Subjects to be covered

(Professor Kozikowski) Cytochrome P450, metabolism mechanisms HDACs and Inhibitors cancer applications Kinases phosphate transfer, inhibitor development for cancer some SAR studies

Metabolism
Sum of processes by which particular substances are handled by the body. From the Greek, metabole -- change

Drug Metabolism Cytochrome P450

Substrates 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 functionalization reactions; And Phase II, which are the conjugation reactions.

Drug Metabolism
Foreign organism elicits antibody response Low molecular weight xenobiotics nonspecific enzymes convert them into polar molecules for excretion Enzymatic biotransformations of drugs drug metabolism

Principal site of drug metabolism is the liver; also kidneys, lungs, GI tract Pathway of Oral Drugs
take via mouth absorbed through small intestine or stomach bloodstream

liver (first metabolized)

Drug metabolism by liver enzymes first-pass effect

Example of Phase I and II

Phase I Phase II

OH

O-SO3H

Drug Metabolism
Drug metabolism is desirable once drug has reached site of action may produce its 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.

Metabolism Studies Can Be a Useful Lead Modification Approach Approach: 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 H C arrhythmias. R
3

HCl

CH3 OH

HO

terfenadine HCl (R = CH3 ) fexofenadine HCl (R = COOH) 7.4

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 of administration. For example, the antiarrhythmia drug verapamil is 16 times more potent when administered i.v. than orally.

One enantiomer can be metabolized to the other.

CH 3

COOH

ibuprofen (Advil) 7.10

Inactive (R)-isomer is metabolized to active (S)-isomer No need to use a single enantiomer

Drug Metabolism
The Phase I reactions create a reactive functional group on the molecule so that it can be attacked by Phase II enzymes. Phase II reactions are the true detoxification pathways and give rise to products that account for the bulk of the inactive, excreted products of a drug. Many of the enzymes involved in drug metabolism are principally involved in the metabolism of, or are capable of metabolizing, endogenous compounds.

Phase I/II
Drug metabolism reactions two categories Phase I transformations introduce or unmask a functional group, e.g., by oxygenation or hydrolysis Phase II transformations generate highly polar derivatives (called conjugates) for excretion

Phase I and Phase II Reactions

Table 1. Reactions classed as phase I or phase II metabolism.
Oxidation Reduction Hydrolysis Hydration Dethioacetylation Isomerization Glucuronidation/glucosidation Sulfation Methylation Acetylation Amino acid conjugation Glutathione conjugation Fatty acid synthesis

Phase I Transformations
Oxidative Reactions
Late 1940s, early 1950s Metabolism of 4-dimethylaminoazobenzene shown to require O2 and a reducing system (NADPH). Called a mixed function oxidase. One atom of O from O2 is incorporated into product; a heme protein is involved. Cytochrome P450 family of heme enzymes that catalyzes the same reaction on different substrates (isozymes)

Phase I
A diverse array of reactions are performed by the microsomal 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 atom of oxygen is transferred to the substrate, and the other undergoes a two electron reduction and is converted to water. Cytochrome P450 represents a family of enzymes that catalyze the same reaction on different substrates. The related enzymes are called isozymes.

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 NADPHcytochrome P-450 reductase, a flavoenzyme that contains one molecule of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).

P-450
Heme, or protoporphyrin IX, is an iron(III) containing porphyrin cofactor for a large number of mixed function oxygenases, particularly those belonging to the P-450 family. Molecular oxygen binds to the heme cofactor after reduction of Fe3+ to Fe2+ and is converted to a reactive form which is used in a number of oxygenation reactions. The mechanism is still under debate. NADPH is required in the heme dependent enzymes to reduce the flavin coenzymes used to transfer electrons to the heme and heme-oxygen complex.

Heme

N N Fe3+ N N

CO 2 H HO 2 C

Protoporphyrin IX

Reactions Catalyzed by Cytochrome P450

Table 7.1
Functional Group Product

R R' R R'

R O R' R R'

OH

ArCH 2R R R O R CH2R' R R

ArCHR OH CH2R' CHR' R O CHR' OH OH

RCH2 R'

RCHR' OH

RCH2 -X-R' ( X = N, O, S, halogen ) R-X-R' ( X = NR, S )

RCH-XR' OH

RCHO + R'XH

R X R' O

Site of Reactions Catalyzed by P450

Site 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

Iron-Oxo Species
H N N O H N

H-B + R-H R-H

N N FMNH FMN N

R-H
N
FMN

R-H O2
N

R-H
III

Fe
S

III

N N

Fe
S

N FMN

FMN N

ON

Fe III
N S

FeIII
S FAD FADH -

Fe II
S

NAD(P)H

NAD(P) +

ROH
B-H N N O N N O N N O N N O+

R-H
N

FeIV
N S

OH N

FeV
N S

Fe
S

IV

Fe
S

III

-H 2 O

Fe III
N S

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

Heme Dependent Oxidations

R' R'' N R H R' R'' R R' R'' OH N
IV

R OH

O
N

Fe
N

Fe
N

IV

oxygen rebound
N N

N S S

FeIII
S

R'

R'

R'

O N N N N N S

O N

R''

O N N N N

FeIV
N

FeIV
N S

FeIII
S

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.
Ph Ph O N N O Me N Me Ph ON N Ph O Me N CH2 H Ph Ph HO N N O Me N CH2

Fe4+
N N

Fe3+
N N

Fe3+
N N

Ph Ph

Me N H

-HCHO

Ph Ph

Me N C H2 OH N

Fe3+
N

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

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 H N N(Et)2 O CH3

CH3 H N N(Et)2 O CH3 OH

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 isolated naphthalene 1,2-oxide from microsomal oxidation of naphthalene.

+ O2 + NADPH + H

P450

+ NADP+ + H2O

Arene oxide Formation

Kinetic isotope studies indicate that direct arene epoxidation is unlikely, but rather an activated heme iron-oxo species may add to the aromatic ring, similar to the reaction with alkenes. A tetrahedral intermediate is formed that can rearrange via 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 glutathione catalyzed by glutathione S-transferase to form a hydroxy sulfide, as well as react with macromolecular nucleophiles.

Addition-Rearrangement Mechanism
R H R N N N O N
IV

+
O

N N

Fe
S

Fe
S

IV

electron transfer

FeIII
N S

R N

N N

FeIII
O OH O S

Reactions of Arene Oxides

R

epoxide hydrolase H 2O
R R

OH HO

glathione S-transferase GSH

H O H GS OH

macromolecular nucleophile

OH X

Glutathione
The tripeptide glutathione is found in virtually all mammalian tissues. It contains a potent nucleophilic thiol group, and its function appears to be to scavenge harmful electrophiles that are ingested or produced by metabolism. Drug toxicity can result from the reaction of cellular nucleophiles with electrophilic 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 glutathione S-transferase occur nonenzymatically as well, but at a slower rate.
NH3+ H N -O2C O HS N H O CO2-

NIH Shift
Rearrangement of an arene oxide to arenol is known as the NIH shift. 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

P-450 O 2 , NADPH
D D R H O -O

+
D

H D O
+

:B
D HO

H The NIH Shift.

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 more deprotonation 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 R

+
H O D -O D HO H

Deprotonation Pathway.

:B

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.
CH 3 NH 2 CH 3 NH 2 O -O Cl CH 3

+
NH 2

Cl

Cl

CH 3 NH 2 Cl

CH 3 NH 2 Cl

HO

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 is hydroxylated. The antipsychotic chlorpromazine undergoes hydroxylation at the 7-position.
R HO 2 C SO 2 N(n-Pr)2 N Cl S

Probenecid
NMe 2

This reaction is electrophilic aromatic substitution Favors electron-donating substituents No aromatic hydroxylation e- withdrawing
HOOC SO2N(CH2CH2CH3)2 probenecid uricosuric 7.24 agent

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. COOH
COOH NH Cl Cl

O Cl

diclofenac 7.21

Cl fenclofenac 7.22

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.
R3 R2

dogs

HN O N H O

R1

humans

Meta-Hydroxylation
Meta-hydroxylation may be catalyzed by an isozyme of P-450 that works through a different mechanism. Metabolite of chlorobenzene is 3chlorophenol; however, neither 3- nor 4chlorophenol oxide afford 3-chlorophenol in the presence of rat liver microsomes. Direct insertion mechanism may occur in this case.

