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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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Aliphatic Hydroxylation
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
Aliphatic Hydroxylation
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Aliphatic Hydroxylation
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
oxidativeN-dealkylation
N-oxidation
oxidativeN-dealkylation
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=18343a6f99c2f23f2d5a441667157db47/31/2019 4.11.Drug Metabolism Cytochrome P450
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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 >