stereochemical course

of the oxidation; 2S-metoprolol gives 1R,2S/1S,2R metabolite ratio

of 9.4, whereas 2S gives a ratio of 1R,2S/1S,2S of 26.

OHN

MeO

Hs HR

OH

antihypertensive drug metoprolol

hydroxylation takes place here, 1'R-hydroxylation is preferred

21'

Stereochemistry at C-2 will affect how the molecule binds in P450, whichdetermines which H is closest to the heme iron-oxo species.


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Hydroxylation beta to a CarbonylGroup

flutamide7.43

H3 C CH3

OHN

F3 C

NO2

H3 C CH3

OHN

F3 C

NH2

H3 C CH2

OHN

F3 C

NH2

OH

7.44

H3 C

OHN

F3 C

NH2

7.45

H2 C

OHN

F3 C

NH2

OH

CH3

OHN

F3 C

NH2

7.46

-CH2O -CH2O

Scheme 7.15

Oxidations of Carbon-Nitrogen


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Systems

RCR'

O

RC

O

NH2 RC NHOH

O

ArN O

NH4+

ArNH2

-H2 O

ArNHOH ArNO2

RC NOH

+

R

RC NH

R

RCHNH2

R'

RCHNH2

R'

RCHNHOH

R'

RCHNO2

R'

Table 7.3

Oxidations of 1 amines and amides

Oxidation of C-N Systems


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Oxidation of C-N Systems In this preferential oxidation, oxidation by P50 occurs alpha to a

heteroatom like N, O, or S. With amines hydroxylation leads to an

aminal that is broken down to a dealkylated amine plus an aldehyde.

This results in dealkylation of the amine, or to deamination when the

substrate loses an amino group.

RN

RN

OHR

N

O

+

R' R' R'

H

RN

R'

RN

R'

OH

OH

minor products

+

privileged attack

Oxidative Deamination


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O da e ea a o

amphetamine7.48

NH2

P450

18O2

Ph18O

+ NH3

Flavin monooxygenases and P-450


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Primary amines are also converted by N-oxidation to thecorresponding hydroxylamines catalyzed by flavin

monooxygenases. IN GENERAL - Basic amines (pKa 8-11) are oxidized by

flavoenzymes and non-basic nitrogen compounds likeamides by cytochrome P-450, while intermediate basicity

compounds like aromatic amines are oxidized by both. Amphetamine is converted by N-oxidation to

corresponding hydroxylamine, oxime, and nitro

compounds.

Amphetamine Oxidation


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NH2

flavin mono-

oxygenase NH

H

OH

B:

NH

N

OH

N

H

OHOH+

B:

NN+

OHHO OH

NO2

:B

H+

O+ NH2OH

Reaction Mechanism of Flavin Monooxygenases


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The flavin monooxygenases incorporate an oxygen from molecular

oxygen into their substrates. Flavin is converted to its reduced form

by NADH of NADPH, and this then initiates the oxygenation reaction

through formation of an intermediate flavin hydroperoxide.

N

N

NH

N O

OHHO

B:

N

N

NH

N O

O

N

N

NH

N- O

OH

R

R

N

H H

R

O

NH2

R

-H2O

N

N

NH

N O

OH

R

O

HO

R-NH2

B+-H

O2

NH

N

NH

N O

OH

R

O

R-NH2

OH

+

R-NHOH

Secondary Amine Oxidation


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Secondary amines are metabolized by oxidative N-

dealkylation, oxidative deamination, and N-oxidation.

The aldehyde metabolites can be further oxidized by

aldehyde oxidases or dehydrogenases to corresponding

carboxylic acids. Secondary hydroxylamine formation iscommon, and further oxidation to nitrone can take place.

F3C

HN

F3C

N

F3C

N+HO -O

N-Oxidation of Fenfluramine to its Nitrone.

Metabolism of 2 Amines and Amides

Table 7 4


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+

+

+

+RCH

R'

RC

O

RCO

NHCH2R'

RCH2 NHR'

R C

RC NH2

O

O

R'

RCH N

HCR2

O

RCH2N

OH

Ar NR'

OH

RC NR'

OH

O

O-

NHR'

R' R'

RCH NR'

NHCH2R2

NHCH2R2

NH2

NH2CH2R2

OHCR'

ArNHR'

RCH

R'

RCH

R'

oxidativeN-dealkylation

oxidativedeamination

N-oxidation

difference iswhich side of Nis oxidized

Table 7.4

Oxidation of Propranolol


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O NH

OH

O NH

OH

OH

O NH 2

OH

O

O N

HOH

OH

O

H2N

OH

O

O

OH

O

OH

+

Oxidative Metabolism of the beta-Blocker Propranolol.