Further Reactions of Arene Oxides

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 give trans-dihydrodiols. The reaction involves general base-catalyzed nucleophilic attack of water, with attack occurring from the backside at the less sterically hindered side. The trans dihydrodiol product can be oxidized to catechol; the catechols are further oxidized to ortho-quinones or semiquinones
R R R

OH HO HO

OH O R

OH O

Mechanism of Epoxide Hydrolase Hydration of Arene Oxide

R R R [O] OH O O O H HO O H B: O O O OR OH HO R OH OH R R

Anti-attack

OH

GSH Reactions
Glutathione S-transferase is another enzyme that protects cell from electrophilic arene oxide metabolites. The adducts can undergo rearrangement upon dehydration.

O GSH P450 O GSH

HO SG

SG OH

OH SG

SG OH

only isomer not detected

Carcinogens
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 malignant cellular transformation. The arene oxide reacts with DNA to form a covalent adduct with the C-2 amino group of guanosine

HO O O N N R HO NH N NH O OH

HO OH

HO OH

Alkene Epoxidation
Alkenes also undergo epoxidation by P-450, and are more reactive than aromatic systems. The anticonvulsant agent carbamazepine is converted to its epoxide (the metabolite may be responsible for anticonvulsant activity). This is converted in turn to the trans diol by epoxide hydrolase, and then conjugated to the glucuronide by UDPglucuronosyltransferase.
O HO OH

epoxide
N H 2 NOC N H 2 NOC

hydrolase

N H 2 NOC

HO

OG

UDP GT

N H 2 NOC

Aliphatic Hydroxylation
Another very common reaction is hydroxylation in the aliphatic side chain as shown in the case of pentobarbitone. In practice, a nonactivated alkyl group undergoes and -1 oxidation. n-Hexadecane is oxidized to hexadecanol in the liver, and this then further oxidized to hexadecanoic acid.
O HN O HN O CH 3 (CH 2 )2 -CH 3 CH 3 O HN O CH 3 OH HN O CH 3

Pentobarbitone

Aliphatic Hydroxylation
In the case of the sedative-hypnotic (-)-glutethimide this is oxidized at the -1 position (ethyl group)but enantiomer difference in metabolism.

hydroxylation here for (+)-isomer

R O Ph R'

hydroxylation here for (-)-isomer

N O H glutethimide (R = R' = H) 7.40

sedative/hypnotic

Preferential Oxidation
In the case of activated sp3 carbon atoms in allylic, propynylic, or benzylic positions, these activated carbon atoms are preferentially hydroxylated. Carbons adjacent to carbonyl or imine groups can also be oxidized. (ease of oxidation parallels C-H bond dissociation energies
OH

major

minor

HO

R HO OH O R X

R X

+
X-H

OH R R

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.
OH O

H N

2 1'
MeO Hs HR

antihypertensive drug metoprolol hydroxylation takes place here, 1'R-hydroxylation is preferred

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

Hydroxylation beta to a Carbonyl Group

Scheme 7.15
OH H3 C HN CH3 O H3 C HN CH3 O H3 C HN CH 2 O -CH2O F3 C NO2 flutamide 7.43 F3 C NH2 F3 C NH2 7.44 F3 C NH 2 7.45 F3 C NH2 H3 C HN O OH H2 C HN O -CH 2O F3 C NH2 7.46 HN CH 3 O

Oxidations of Carbon-Nitrogen Systems

Table 7.3

Oxidations of 1 amines and amides

RCHNH2 R' RCHNH2 R' ArNH2 RC NH2 O RC O R' + -H2 O NH4+

RCHNHOH R' ArNHOH

RC NH R ArN O

RC NOH R ArNO 2

RCHNO2 R'

RC NHOH O

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.

privileged attack
OH R R N R' R N R' N R' H O

OH R N R'

+ minor products

N R' OH

Oxidative Deamination

P450 NH2 amphetamine 7.48

18O 2

Ph
18O

+ NH3

Flavin monooxygenases and P-450

Primary amines are also converted by N-oxidation to the corresponding hydroxylamines catalyzed by flavin monooxygenases. IN GENERAL - Basic amines (pKa 8-11) are oxidized by flavoenzymes and non-basic nitrogen compounds like amides 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
B:
H

N HO OH

N O

H+

B:
H

flavin monoNH 2

oxygenase

NH OH

NH

H+

NO 2

HO

N+

OH

:B

OH

+ NH 2 OH

Reaction Mechanism of Flavin Monooxygenases

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.
R N N NH N H H O O NH 2 N R R N N NH N H HO O NH NH H OH O O O N R N N O R N N NH O O R N NNH N H O O

-H 2 O

O2

B +-H

H HO

B:

R-NH 2

R-NHOH

R-NH2 +

Secondary Amine Oxidation

Secondary amines are metabolized by oxidative Ndealkylation, oxidative deamination, and N-oxidation. The aldehyde metabolites can be further oxidized by aldehyde oxidases or dehydrogenases to corresponding carboxylic acids. Secondary hydroxylamine formation is common, and further oxidation to nitrone can take place.

F3C HN

F 3C HO N

F3C -O N+

N-Oxidation of Fenfluramine to its Nitrone.

Metabolism of 2 Amines and Amides

Table 7.4
O oxidative N-dealkylation RCH NHCH2R2 R' RCH oxidative deamination R' RC NHCH 2R' O RC NH 2 O RCH NR' OH RCH 2N R' OH NR' ORCH N R' + + OHCR' NHCH2R2 RCH NH2 R' O R C R' + NH2CH2R2 difference is which side of N is oxidized + HCR 2

RCH2

NHR'

N-oxidation

ArNHR' RC NHR' O

Ar

OH RC NR' O

Oxidation of Propranolol
OH O OH N H O OH N H O OH O NH 2

OH O OH N H O

Aldehyde dehydrogenase

O O OH O H

OH

+ HN 2 Oxidative Metabolism of the beta-Blocker Propranolol.

Oxidation of 3 Amines and Amides

Table 7.5
O HC R2 oxidative N-dealkylation RCH NCH 2R2 R' CH 2R 3 R3N N-oxidation R ArNR' R' RC NCH2R2 O RCH NHCH2R3 R' R3 N+ O+

R ArN+ R' ORC NHR' O +

oxidative N-dealkylation

O HC R2

No oxidative deamination

Tertiary Amine Oxidation

Tertiary amines are metabolized by oxidative dealkylation and N-oxidation. The tricyclic antidepressant imipramine is metabolized to the secondary amine desmethylimipramine.

N N Me Me

N N H Me

Scheme 7.22
CH3 N H3 C H deprenyl 7.57 P450 NHCH 3 H3 C H metamphetamine 7.58 P450 NH2 H3 C H amphetamine

(S)-(+)-deprenyl (S)-(+)-metamphetamine (S)-(+)-amphetamine weak MAO B inhibitor undesirable 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. The immonium ion intermediate is very electrophilic, and can be trapped by cyanide in vitro.
N N CH3 N N CH3 OH N N+ CH3

CNN

N CH3

CN

HO N N CH3 O N N CH3 O

O HN N CH3 OH

-OHN N CH2OH N N+ CH2 N N H2C CN

-HCHO

N H N

Routes of Nicotine Metabolism.

N-Oxide Formation
N-oxidation of tertiary amines gives the N-oxides that are chemically stable. These do not undergo further oxidation. The antihistamine cyproheptadine is converted to the N-oxide in dogs.

N CH 3

N+

O-

CH 3

Tertiary Aromatic Amines

Tertiary aromatic amines are N-oxidized by a flavoprotein monooxygenase and by cytochrome P-450. The P-450 N-oxidation appears to occur when there are no alpha hydrogens are available for abstraction.
R' N+ OO N N ON
4+

R' R N R' R N

R' R'

R'

Fe
N

Fe
N

3+

Fe
N

3+

Two Mechanisms for N-Demethylation of Tertiary Aromatic Amines

CH3

Ar

N CH3

flavin monooxygenase Ar

CH3 N+ OCH3

CH2-

Ar

N+

OH CH3

P450
HO CH2

OHAr
N

CH2

-HCHO Ar
N

H CH3

Ar

N+ CH3

CH3

Mechanism of Carbinolamine Formation

Based on low intrinsic isotope effects by P450, direct H abstraction mechanism was excluded.
Scheme 7.28
R Ar N CH3 .. O N N Fe 4+ N N +. R Ar N CH 2 +. H ON N 3+ Fe +. N N R Ar N CH2 +. Ar R N CH .. . 2 HO N N R N CH2 OH Fe 3+ N N

Ar

Metabolic Activation of Amines

The oxidation of primary and secondary aromatic amines leads to the generation of reactive electrophilic species that form covalent bonds to cellular macromolecules. Bond formation to proteins, DNA and RNA is known.
H N R' R OH N R' OX

X+
R Y

R'

X = acetyl or sulfate -OX

H N

H-B +
R' R N R'

:B

Amide Oxidation
Amides are also metabolized by oxidative Ndealkylation and N-oxidation. Diazepam undergoes extensive demethylation.
Me N

Cl

Aromatic Amide Oxidation

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

H O R N OH R X O X

O N H

-OX

NH2 R R

NH H X

Oxidations of Carbon-Oxygen Systems Oxidative O-Dealkylation

Same mechanism as oxidative N-dealkylation O-Demethylation is rapid; as increase alkyl chain length, O-dealkylation gets faster up to propoxyl, then rate decreases. Cyclopropyl gives ethers with longer plasma half lives.

Oxidative O-Dealkylation
CH 3 N H H

RO

OH

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

analgesic O-Demethylation is rapid

Regioselective O-Demethylation
In dogs O-demethylation only here
RO OH CH3 NH 2 CH 3O methoxamine (R = CH3 ) 7.81

blood pressure maintenance

Oxidation on the Carbon Next to a Lactone Oxygen

Scheme 7.34
O O O O OH

rofecoxib 7.82

SO2CH3 7.83

SO2CH3

Sulfur Oxidation
S-oxidation to sulfoxides is catalyzed by both flavin monooxygenase and by cytochrome P-450. The flavin enzyme produces only the sulfoxide while P-450 gives both S-dealkylation products and sulfoxides probably via the sulfenium cation radical.
R RCH 2 R RCH 2 S RCH 2 S R S+ O-

O rebound

O N N N

ON

H+ transfer
3+

Fe
N

4+

Fe
N

R RCH S

N OH R N

RCHO + -SR

RCH HO

S N

Fe

3+

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

SMe

Me

Thioridazine, antipsychotic

H N S N albendazole 7.87 NHCO2CH3

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 proteins.
Scheme 7.36
O Cl Cl OCH2COOH S P450 O Cl Cl OCH2COOH S OS HS OH OS 7.90 O Cl Cl OCH2COOH OH

tienilic acid 7.89

added in vitro to mimic a liver protein cysteine residue

Oxidation of Sulfoxide to Sulfone

O OS N CH3 N O O S O CH3

oxisuran 7.91 immunosuppressive

Desulfuration (C=S C=O)

O HN X N H 7.85 thiopental (X = S) - anesthetic pentobarbital (X = O) - sedative CH3 H O

Other oxidations brought about by P-450 are oxidative dehalogenation, oxidative aromatization, and conversion of arenols to quinones. The anesthetic haloethane is metabolized to trifluoroacetic acid. The intermediate acid chloride that is formed can bind covalently to liver microsomes.
H OH O Br Cl O

P450
F 3C Cl Br

F 3C

-HBr

F3 C Cl

H2 O

F 3C OH

Additional Mechanistic Considerations

Archives of Biochemistry and Biophysics Volume 409, Issue 1 , 1 January 2003, Pages 72-79 Many of the qualitative results from studies of P-450 catalyzed hydroxylation 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 labeled cyclohexene. These results indicate that a radical in a radical pair that recombined rapidly was formed. Bicyclo[4.1.0]heptane underwent oxidation at the cyclopropylcarbinyl position without ring opening. This was considered to exclude a cationic intermediate; however, this presupposes that only the carbocation can undergo rearrangement.

Radical Clock
Relatively large hydrogen-deuterium kinetic isotope effects in P450 catalyzed hydroxylations were also taken as evidence that radical 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 a known 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 product distribution and the known rate constant for rearrangement (kR).

Bicyclo[2.1.0]pentane Rearrangement Bicyclo[2.1.0]pentane is hydroxylated to give unrearranged product, and rearranged cyclopent-3-ol, the later presumably formed by ring opening of the bicyclo[2.1.0]pent-2-yl radical. Subsequent studies determined the rate constant for ring opening of the radical, and this value could be used with the results to give a radical rebound rate constant of 1.4 x 1010s-1, or a lifetime of (t = 1/k) of 70 ps.

Mechanistic Picture Not Clear

A dozen radical clocks were used in studies of P450 2B1 and 2B4 hydroxylations, but no consistent trends were found.A plot of the logarithm of the ratio of unrearranged to rearranged alcohol products versus the logarithm of the radical rearrangement rate constant, which should have 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 than correlated.In the context of a single pathway for hydroxylation involving abstraction and rebound, most of the results had to be explained as special cases.which means that the mechanistic picture is not clear.

Cationic Intermediate?
In order to test for the possibility of a cationic intermediate, a substrate was needed that gave different products for a radical pathway versus a cationic pathway. Studies were made using a hypersensitive radical probe, such as use of a cyclopropyl system. A cyclopropylcarbinyl radical ring opens to give predominantly the benzylic radical products (> 50:1), and incipient cyclopropylcarbinyl cations rearrange to give only products derived from the oxonium ion.

No Discrete Radicals
The amounts of radial-derived products from probes 11 were very small, even though the radicals derived from these substrates 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 in the range of 0.08 to 0.2 ps, which are too short for true radical intermediates, but correspond to lifetimes of transition states. Thus, probes like 11 indicate that no discrete radicals were formed in the hydroxylation reactions.

Homocubanol System
For substrate 12, the amount of cation-derived product homocubanol varied form 0 to 30% of the total amount of oxidation at the methyl position. The radical reacts by cleavage of the cube bonds. The large variance in the amount of cation-derived product suggests a complex mechanistic picture. This strong evidence for cations of some type provides a possible explanation as to why radical clock studies had given inconsistent radical lifetimes. All of the radical lifetimes determined from probes that did not give different products from radical and cationic intermediates can be understood if cations are formed in the oxidations. Results from such probes can only be used 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 hydroxylations. In most cases of radical clocks, a cationic rearrangement would give the same products as the radical intermediates. There is a question as to how cationic intermediate is generated; in case of 11, the radical lifetime is too short for this radical to be oxidized. Thus, the direct insertion of OH+ would have to take place. This means oxidation by a precursor to the iron-oxo species, either the hydroperoxo-ion or iron-complexed hydrogen peroxide 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+.

Mutated Enzyme
Besides the iron-oxo species, the hydroperoxo-iron intermediate may play 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 the peroxo-iron or hydroperoxo-iron species. This residue was thus mutated and reactions examined. Probe 7 was examined using both wild type that contains a short N-terminal deletion and mutant enzymes. Probe 7 can be oxidized at either the methyl group or the phenyl group. Increased amounts of phenol were produced by the mutants. 85% methyl oxidation for 2B4 reduced to 44% for 2B4 T302A and 81% methyl oxidation for 2E1 reduced to 33% for 2E1 T303A. Changes ascribed to amounts of oxidation brought about by iron-oxo for hydroperoxo-iron species. Also, with probe 13, more rearranged product was found with 2E1 T303A (38%) versus 2E1 (9%). The phenol forming reaction is suppressed by the trifluoromethyl group.

Two Spin States

Another suggestion has been made that the iron-oxo species has different spin states, and that reaction via different spin states could have different outcomes. There is a low spin doublet state, and a high spin quartet state. After reaching the TS for abstraction, the energetics of the two pathways diverge. The reaction on low-spin surface proceeds through a radial-like species that collapses with no barrier to give alcohol productthis is effectively an insertion. The reaction on the high spin surface has a considerable barrier to collapse, and this pathway gives a true radical intermediate. The reactions on the high spin surface would give radicals that could rearrange.

P450 oxidations are thus complex.

It seems possible that the hydroxylation reactions could be explained by the qualitative picture in Fig.4, where two electrophilic oxidants exist and two spin states of ironoxo are reactive. In this model, the early oxidant reacts by insertion of OH+ to give protonated alcohols as first-formed products, and these species are the origins of cationic rearrangement products.The low-spin iron-oxo ensemble reacts by insertion of oxygen in a process that resembles the oxene insertion pathway proposed many years ago.The high-spin iron-oxo species abstracts hydrogen to give a radical intermediate in a process that resembles the oxygen-rebound pathway.

HDACs
Histone deacetylase enzymes have been divided into three distinct structural classes, operate by zinc-dependent (class I/II) or NADdependent (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 is made up of SIRT 1-7 (sirtuins). Class I/II histone deacetylase (HDAC) enzymes are an emerging therapeutic target for the treatment of cancer and other diseases.