Aldehyde

dehydrogenase

Oxidation of 3 Amines andAmides


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Amides

+

+

+

RCH NCH2R2

R' CH2R3

RC

O

NCH2R2

R'

ArNR'

R

RCH

R'

NHCH2R3

ArN R'

R

O-

RC

O

NHR' HC R2

O

R2HC

O

R3 N+ O-R3N


N-oxidation


Table 7.5

No oxidative deamination

Tertiary Amine Oxidation


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y

Tertiary amines are metabolized by oxidative

dealkylation and N-oxidation. The tricyclic antidepressant

imipramine is metabolized to the secondary aminedesmethylimipramine.

N

NMe

Me

N

NMe

H

Scheme 7.22

CH


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deprenyl

7.57

metamphetamine

7.58

P450 P450N

H3 C

CH3

H3 C

NHCH3

H H H3 C

NH2

H

amphetamine

(S)-(+)-deprenyl (S)-(+)-metamphetamine (S)-(+)-amphetamine

weak MAO B inhibitorundesirable CNS stimulant

(R)-(-)-deprenyl (R)-(-)-metamphetamine (R)-(-)-amphetamine

potent MAO B inhibitor

weak CNS stimulant

Therefore only the (R)-(-)-isomer is used

Nicotine (cyclic tertiary amine) metabolism is shown below. Theimmonium ion intermediate is very electrophilic, and can be trapped by

id i it


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cyanide in vitro.

N

N

CH3 N

N

CH3 N

N+

CH3 N

N

CH3

OH CNCN-

N

N

CH3

O

N

N

CH3

O

HO

N

HN

CH3

OH

O

N

N

CH2OH N

N+

N

N

H2CCH2

CN

N

NH

Routes of Nicotine Metabolism.

-HCHO

-OH-

N-Oxide Formation


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N-oxidation of tertiary amines gives the N-oxides that are chemicallystable. These do not undergo further oxidation. The antihistaminecyproheptadine is converted to the N-oxide in dogs.

N

CH 3

N+

CH3

O-

Tertiary Aromatic Amines


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Tertiary aromatic amines are N-oxidized by a flavoproteinmonooxygenase and by cytochrome P-450. The P-450 N-oxidationappears to occur when there are no alpha hydrogens are available

for abstraction.

N

N

N

NFe4+

O

+

N

N

N

NFe3+

O-

+

N

N

N

NFe3+

R N

R'

R' R N

R'

R'

R

N+

R'

R'

O-

Two Mechanisms for N-Demethylation of Tertiary

Aromatic Amines


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Aromatic Amines

Ar N

CH3

CH3

Ar N+ O-

CH3

CH3

Ar N

CH2

CH3

Ar N

H

CH3

HO

Ar N+ OH

CH2-

CH3

Ar N+

CH2

CH3

OH- -HCHO

P450

flavinmonooxygenase

Mechanism of Carbinolamine


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Formation

Scheme 7.28

Based on low intrinsic isotope effects by P450, direct

H abstraction mechanism was excluded.

...

.+

..

+ .

.++ .

N N

N NFe4+

O

NAr

N N

N NFe3+

O-

NN

N NFe3+

HO

N CH2Ar

R

H

N CH2

N

R

Ar CH2 N CH2Ar

R

Ar

R

OH

R

CH3

Metabolic Activation of Amines


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The oxidation of primary and secondary aromatic amines leads tothe generation of reactive electrophilic species that form covalentbonds to cellular macromolecules.

Bond formation to proteins, DNA and RNA is known.

NR'

H

R

NR'

OH

R

NR'

OX

R

X+

Y

NR'

RY

HN

R'

RY

H

:B

H-B+

-OX

X = acetyl or sulfate

Amide Oxidation

Amides are also metabolized by oxidative N-


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Amides are also metabolized by oxidative N

dealkylation and N-oxidation.

Diazepam undergoes extensive demethylation.

N

N

Cl

OMe

Aromatic Amide Oxidation


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N-oxidation of aromatic amides can lead to electrophilic intermediates 2-acetylaminofluorene undergoes P-450 catalyzed oxidation to the N-hydroxy analog that can lead to an electrophilic species after activation ofOH by N,O-acyltransferase.

N

OH

R

NOH

H

R

NH

O

R

NH

R

X

NH2

RX

H

-OX

O

XX O

X

O

NH

O

Oxidations of Carbon-Oxygen Systems

Oxidative O-Dealkylation


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y

Same mechanism as oxidative N-dealkylation

O-Demethylation is rapid; as increase alkyl chainlength, O-dealkylation gets faster up to propoxyl,

then rate decreases.Cyclopropyl gives ethers with longer plasma half

lives.

Oxidative O-Dealkylation


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codeine (R = CH3)morphine (R = H)7.80

O

HHCH

3N

OHRO

analgesicO-Demethylation is rapid

Regioselective O-Demethylation


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methoxamine (R = CH3)

7.81

CH3O

NH2

OHROCH3

In dogs O-demethylation only here

blood pressure maintenance

Oxidation on the Carbon Next to a


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Lactone Oxygen

O

SO2CH3

O O

SO2CH3

O OH

rofecoxib7.82

7.83

Scheme 7.34

Sulfur Oxidation

S-oxidation to sulfoxides is catalyzed by both flavin monooxygenaseand by cytochrome P-450.


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y y

The flavin enzyme produces only the sulfoxide while P-450 givesboth S-dealkylation products and sulfoxides probably via thesulfenium cation radical.

N

N

N

N

Fe4+

O

+

RC H2 S

R

RC H2 S

R

N

N

N

N

Fe3+

O-

+

RC H2 S+

R

RCH S

R

O rebound

H+ transfer

N

N

N

N

Fe3+

OH

+RCH S

R

HO

RCHO + -SR

O-

Thioridazine Oxidation


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Thioridazine is oxidized on both sulfur atoms to thesulfoxides; the S(O)Me metabolite (without ring oxidation)is twice as potent as the parent compound and is alsoused as an antipsychotic drug

N

S

N

SMe

Me Thioridazine, antipsychotic


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albendazole7.87

N

HN

NHCO2CH3

S

antihelmintic agent

Gives both S-dealkylation

and S-oxidation metabolites

Thiophenes are converted to thiophene S-oxides,

which are electrophilic and can bind to liver


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proteins.

Scheme 7.36

S

OCl Cl

OCH2COOHP450

S

OCl Cl

OCH2COOH

tienilic acid7.89 O-

HS

OH

S

OCl Cl

OCH2COOH

O-S

OH

7.90

added in vitro to mimic

a liver protein cysteine

residue

Oxidation of Sulfoxide to Sulfone


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immunosuppressive

oxisuran7.91

N

O

SCH3

O-

N

O

SCH3

O

O

Desulfuration (C=S C=O)


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7.85

HN

NH

O

CH3O

H

X

thiopental (X = S) - anesthetic

pentobarbital (X = O) - sedative

Other oxidations brought about by P-450 are oxidative


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g ydehalogenation, oxidative aromatization, and conversion of

arenols to quinones. The anesthetic haloethane ismetabolized to trifluoroacetic acid. The intermediate acid

chloride that is formed can bind covalently to livermicrosomes.

F3C Br

H

Cl

F3C Br

OH

Cl

F3C

O

Cl

P450 -HBr

H2O F3C

O

OH

Additional Mechanistic ConsiderationsArchives of Biochemistry and BiophysicsVolume 409, Issue 1 , 1 January 2003, Pages 72-79