Tumorigenesis
These enzymes, as part of multiprotein complexes, catalyze the removal of acetyl groups from lysine residues on proteins, including histones. Tumour cells must be able to circumvent APOPTOSIS, replicate indefinitely and sustain growth and survival by maintaining a sustainable oxygen and nutrient supply. Mutations that result in constitutive activation of ONCOGENES or functional inactivation of TUMOUR-SUPPRESSOR GENES are important tumorigenic events. Moreover, aberrant transcription of the genes that are needed to initiate the host antitumour immune response and induce neovasculature can result in tumour immune escape and ANGIOGENESIS events that are essential for cancer progression

Chromatin Remodelling
There has been a rapid advancement in our understanding of the molecular processes that lead to the activation or repression of transcription, and chromatin architecture has emerged as the foundation for gene regulation. CHROMATIN REMODELLING which is controlled by factors that relocate nucleosomes and alter nucleosome structure after post-translational modification of histone tails directly affects gene expression. In cancer, the molecular processes that lead to inappropriate expression of genes due to altered chromatin structure are now being identified, and aberrant acetylation of histone tails has been strongly linked to carcinogenesis.

Reprogram Transcription
Thus, targeting the transcriptional lesions that lead to neoplasia provides an opportunity for therapeutic intervention at the very apex of the transformation process. Such therapies could affect several molecular programs, and would therefore be more powerful than targeting the end stages of a single disrupted molecular pathway. Understanding how gene expression can be regulated opens the way for new molecular tools to reprogram transcription and inhibit tumour-cell growth and progression.

Condensed and Open Chromatin

All of the human genome is packaged into chromatin, which is a dynamic macromolecular complex that consists of DNA, histones and non-histone proteins. Nucleosomes form the basic repeating unit of chromatin, and consist of DNA wrapped around a histone octomer that is formed by four histone partners an H3H4 tetramer and two H2AH2B dimers.The linker histone H1 stabilizes the higher-order folding by electrostatic neutralization of the linker DNA segments through a positively charged carboxy-terminal domain. So, the dynamic higherorder structure of nucleosomes defines distinct levels of chromatin organization and, subsequently, gene activity (FIG. 1). In general terms, condensed chromatin mediates transcriptional repression, whereas transcriptionally active genes are in areas of open chromatin.

Covalent Modification of Histone Tails

Indeed, histone tails, particularly H3 and H4, are targeted for various post-translational modifications, including acetylation, phosphorylation and methylation (FIG. 1). Covalent modification of core histone tails by histone acetyltransferases (HATs), HDACs, methyltransferases and kinases offers a mechanism by which upstream signalling pathways can converge on common targets to regulate gene expression. In addition, chromatin structure can also be regulated by protein complexes that use ATP hydrolysis to reposition nucleosomes. Although ATP-dependent chromatin remodellers were initially characterized as factors that promote gene activation, it is now known that they can cooperate with either HATs or HDACs, and therefore have dual roles in activating and repressing transcription. So, chromatin can be remodelled by the integrated activities of histone-tail-modifying enzymes and ATP-dependent factors.

Chromatin Structure

H1
Linker histone

HISTONES
are highly conserved, small, basic proteins
helix

H2A H2B
Core histones

H3 H4
N

variable

conserved

Histone acetylation is a reversible modification of lysines in the N-termini of the core histones. Result: reduced binding to DNA destabilization of chromatin

Histone octamer assembly

H3-H4 tetramer

Histone octamer

H2A-H2B dimer

Histone octamer forms a ramp for DNA

H4 H3 H2A H2B

white green light blue dark blue

red: + (arg, lys)

orange: -OH (ser, thr)

Histone octamer organizes 145 bp of DNA

> 11 nm < 6 nm > <
Each core histone dimer has 6 DNA binding surfaces that organize 3 DNA turns; The histone octamer organizes 145 bp of DNA in 1 3/4 helical turn of DNA: 48 nm of DNA packaged in a disc of 6 x 11nm

Chromatin fibers

30 nm chromatin fiber

11 nm (beads)

+ charged N termini (bind DNA on neigboring nucleosomes) HIGH level of histone H1

highly acetylated core histones (especially H3 and H4) Reduced level of histone H1

NO gene transcription

Gene transcription possible

Transcriptional Repression Mediated by HDACs

Various biological data show that inappropriate transcriptional repression mediated by HDACs is a common molecular mechanism that is used by oncoproteins, and alterations in chromatin structure can impinge on normal cellular differentiation, which leads to tumour formation.

HDAC Inhibitors
HDAC inhibitors. Various structurally diverse compounds can bind to HDACs and induce histone acetylation. Crystallographic studies using trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) indicate that these compounds inhibit HDAC activity by interacting with the catalytic site of HDACs, thereby blocking substrate access. Almost all HDAC inhibitors can induce cell cycle arrest, differentiation or apoptosis in vitro, and many have potent antitumour activities in vivo.

HDAC-Is
Short-chain fatty acids such as phenylbutyrate and phenylacetate, and the anti-epileptic drug valproic acid, inhibit HDAC activity and affect the expression of numerous genes with disparate cellular functions. These agents have been assessed in the clinic, but suffer from a short plasma half-life and the relatively high (millimolar) concentrations that are necessary for their action. Hydroxamic acids such as TSA, SAHA (rat liver HDAC 12 nM vs. 165 nM), m-carboxy cinnamic acid bishydroxamic acid (CBHA) and oxamflatin (15.7 vs TSA at 1.44 nM IC50) can be used at micromolar and nanomolar concentrations, and generally have longer in vivo half-lives and bioavailabilities than short chain fatty acids.

CO2H

Valproic Acid

Histone Deacetylase Like Protein

In an effort to understand the importance of the methyl-substituted olefinic linker in TSA hybrids, the synthesis and evaluation of the compounds 7 and 8 has been performed. These agents, along with related analogue 6, demonstrate a highly sensitive and limited SAR profile. For example, the sequential addition of a methyl group (7) and two double bonds (8) caused a 2.3- and 33-fold reduction in activity, respectively, relative to the linear alkyl (6). Amide bond region of 9 is displaced from ketone part in TSA, and the dimethylaminobenzoyl group is shifted, resulting in less efficient interaction with enzyme surface. Bioorganic & Medicinal Chemistry Letters 13 (2003) 18611864

SAHA Analogs
Heterocyclic derivatives with the optimized linker (19e and 19f) were also prepared and found to be active. Para-substituted aryl derivatives (19h-j) were more active than ortho-substituted derivative 19g, and it was also shown that increased hydrophobicity in the recognition domain could substantially enhance activity (19k and 19l). The ketone functionality was also replaced with oxime (19m,n), alcohol (19s), alkene (19o,q), and alkane (19r) linkages. All of these compounds were shown to be effective inhibitors of HDAC, with those carrying sp3 centers (19r,s) displaying attenuated activity relative to the sp2-bearing analogues (19k,m,o,q). The corresponding carboxylic acids were also tested and found to be inactive with the notable exception of 19t.

Cyclic Peptides
Cyclic peptide containing HDAC inhibitors are the most structurally complex. The inhibitors containing the (S)-2amino-9,10-epoxy-8-oxodecanoic acid (L-Aoe) group may bind irreversibly to HDAC. From chemical studies in which reduction of the epoxide group of trapoxin A was carried out, it has been suggested that this compound binds irreversibly to HDAC.

Tetrapeptides active without epoxide group

Within the subclass of cyclic tetrapeptides classified as reversible inhibitors are active variants of the Aoe containing natural products lacking the epoxy ketone functionality. These agents demonstrate that the potentially reactive epoxy ketone moiety is not required for HDAC inhibition. Apicidin (63), a fungal metabolite, contains an (S)-2-amino-8-oxodecanyl side chain lacking an epoxide. It has broad spectrum activity (ranging from 4 to 125 ng/mL) against the apicomplexan family of parasites, presumably via inhibition of protozoan histone deacetylases (IC50 ) 1-2 nM), and demonstrates efficacy against Plasmodium berghei malaria in mice. Other fungal metabolites devoid of the reactive epoxy ketone have been characterized, including numerous apicidin analogues, desepoxy-trapoxin analogue 64, and structurally related chlamydocin analogues 65, 66, and 67. Compounds 64 and 66 were characterized in vitro using antiproliferation assays with human Jurkat and HT-29 tumor cells lines. Compound 64 exhibited IC50 values of 152 and 158 ng/mL, respectively, and 66 exhibited IC50 values of 11 and 14 ng/mL, respectively, in these assays.

Apicidin Side Chain Analogs

A systematic study of the activity of various side chain analogues of apicidin was made; the alpha-hydroxy ketone, hydroxamic acid, and epoxide showed improved activity over apicidin. Interestingly, the methyl ester is also active, showing importance of the tetrapeptide.