Many of the qualitative results from studies of P-450 catalyzed hydroxylation
http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=6701&_auth=y&_acct=C000013678&_version=1&_urlVersion=0&_userid=186797&md5=18343a6f99c2f23f2d5a441667157db4http://www.sciencedirect.com/science?_ob=IssueURL&_tockey=%23TOC%236701%232003%23995909998%23367912%23FLA%23display%23Volume_409,_Issue_1,_Pages_1-241_%281_January_2003%29%2BMSpecial_Issue_dedicated_to_R.W._Estabrook%2BMEdited_by_Dr._Michael_Waterman_and_Dr._H._Sies%23tagged%23Volume%23first%3D409%23Issue%23first%3D1%23Pages%23first%3D1%23last%3D241%23date%23%281_January_2003%29%23specissname%23Special_Issue_dedicated_to_R.W._Estabrook%23specisseditor%23Edited_by_Dr._Michael_Waterman_and_Dr._H._Sies%23&_auth=y&view=c&_acct=C000013678&_version=1&_urlVersion=0&_userid=186797&md5=7da9809989fbc545bc54fba50ac92394http://www.sciencedirect.com/science?_ob=IssueURL&_tockey=%23TOC%236701%232003%23995909998%23367912%23FLA%23display%23Volume_409,_Issue_1,_Pages_1-241_%281_January_2003%29%2BMSpecial_Issue_dedicated_to_R.W._Estabrook%2BMEdited_by_Dr._Michael_Waterman_and_Dr._H._Sies%23tagged%23Volume%23first%3D409%23Issue%23first%3D1%23Pages%23first%3D1%23last%3D241%23date%23%281_January_2003%29%23specissname%23Special_Issue_dedicated_to_R.W._Estabrook%23specisseditor%23Edited_by_Dr._Michael_Waterman_and_Dr._H._Sies%23&_auth=y&view=c&_acct=C000013678&_version=1&_urlVersion=0&_userid=186797&md5=7da9809989fbc545bc54fba50ac92394http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=6701&_auth=y&_acct=C000013678&_version=1&_urlVersion=0&_userid=186797&md5=18343a6f99c2f23f2d5a441667157db4


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reactions is consistent with abstraction-rebound pathway. Partial isotopic scrambling of deuterium was observed in hydroxylation of

norbornane, and allylic shift occurred in hydroxylation of labeledcyclohexene. These results indicate that a radical in a radical pair thatrecombined rapidly was formed.

Bicyclo[4.1.0]heptane underwent oxidation at the cyclopropylcarbinyl positionwithout ring opening. This was considered to exclude a cationic intermediate;however, this presupposes that only the carbocation can undergorearrangement.

Radical Clock Relatively large hydrogen-deuterium kinetic isotope effects in P450

catalyzed hydroxylations were also taken as evidence that radical


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y y y

intermediates are produced, although any type of C-H functionalization

reaction should have a KIE.

Most convincing evidence found from radical clock study. A radical

clock study involves use of a substrate that gives a radical with aknown rate constant for rearrangement.

As in Scheme below with the cyclopropylcarbinyl radical, if the radical

is trapped by reagent X-Y in competition with rearrangement, then the

rate constant for trapping kT can be determined from the productdistribution and the known rate constant for rearrangement (kR).

Bicyclo[2.1.0]pentane Rearrangement

Bicyclo[2.1.0]pentane is hydroxylated to give


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unrearranged product, and rearranged cyclopent-3-ol, thelater presumably formed by ring opening of thebicyclo[2.1.0]pent-2-yl radical. Subsequent studiesdetermined the rate constant for ring opening of theradical, and this value could be used with the results togive a radical rebound rate constant of 1.4 x 1010s-1, or alifetime of (t = 1/k) of 70 ps.

Mechanistic Picture Not Clear


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A dozen radical clocks were used in studies of P450 2B1and 2B4 hydroxylations, but no consistent trends werefound.A plot of the logarithm of the ratio of unrearrangedto rearranged alcohol products versus the logarithm ofthe radical rearrangement rate constant, which shouldhave a slope of unity if the rebound rate constant is the

same for all radicals, had a slope of 0.2 +/- 0.4. The correlation coefficient (r) for this plot was 0.3, which

indicates that the data are more likely uncorrelated thancorrelated.In the context of a single pathway forhydroxylation involving abstraction and rebound, most ofthe results had to be explained as special cases.whichmeans that the mechanistic picture is not clear.

Cationic Intermediate?


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In order to test for the possibility of a cationic intermediate, asubstrate was needed that gave different products for a radicalpathway versus a cationic pathway.

Studies were made using a hypersensitive radical probe, such

as use of a cyclopropyl system. A cyclopropylcarbinyl radicalring opens to give predominantly the benzylic radical products(> 50:1), and incipient cyclopropylcarbinyl cations rearrange togive only products derived from the oxonium ion.