FK228
FK228 (52), which was discovered from a fermentation broth of Chromobacterium violaceum, differs structurally from other constituents of the cyclic peptide class. FK228 contains a unique bicyclic structure with four amino acids and a betahydroxyamide moiety, which collectively form a 16-membered lactone with a disulfide bridge. FK228 is currently the only member of the cyclic peptide class under clinical investigation. Furumai and co-workers have investigated the longstanding question of FK228s mode of action. It has been postulated that the disulfide bond is reduced in the cellular environment, releasing the free thiol analogue as the active species. To address this issue, FK228 (52) was converted to its reduced form, redFK (73), in the presence of DTT and evaluated for its inhibitory activity against various HDACs prepared from 293T cells (Table 18). The reduction of the intramolecular disulfide bond of FK228 greatly enhanced its inhibitory activity (73 vs 52). This reduction was shown to occur in the cellular environment via participation of glutathione. HDAC activity is significantly lowered after capping of the two sulfhydryl groups via methylation, lending further support to the active species of FK228 being 73. RedFK (73) is more active against HDAC1 and HDAC2 compared to HDAC4 and HDAC6. HDAC1 is reversibly inhibited by 73, and a cysteine to serine HDAC1 mutant is still sensitive to 73, suggesting the 73 is not covalently bound to the conserved Cys-151 in the active site.

FK228
Computer modeling suggests that the thiol of the four-carbon side chain passes down the HDAC tubelike channel toward the active site residues and interacts with the zinc via a water molecule. Therefore, FK228 (52) acts as a potential prodrug by entering the cell and then being reduced in the cellular environment to 73, generating the sulfhydryl groups responsible for inhibiting HDAC activity.

FK228 interaction with Zinc

Other zinc binders

Alternative metal binding groups have been studied, but as can be seen from the following table the best of these requires micromolar concentrations.

SAHA Analogs with thiol groups: JMC, 2005, Suzuki et al.,

Potent Cell Growth Inhibition by Pro-Drug Forms of Thiols

HDAC X-ray Structure

The crystal structure of histone-deacetylase-like protein (HDLP, Figure 1), a homologue of mammalian class I/II HDAC with ~35% sequence identity, was solved separately with the HDAC inhibitors SAHA (1) and (R)-trichostatin A (TSA, 2) (Figure 2) bound to the active site. The structural findings revealed by these studies led to a proposed mechanism of the hydrolysis of acetyllysine by the class I/II HDAC enzymes based on the observation that the HDLP active site has features of both metallo and serine protease active sites and the hypothesis that these hydroxamate inhibitors act as substrate analogues. These studies demonstrated that both SAHA (1) and TSA (2) make contact with the same residues in the rim, channel, and active site regions of the protein. In addition, hydrogen bonding between the inhibitor hydroxamic acid functionality and the imidazole groups in the histidine (H131, H132)-aspartate (D166, D173) salt bridges, along with hydrogen bonding of the active site tyrosine (Y297) to the hydroxamate carbonyl, provides further rationale for the potent inhibitory activity of these agents (Figure 3).

HDLP

TSA Binding
TSA is a natural product and was one of the first compounds to be shown to function as an inhibitor of HDAC. TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme. In the crystal structure of the HDLP-Zn2+ -TSA complex, TSA binds by inserting its long aliphatic chain into the HDLP pocket, making multiple contacts to the tube-like hydrophobic portion of the pocket. The hydroxamic acid group at one end of the aliphatic chain interacts with zinc in a bidentate fashion and also contacts active site residues. The aromatic dimethylamino-phenyl group at the other end of the TSA chain makes contacts at the pocket entrance and in an adjacent surface groove, capping the pocket. The length of the aliphatic chain appears to be optimal for spanning the length of the pocket and allowing contacts both at the bottom and at the entrance of the pocket.

TSA-HDLP Interactions

Mechanism of Deacetylation.

In proposed mechanism for deacetylation, by analogy to the zinc proteases, the carbonyl oxygen of the N-acetyl amide bond could bind the zinc, and the carbonyl carbon could be positioned in close proximity to the water molecule. The zinc ion could polarize the carbonyl group so that the carbon is a better electrophile, and could also help orient the water molecule. The nucleophilicity of the water molecule would be increased by the negative charge of the buried Asp 166-His 131 charge-relay system to which the water is hydrogen bonded. This is analogous to the activation of a serine hydroxyl by a buried charge-relay system in the serine proteases, and to the activation of a water molecule by a glutamic acid in the zinc proteases. The nucleophilic attack of the water on the carbonyl carbon would result in a tetrahedral carbon. This oxyanion intermediate could be stabilized by two zinc-oxygen interactions, in a manner analogous to the zinc proteases, and possibly by a hydrogen bond from the Tyr 297 hydroxyl group (Fig. 5). In the final step, the carbon-nitrogen bond of the intermediate would break, and the nitrogen of the scissile bond would accept a proton from the exposed Asp 173-His 132 charge relay, yielding the acetate and lysine products (Fig. 5).

HDLP Catalytic Mechanism

Analogy of Lysine and TSA

The structures of the HDLP-Zn2+-TSA and HDLP-Zn2+SAHA complexes support the overall analogy of the aliphatic chain and hydroxamic acid groups of these inhibitors to the lysine side chain and the acetyl group of the HDAC substrate. The branched C8 carbon, where the TSA structure bends as it enters the pocket, may approximate the Ca of the lysine amino acid. This positioning of the Ca would allow the aliphatic portion of the lysine side chain to span the tube-like portion of the pocket and place the acetylated amino group into the polar bottom of the pocket. The polypeptide backbone could interact with surface residues and in the shallow grooves around the pocket, including the groove where the TSA cap binds (Fig. 3d).

Human HDAC8
Crystal structure of HDAC8 in complex with TSA is now known. Interestingly, this shows two TSA molecules binding, one to the active site and one to a second pocket.

HDAC8

Schematic diagram showing binding of hydroxamate inhibitors to HDAC8.

Protein Kinases Kinetic and Catalytic Mechanisms of Protein Kinases Joseph A. Adams* 2271 Chem. Rev. 2001, 101, 2271-2290

The phosphorylation of proteins triggered in response to extracellular signals represents a universal mechanism for the cellular control of many different processes, including metabolic pathways, cell growth and differentiation, membrane transport, and apoptosis. The reverse reaction of dephosphorylation is catalyzed by phosphatases.

Kinases
The phosphorylation of protein targets is catalyzed by a large family of homologous proteins known as protein kinases. These catalyze the transfer of the terminal (or gamma) phosphate of ATP to specific residues of protein substrates. The protein kinase family is made up of two major subfamilies; the protein tyrosine kinases and the protein serine-threonine kinases. Also, recently, histidine kinases that phosphorylate an imidazole nitrogen on a histidine residue have emerged as signaling enzymes.

Phosphorylation of a Kinase Can Activate or Inhibit its Kinase Activity Phosphorylation Can Be: By Another Similar Kinase, Forming a Signaling Cascade By the Same Kinase Acting on Itself Autophosphorylation

PTKs
The protein tyrosine kinases activate numerous signaling pathways within cells, leading to cell proliferation, differentiation, migration, and metabolic changes. The PTKs include receptor tyrosine kinases (RTKs, such as the insulin receptor tyrosine kinase, IRK, or the epidermal growth factor receptor kinase, EGFR-K) and non-receptor kinases (NRTKs) which include Src, JAKs, and Abl among others. .The RTKs are transmembrane glycoproteins comprised of three major domains: an extracellular receptor domain, a membrane spanning linker domain, and a cytoplasmic domain which contains the catalytic kinase activity.

RTKs and NRTKs

The RTKs are typically activated following binding of a ligand to the receptor domain. The NRTKs are cytoplasmic proteins and are components of the signaling cascades triggered by RTKs and other cell surface receptors such as G-protein coupled receptors and T-cell receptors. In addition to the PTKs, a large number of serinethreonine kinases have been identified, one example being the mitogen-activated protein kinase (MAPK). It has been estimated that there are approximately 600 human kinases.

Tyrosine Kinases
Non-receptor tyrosine kinases: many receptors lack a selfcontained kinase function. These cell surface proteins can, nonetheless, recruit the aid of other intracellular protein kinases in order to transduce a signal. Examples of this type of kinase would constitute a list too long to mention here, but include the src family of tyrosine kinases (src, p56lck, p59lyn, etc.) and numerous serine/threonine kinases. Receptor tyrosine kinases: some receptors possess an intrinsic kinase activity that, when bound to their ligand, is turned on. This can lead to subsequent activation of downstream signalling components and propagation of an intracellular signal. Examples of this type of receptor include those for growth factors (epidermal growth factor [EGF], platelet-derived growth factor [PDGF], and insulin). Our discussions in this lecture will focus on the second type of tyrosine kinase mentioned above, the so-called receptor tyrosine kinases (or RTKs).

RTK

Structural Features
There are four common structural features shared among the different RTKs described thus far: An extracellular ligand binding domain, which promotes specific, high-affinity binding of ligand to the receptor. A single transmembrane domain, consisting of a hydrophobic stretch of amino acids, that traverses the cell membrane. A cytoplasmic tyrosine kinase domain that contains the ligand-induced catalytic activity of the receptor. Some RTKs (i.e., PDGF receptors) possess a tandem repeat of two catalytic domains instead of a single domain. Regulatory domains or kinase insert regions that are sites for autotransphosphorylation. Clustered within these regions of the receptor are multiple tyrosine residues that, when selfphosphorylated by the receptor tyrosine kinase, provide binding sites for other signal transduction proteins that possess so-called SH2 domains.

RTK ligands
Ligands for RTKs are small soluble proteins that typically work in an autocrine or paracrine manner.
Paracrine signalling is a form of signalling in which the target cell is close to the signal releasing cell, Autocrine signalling is a form of signalling in which the target cell is the secretory cell itself.

Thus, these proteins are synthesized by cells that are in close range to their RTK target. Growth factors, such as PDGF, can dimerize and may aid in receptor dimerization (see below) which is crucial for increased RTK activity. Some RTK ligands can exist in a membrane-bound form. Activation of RTKs by such ligands can occur during direct cell-to-cell contact.

PDGF dimer

Numerous studies that primarily focused on ligand-induced RTK activation have clearly delineated at least two key events that are, in many cases, required for normal receptor function and propagation of an intracellular signal. These are: Receptor dimerization Receptor autotransphosphorylation

Receptor Autophosphorylation

A hallmark of RTKs is their ability to, upon ligand binding, undergo receptor dimerization in which two receptor proteins form a noncovalent complex via lateral movement through the cell membrane. This process enables the protein tyrosine kinase domains of the two receptors to be close together, allowing for cross-phosphorylation of the adjacent receptor protein. The targets of this autotransphosphorylation are tyrosine residues found both in the catalytic domain and regulatory regions of the receptor. A functional tyrosine kinase domain is absolutely required for mediating biological responses of RTKs.

Receptor autotransphosphorylation does at least two things:

It further stimulates the kinase activity of the catalytic domain leading to additional autophosphorylation as well as phosphorylation of other signalling proteins involved in RTK pathways. It provides docking sites for downstream signalling proteins (i.e., Grb2, PI3-kinase, phospholipase C, etc.) that recognize phosphotyrosine (P-tyr) residues. These signal transducers contain conserved amino acid sequences, called src homology 2 (SH2) domains, that recognize and bind P-tyr residues found on RTKs.

Ras-Raf-MAP kinase pathway. The mitogen-activated kinases (MAPKs) represent a family of growth regulatory proteins that have been implicated in cell proliferation, stress response, and gene transcription. Several MAP kinase pathways have been described; however, we will focus on one pathway (and perhaps the best known) stimulated by RTKs: the Ras-Raf-MAP kinase pathway (refer to figure and discussion below).

RTKs and Gene Transcription

As we discussed previously, RTK activation by its ligand results in receptor dimerization and autophosphorylation of a number of tyrosine residues located within the receptor catalytic and regulatory domains. The binding sites created by phosphorylation of many of these tyrosines promotes the binding of the adaptor protein, Grb2 (pronounced Grab2), through its single SH2 domain. An adaptor protein often lacks catalytic activity but contains several different protein binding motifs that link upstream components with other intracellular signal transduction proteins. Grb2 also has two src homology 3 (SH3) domains that bind prolinerich sequences found on target proteins. Both SH2 and SH3 domains, then, serve as common binding motifs found in many proteinprotein interactions. In our particular case, a guanine nucleotide exchange factor (GEF), called Sos (for Son of Sevenless, which was a name adopted from the R7 photoreceptor pathway, discussed below), binds to Grb2.

RTKs and Gene Transcription

As a GEF, Sos causes the stimulation of the small G protein, Ras, by promoting loss of GDP and binding of GTP to Ras. Once Ras is activated, serial activation of a kinase cascade can be initiated. GTP-bound Ras binds to and activates a serine/threonine kinase, Raf. Stimulation of Raf enables this kinase to serine phosphorylate the dualspecificity kinase (dual specificity means it can phosphorylate both tyrosines and serine/threonine residues), MEK. One target for MEK is another serine/threonine kinase, MAP kinase. In order for MAP kinase to be fully active, MEK needs to phosphorylate key tyrosine and threonine residues found on MAP kinase. Activated MAP kinase translocates from the cytosol to the nucleus where it phosphorylates and activates a number of transcription factors, including cmyc, c-fos, and c-jun. Expression of genes involved in cell cycle progression ensues. Transcription factors are proteins that can bind to specific regulatory sequences of DNA and promote gene expression. One common mode of transcription factor activation is through their phosphorylation. Phosphorylation of transcription factors often leads to dimerization of these proteins which is required for their activity.

Kinase Structure
All kinases contain a structurally conserved catalytic domain (200-250 amino acids) which was first elucidated for the cyclic AMPdependent kinase PKA. The organization of the catalytic domain is well understood. It is composed of two major domains (N- and Cterminal domains), that are further subdivided into 11 subdomains. The two domains are linked by a polypeptide chain that acts as a hinge the two domains can rotate with respect to one another upon binding ATP and/or substrate.

PKA

Activation Loop
Many PKs require phosphorylation on one or more of the serine, threonine, or tyrosine residues that are located in a segment running from the conserved DFG motif to the conserved APE motif (activation loop). Phosphorylation of residues in the activation loop causes conformational changes in the protein that lead to correct positioning of substrate binding residues and catalytic residues, and relief of steric blocking to allow access of substrates to the catalytic site.

Kinase Regulation
Other ways in which kinases are regulated include control by additional subunits or domains that might act in response to second messengers (cAMP binding to the regulatory subunit of PKA or Ca2+-calmodulin binding to calmodulin dependent protein kinase), control by additional subunits whose level of expression varies depending on the functional state of the cell (cyclin regulation of the CDKs), control by additional subunits that target the kinases to different molecules or subcellular localizations (the Src homology 2 and 3 (SH2 and SH3) domains of SRC kinases, and control by additional domains that inhibit kinase activity by autoregulatory processes (myosin light chain kinase).

ATP Binding
ATP binds in a cleft between the two major domains and is anchored to the enzyme by hydrogen bonds between the adenine group and residues of the linker region, and the ribose ring and residues at the start of the C-terminal domain. The triphosphate group is coordinated by two metal ions that are ligated by Asp and/or Asn residues located in the DFG motif and the catalytic loop. Also, polar interactions with several residues of the glycinerich loop, the conserved DFG motif and the catalytic loop further stabilize the phosphates and the transition state during the phosphotransfer reaction.

Key Interactions

Interactions in the Active Site

Asp 184 is a strictly conserved residue and interacts with Mg1, which chelates beta and gamma phosphate groups; chelation helps position terminal phosphate for transfer or to mask the charge of the phosphate group, thereby reducing charge development in reaction transition state or limiting electrostatic repulsion for an incoming nucleophile. Lys-72 interacts with the alpha and beta phosphate groups; its primary function is to facilate phosphoryl group transfer. Asp-166 may direct the hydroxyl for attack on the gamma phosphate of ATP. Lys-168 also supports phosphoryl group transfer and possibly peptide binding.

Activation Loop
It has been suggested that the activation loop limits access of substrates to the active site. Loop phosphorylation is thus expected to impair substrate and/or ATP binding but to have little effect on phosphoryl transfer. Figure below shows relative effects of activation loop phosphorylation on the kinetic mechanisms of 4 PKs. Activation loop phosphorylation enhances substrate binding for ERK2 and cyclin A-cdk2 but has no effect for PKA and v-Fps. Binding affinity of ATP is impaired only for PKA. Loop phosphorylation increases rate of phosphoryl transfer by 2-4 orders of magnitude. The activation loop is thus not behaving as a pure competitor for either ATP or substrate.

Phosphorylation of Activation Loop

Loop Region
Data from structural and kinetic studies do not agree on a general mode of regulation through the activation loop of protein kinases. There is little sequence conservation in loops, and requirement for phosphorylation is not universal. Some kinases like phophorylase kinase do not have a phosphorylation site in this loop region.

TS for Phosphorylation
Support for a dissociative-like transition state for the PKs has come from x-ray and NMR studies. PKA has been co-crystallized in a ternary complex with ADP and a 20residue phosphopeptide. The distance between the attacking nucleophile (serine oxygen) and the leaving group (beta-gamma bridging oxygen of ATP) can be used to calculate the bond order of the incoming oxygen. Such calculations predict 91.6% dissociative and 8.4% associative character. In general various studies demonstrate that the transition state for phosphoryl transfer possesses a high degree of dissociative character.