No Discrete Radicals

Th f di l d i d d f b 11


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The amounts of radial-derived products from probes 11 werevery small, even though the radicals derived from thesesubstrates rearrange with rate constants greater than 5 x 1011

s-1. From the small amount of rearranged products, one

calculates radial lifetimes in P450-catalyzed hydroxylations inthe range of 0.08 to 0.2 ps, which are too short for true radicalintermediates, but correspond to lifetimes of transition states.Thus, probes like 11 indicate that no discrete radicals were

formed in the hydroxylation reactions.

Homocubanol System

F b t t 12 th t f ti d i d d t h b l i df 0 30% f h l f id i h h l i i Th


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For substrate 12, the amount of cation-derived product homocubanol variedform 0 to 30% of the total amount of oxidation at the methyl position. Theradical reacts by cleavage of the cube bonds. The large variance in theamount of cation-derived product suggests a complex mechanistic picture.

This strong evidence for cations of some type provides a possibleexplanation as to why radical clock studies had given inconsistent radicallifetimes. All of the radical lifetimes determined from probes that did not givedifferent products from radical and cationic intermediates can be understoodif cations are formed in the oxidations. Results from such probes can only beused to set an upper limit on radial lifetimes.

Cationic Intermediate Growing radical lifetime quantitation problem led to reconsideration

that cationic intermediates may be involved in the P450 catalyzed

h d l ti I t f di l l k ti i


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hydroxylations. In most cases of radical clocks, a cationicrearrangement would give the same products as the radicalintermediates. There is a question as to how cationic intermediate isgenerated; in case of 11, the radical lifetime is too short for thisradical to be oxidized. Thus, the direct insertion of OH+ would haveto take place. This means oxidation by a precursor to the iron-oxospecies, either the hydroperoxo-ion or iron-complexed hydrogenperoxide would take place.

Another source of cationic rearrangement products would

be solvolytic type rearrangement reactions of protonated

alcohols --- these are formed by insertion of OH+.


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Mutated Enzyme

Besides the iron-oxo species, the hydroperoxo-iron intermediate mayplay a role. There is a threonine residue in the active site of P450

enzymes that is thought to play a role in the protonation reactions of


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enzymes that is thought to play a role in the protonation reactions ofthe peroxo-iron or hydroperoxo-iron species. This residue was thusmutated and reactions examined. Probe 7 was examined using bothwild type that contains a short N-terminal deletion and mutantenzymes. Probe 7 can be oxidized at either the methyl group or thephenyl group. Increased amounts of phenol were produced by themutants. 85% methyl oxidation for 2B4 reduced to 44% for 2B4T302A and 81% methyl oxidation for 2E1 reduced to 33% for 2E1T303A. Changes ascribed to amounts of oxidation brought about byiron-oxo for hydroperoxo-iron species. Also, with probe 13, morerearranged product was found with 2E1 T303A (38%) versus 2E1

(9%). The phenol forming reaction is suppressed by the trifluoromethylgroup.

Two Spin States


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Another suggestion has been made that the iron-oxospecies has different spin states, and that reactionvia different spin states could have different

outcomes. There is a low spin doublet state, and ahigh spin quartet state. After reaching the TS forabstraction, the energetics of the two pathwaysdiverge. The reaction on low-spin surface proceeds

through a radial-like species that collapses with nobarrier to give alcohol productthis is effectively aninsertion. The reaction on the high spin surface hasa considerable barrier to collapse, and this pathway

gives a true radical intermediate. The reactions onthe high spin surface would give radicals that couldrearrange.

P450 oxidations are thus complex.

It seems possible that the hydroxylation reactions could be explainedby the qualitative picture in Fig 4 where two electrophilic oxidants


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by the qualitative picture in Fig.4, where two electrophilic oxidantsexist and two spin states of ironoxo are reactive.

In this model, the early oxidant reacts by insertion of OH+ to giveprotonated alcohols as first-formed products, and these species are

the origins of cationic rearrangement products.The low-spin iron-oxoensemble reacts by insertion of oxygen in a process that resemblesthe oxene insertion pathway proposed many years ago.The high-spiniron-oxo species abstracts hydrogen to give a radical intermediate in aprocess that resembles the oxygen-rebound pathway.

HDACs


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Histone deacetylase enzymes have beendivided into three distinct structural classes,

operate by zinc-dependent (class I/II) or NAD-dependent (class III) mechanisms.

There are 18 HDACS in humans and there are

many splice variants. Class I includes 1,2,3,8,Class II includes 4,5,6,7,9,10 and 11. Class III ismade up of SIRT 1-7 (sirtuins).