Phosphotransfer

Magnesium Role
Protein kinases require an essential Mg2+ for catalysis, This metal ion chelates the phosphates of ATP and Asp-184 in PKA. Mg(1) is considered the primary metal since it is visible in the crystal at low concentrations and may gain entrance to the active site as Mg-ATP complex. The second metal is also observed in the active site and chelates the phosphates of ATP, Asp184 and Asn-171. The dissociation constant of second metal binding event (~2 mM) is 2 orders of magnitude higher than the first, so this secondary site is only partially occupied under physiological metal concentrations. Overall role of metal ions in the active site of kinases is facilitation of phoshorylation, but discrete mechanism of involvement for each metal is not shared throughout the family.

Role of Asp-166
Protein kinases accelerate phosphorylation rates by 9-11 orders of magnitude. The large enhancement probably stems from a number of factors, but participation of a general base catalyst has been given special attention. Second order rate constant kcat/Km for phosphorylation of Kemptide by PKA is pH sensitive. A plot of this parameter vs. pH is bellshaped with the acidic limb being ascribed to ionization of a general base catalyst. The conserved aspartate in the catalytic loop of PKA forms a hydrogen bond with the substrate peptide so it is ideally poised for proton abstraction. Other data suggest that the acidic limb is involved in substrate binding and does not correspond to the ionization of Asp-166. Overall, various data are not consistent with a general base mechanism and are supportive of a dissociative mechanism. The aspartate group may thus play a positioning function with or without participation of the metal ion.

Roles of Asp-166 and Mg2+

Substrate binding site

In contrast to well defined ATP-binding site, substrate binding site of kinases are shallow depressions. Substrate recognition and binding depends in part on local residues, but short peptide sequences do not exploit complete binding capacity offered by the protein kinases. This suggests that kinases might use distal recognition elements for efficient phosphorylation.

Kinase Inhibition
Kinase inhibition. Physiologically, at least three inhibition mechanisms have been identified. A pseudo-substrate mechanism, an adenine mimetic mechanism, and a mechanism that involves locking the enzyme into an inactive form. Much of the synthetic effort has been directed to adenine mimetics, although some inhibitors directed to noncatalytic domains of PTKs exist. The protein substrate binding site has not been extensively exploited for inhibitor design. The substrate competitive inhibitors are likely to be more selective. Also, high ATP concentrations can reduce the efficacy of the ATP mimics.

ATP binding cleft

Protein kinases share common sequences and structural homology in their ATP-binding site. Only a fraction of all known kinases are screenable at present. Based upon numerous x-ray structures of small molecule inhibitors bound to kinases, have provided a clear description of the ATP-binding cleft. There are regions within this binding cleft that are not occupied by ATPthese are called the hydrophobic regions 1 and 2; some of these show structural diversity between members of the kinase family.

ATP binding cleft

Structural Basis for Selectivity

In some cases structural biology efforts showed that compounds known to be selective for a specific kinase or kinase class targeted the poorly conserved areas of the ATP binding site, thus providing a structural basis for observed selectivity.

Cdk2 Inhibitor
Cyclin dependent kinases are associated with the regulation of cell cycle progression. Using virtual screening methods, dimethylthiazoloaminopyrimidine was identified as a lead compound, and shown to possess moderate CDK2 inhibitory activity. It is believed that CDK2 inhibition may provide a way of interfering selectively with cancer cells leading to apoptosis. Upon inspection of this compound in the ATP pocket of a CDK2 complex crystal structure, it was found to bind in a similar fashion as ATP itself.

Cdk2 Inhibitor
Three H-bonds between the ligand and the backbone of CDK2 residues Glu181 and Leu83 were observed, interactions that are conserved in most kinase inhibitor complexes. In the ATP structure, the guanine amino group donates a H-bond to the carbonyl of Glu81, whereas N1 and H2 of the purine system accept and donate H-bonds to the amide NH and carbonyl of Leu83, respectively. The thiazole forms similar H-bonding contacts through the NH2 (Glu 81 CO), pyrimidine N1 (Leu83 NY), and H6 (leu83 CO). The dimethylthiazole ring overlaps roughly with space occupied by the ribose system in ATP. Not unexpectedly, dimethylation of the amino group on the pyrimidine ring abolishes biological activity.

H-bonding interactions in Cdk2

Further modification of pyrimidine inhibitor of Cdk2 Introduction of an aromatic substitutent on the amino group was found to improve activity, with the aniline derivative 11 being almost 100-fold more potent in inhibition of substrate phosphorylation by CDK2 compared to 8. It was possible to get a crystal structure on 20 and this showed a different binding mode compared to 8. The pyrimidine ring of 20 is in the same plane but flipped 180 degrees compared to 8. The phenyl group of 20 points out toward the entrance of the binding cleft in an area not occupied by ATP.

SAR
To further improve activity, effect of altering substituents on the aniline ring were made. In general electron withdrawing groups in meta or para-position of the aniline ring preserved or enhanced activity. The amino (35) and nitrile (36) substiutents in the aniline meta-position furnished potent compounds. Introduction of a meta (21) or para-hydroxy group (24) was found to maintain potency compared to 11.

SAR of thiazole ring

While the thiazol-4-yl methyl group makes near optimal contacts with Phe80 of CDK2, the thiazol-2-yl methyl group is positioned in vicinity of polar residues, in particular, Asn 132. The introduction of groups capable of H-bonding was thus explored. Replacement (of 2-methyl) with a methylamino group as in 22, 25, and 30 did not change potency appreciably. A primary amino group in place of the methyl preserved activity (26); but in the case of the m-nitroanilino group, the compound was particularly potent. The potency gain was not accompanied by increased cellular activity. The fact that dimethylamino replacement in 29 preserved activity indicates that a H-bond acceptor is favorable in place of the thiazol-2-yl methyl group.

Optimized compounds
Combination of the p-dimethylamino substituent in the aniline portion with favorable meta-anilino substituents such as chloro and nitro led to soluble nanomolar CDK2 inhibitors (38, 39).

X-ray of complex
In the x-ray of the ligand in complex with CDK2, the characteristic H-bonding network involving Glu81 and Leu83 is observed. Leu134 side chain forms strong hydrophobic interactions with the pyrimidine and anilino rings, and the thiazol-4-yl methyl group packs up against the Phe80 ring. The Asp145 carboxyl group forms a polar interaction with the ligand thiazole N atom. The primary amino group of 32 interacts with the side chains of Asp 145 and Asn132. The nitrophenyl group in 32 is disorderd and adopts two conformations in which it either interacts with the carboxyl of Asp86, whereas in the alternative conformation there is a weak H-bond with the side chain of Lys89.

P38 MAP Kinase

Design and Synthesis of 4-Azaindoles as Inhibitors of p38 MAP Kinase J. Med. Chem. 2003, 46, 4702-4713

It is now known that regulation of cytokine biosynthesis including proinflammatory cytokines interleukin-1b and tumor necrosis factor- is regulated through activation of p38 MAP kinase. Both TNF and IL-1 play a role in the onset of rheumatoid arthritis and in the progression of bone and joint destruction associated with RA. The development of orally active small molecule inhibitors of p38 can be an effective way to inhibit bone resorption, inflammation, as well as other immune and inflammatory based pathologies.

Diarylimidazole Inhibitors
The mode of binding of certain lead inhibitors like the diarylimidazole SK&F86002 is known from x-ray work. The 4-pyridyl group hydrogen bonds with the main chain NH of Met109, the 4-fluorophenyl group fills a hydrophobic pocket partly created by specificity residue Thr106, and the nitrogen of imidazole ring interacts with the terminal nitrogen of Lys53. Lys53 is highly conserved in ATP-binding sites, thus targeting this interaction is unlikely to induce kinase selectivity-but can increase potency. It was reasoned that an azaindole ring could incorporate a basic nitrogen that would be within Hbonding distance to Lys53. Compounds in this class were found to be extremely potent with IC50 of 9 being 6 nM compared to 400 nM for SK&F86002.

Azaindole inhibitors

X-ray Structure

SAR
In the crystal structure of 15 bound to p38, the distance between the nitrogen of Lys53 and the nitrogen of the 5-azaindole is 4.3 angstroms compared to 2.8 for structure 9. 15 shows a loss in potency of about 400-fold. Absence of this nitrogen altogether also drops potency as shown with indole 19a. Reduced electron density of the 4-nitrogen in the heterocyclic systems of 22 and 35 may explain their reduced potencies. 24 and 29 may also be poorly active for the same reason, plus the fact that the 6-azaindole nitrogen may cause some repulsive interactions.