Class I/II histone deacetylase (HDAC) enzymesare an emerging therapeutic target for thetreatment of cancer and other diseases.

Tumorigenesis


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These enzymes, as part of multiprotein complexes, catalyze theremoval of acetyl groups from lysine residues on proteins, includinghistones.

Tumour cells must be able to circumvent APOPTOSIS, replicateindefinitely and sustain growth and survival by maintaining asustainable oxygen and nutrient supply. Mutations that result inconstitutive activation of ONCOGENES or functional inactivation ofTUMOUR-SUPPRESSOR GENES are important tumorigenic

events. Moreover, aberrant transcription of the genes that are needed toinitiate the host antitumour immune response and induceneovasculature can result in tumour immune escape and

ANGIOGENESIS events that are essential for cancer progression

Chromatin Remodelling


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There has been a rapid advancement in ourunderstanding of the molecular processes that lead tothe activation or repression of transcription, and

chromatin architecture has emerged as the foundationfor gene regulation. CHROMATIN REMODELLINGwhich is controlled by factors that relocate nucleosomesand alter nucleosome structure after post-translational

modification of histone tails directly affects geneexpression.

In cancer, the molecular processes that lead toinappropriate expression of genes due to altered

chromatin structure are now being identified, andaberrant acetylation of histone tails has been stronglylinked to carcinogenesis.

Reprogram Transcription


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Thus, targeting the transcriptional lesions thatlead to neoplasia provides an opportunity fortherapeutic intervention at the very apex of thetransformation process. Such therapies couldaffect several molecular programs, and wouldtherefore be more powerful than targeting the

end stages of a single disrupted molecularpathway.

Understanding how gene expression can be

regulated opens the way for new molecular toolsto reprogram transcription and inhibit tumour-cellgrowth and progression.

Condensed and Open Chromatin

All f th h i k d i t h ti hi h i


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All of the human genome is packaged into chromatin, which is adynamic macromolecular complex that consists of DNA, histonesand non-histone proteins.

Nucleosomes form the basic repeating unit of chromatin, and consistof DNA wrapped around a histone octomer that is formed by fourhistone partners an H3H4 tetramer and two H2AH2Bdimers.The linker histone H1 stabilizes the higher-order folding byelectrostatic neutralization of the linker DNA segments through a

positively charged carboxy-terminal domain. So, the dynamic higher-order structure of nucleosomes defines distinct levels of chromatinorganization and, subsequently, gene activity (FIG. 1). In generalterms, condensed chromatin mediates transcriptional repression,whereas transcriptionally active genes are in areas of open

chromatin.

Covalent Modification of Histone

TailsI d d hi t t il ti l l H3 d H4 t t d f i


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Indeed, histone tails, particularly H3 and H4, are targeted for variouspost-translational modifications, including acetylation,phosphorylation and methylation (FIG. 1). Covalent modification ofcore histone tails by histone acetyltransferases (HATs), HDACs,

methyltransferases and kinases offers a mechanism by whichupstream signalling pathways can converge on common targets toregulate gene expression. In addition, chromatin structure can alsobe regulated by protein complexes that use ATP hydrolysis toreposition nucleosomes.

Although ATP-dependent chromatin remodellers were initiallycharacterized as factors that promote gene activation, it is nowknown that they can cooperate with either HATs or HDACs, andtherefore have dual roles in activating and repressing transcription.

So, chromatin can be remodelled by the integrated activities ofhistone-tail-modifying enzymes and ATP-dependent factors.

Chromatin Structure


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are

highly conserved

H1

H2A

Linker histone


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highly conserved,small, basic proteins

H2B

H3

H4

helix

variable

conserved

Histone acetylationis a reversible modification

of lysines in the N-termini

of the core histones.

Result: reduced binding to DNA

destabilization of chromatin

Core histones

N


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H3-H4tetramer H2A-H2B

dimer

Histoneoctamer


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H4 white

H3 green

H2A light blue

H2B dark bluered: + (arg, lys) orange: -OH (ser, thr)

>


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Each core histone dimerhas 6 DNA binding surfaces

that organize 3 DNA turns;

The histone octamer

organizes 145 bp of DNAin 1 3/4 helical turn of DNA:

48 nm of DNA packaged in a disc of 6 x

11nm< 6 nm >

Documents

4.11.Drug Metabolism Cytochrome P450