PK properties
Due to poor solubility of 9, efforts were made to improve its physical properties by introducing substituents on the indole nitrogen, as this position does not directly interact with the protein. Small alkyl substituents are preferred based upon the data. Amine and acid bearing substituents were not tolerated. Some of these compounds were found to be more active in vivo in inhibiting LPS-stimulated TNF synthesis.

Metabolism
In studies with hepatic microsomal protein, three sites of oxidative metabolism were identified. The 4pyridyl ring nitrogen and the 4azaindole nitrogen were major sites of metabolic transformation, and hydroxylation of the 6-position of the azaindole ring (45) was observed as a minor metabolite. Mono hydroxyalkylamine derivatives were thus introduced on the pyridine ring with the notion that this may sterically block Noxidation. These were found to be potent both in vitro and in vivo. Compound 42b had the greater plasma exposure.

Synthesis of Amino Substituted 4Azaindoles

Plasma exposure

Mechanism-based design of a protein kinase inhibitor nature structural biology volume 8 number 1 january 2001 37-41
While the nucletotide binding site of kinases has been employed for drug design, the protein substrate binding site has not generally been used. For protein kinases, the direct transfer of the phosphoryl group from ATP to protein substrates occurs, and for such mechanisms designing covalently linked bisubstrate analogs can be a robust approach to inhibitor design. In previous attempst, Gibson linked ATP to the Ser oxygen of a PKA peptide substrate to afford inhibitor compound 1, but this gave an IC50 of 226 micromolar that was competitive versus ATP but noncompetitive versus peptide substrate. Improvements are apparently needed in geometry and electronic character of atoms equivalent to the entering nucleophile and reacting phosphate.

Dissociative TS
Compound 1 has a Ser O-P bond distance (1.7 A) more compatible with a fully associative reaction mechanism for phosphoryl transfer by a protein serine kinase. In this TS, the bond between attacking oxygen and phosphorous atom is largely formed, while bond to the departing ADP would not yet be significantly broken. As evidence of various sorts would indicate that a dissociative transition state is more likely, this would predict that the reaction coordinate distance between the entering nucleophilic oxygen and the attacked phosphorous should be greater than or equal to 4.9 angstroms. This assumes that -phosphoryl group moves toward the entering oxygen and that this nucleophile and ADP are fixed.

Design Strategy
Thus, in designing improved bisubstrate analogs, based upon a dissociative reaction mechanism, a peptide-ATP bisubstrate analog in which the distance between tyrosine nucleophilic atom and -phosphorous was set to about 5 angstroms by a short linker was considered. A second design consideration was based on the fact that the phenolic OH serves as an H-bond donor to the conserved catalytic Asp residue. The tyrosine oxygen was thus replaced by nitrogen to that it could be incorporated into a tether and still hydrogen bond. Compound 2 was thus made and this found to show an IC50 of 10 micromolar at a concentration of 1 mM ATP and 0.3 mM IRS727.

X-ray of IRK
An x-ray structure of this bisubstrate inhibitor bound to the core tyrosine kinase domain of the insulin receptor (cIRK) was obtained. This shows the distance between the phosphorous and aniline nitrogen to be 5 angstroms, the expected reaction coordinate length for a dissociative mechanism. A 3.2 angstrom H-bond between the aniline nitrogen and catalytic Asp (1,132) was also found. The carbonyl oxygen of the linker was coordinated to a Mg2+ in the active site and replacing a water molecule found in the ternary complex. The peptide moiety of the bisubstrate inhibitor binds to cIRK in a manner similar to that observed in the ternary complex. An antiparallel -strand interaction is present between the residues P + 1 to P + 3 of the peptide and Gly 1,169 Leu 1,171 of the activation loop.

X-ray of binary complex A limitation of the present approach is PK properties of peptides and nucleotides; need to identify peptidomimetics and phosphate surrogates.

Legend for X-ray

Stereo view of the interactions between the inhibitor and key catalytic residues. Superimposed are the cIRKbisubstrate inhibitor (binary) complex and the cIRKMgAMP-PNPpeptide (ternary) complex. Oxygen atoms are colored red, nitrogen atoms blue, sulfur atoms green, and phosphorus atoms yellow. Bonds/carbon atoms are colored orange for the binary complex and green for the ternary complex. Bonds and atoms of the ternary complex are semi-transparent. Mg2+ ions and water molecules are represented as purple and red spheres, respectively. Hydrogen bonds and bonds to the Mg2+ are represented as dashed and solid black lines, respectively. Only the modified tyrosine from the peptide moiety of the inhibitor is shown. BSI indicates the bisubstrate inhibitor in the binary complex, and Y(P0) and PNP indicate the substrate tyrosine and AMP-PNP, respectively, in the ternary complex. Selected secondary structural elements (C and 3) are shown. The figure was prepared with GRASP28, BOBSCRIPT29 and MOLSCRIPT30.

p38 alpha MAP Kinase inhibitors using a Fragment-Based Approach. Fragment screening offers an alternative to traditional screening. A fragment based approach using x-ray crystallography has been reported. The fragments identified have weak potency but are efficient binders relative to their size and may represent suitable starting points for lead optimization. Range of interactions like lipophilic, charge-charge, neutral H-bond can drive fragment binding. A library of fragments was created by making use of rings and side chains that make up known drugs. These were studied by virtual screening, and then a library built up for x-ray study. bioassaybased screening methods. Efficient fragment screening requires the soaking of cocktails of fragments into preformed crystals of the target protein.

Secondary Side Chains

Lipophilic/secondary side chains. The properties of the fragments are further modulated by allowing substitution with a set of secondary side chains. Most of these are lipophilic and are intended to pick up hydrophobic interactions in a protein binding site. The secondary side chains for carbon atoms are shown in Figure 4.

Initial Hits
High through-put x-ray crystallography was used to identify low-affinity fragment hits. These fragments exhibit high efficiency binding given their low MWs and limited level of functionality. In this study a structure guided chemistry strategy was employed to transform two such fragment hits into potent compounds having lead like properties. 2-Amino-3-benzyloxypyridine 1 (IC50 1.3 mM) and 3-(2(4-pyridyl)ethyl)indole 2 (IC50 35 mM) were identified from X-ray crystallographic screening of nonphosphorylated p38R MAP kinase (Chart 3).

Fragments Found

Fragment 1 binds to the hinge region of the ATP binding site forming a H-bond with the main chain amide nitrogen of Met109. Pyridine 1 is a competitive inhibitor of p38alpha MAP kinase with respect to ATP. X-ray was solved to 2.2 angstrom resolution. 1 interacts with the hinge region located between the N- and Cterminal lobes of the kinase. The inhibitor makes H-bond interactions through the pyridyl nitrogen to the NH of Met109 and the 2-amino moiety to the His 107 backbone carbonyl. 1 also forms a CH---O H-bond through the pyridyl C6 hydrogen to the backbone carbonyl of Met109. The benzyloxy group also makes a major interaction by filling the lipophilic specificity pocket. To improve upon activity, Examination of the x-ray suggested to improve upon activity by improving inhibitor interactions with the lipophilic specificity pocket.

Modifications
Substitution of the phenyl ring with 2,6-dichloro substitution pattern improved potency to 109 micromolar. Naphthyl substitution further improved activity to 44 micromolar, but for physicochemical reasons the dichlorophenyl compound was advanced. The x-ray structure of 1 also suggested that by accessing the hydrophobic region 2 with appropriate functionality might further increase inhibitory activity. This was the case with 7. Note the 3 indicates that the NH2 group is not necessary for intrinsic activity.

X-ray Fragment 1

Ligand-bound structure

To learn more the known inhibitor 16 was co-crystalyzed with p34alpha MAP kinase. A large conformational change of the residues Asp168Phe169-Gly170 (DFG motif) in the conserved activation loop of the kinase was induced. Overlapping of the crystal structures of 16 with the other inhibitors suggested that substitution at the 2- and 5-positions of the phenyl ring should be carried out. 8, 9, and 10 were thus synthesized with 9 showing 65 nM activity. A large conformational change of the DFG motif is required for binding of these amide and urea derivatives. The Phe169 moves about 10 angstroms to the DFG out conformation. The polar channel formed by Asp168 and Glu71 from the ATP binding site to this allosteric pocket is involved in a H-bond network to both the amide and urea functionalities. The movement of the Phe169 side chain exposes a largely lipophilic pocket into which the morpholine of amide 9 or the t-butyl group of 10 may insert. The morpholine and t-butyl moieties make substantial contacts with neighboring residues of 9 and 10, and they still maintain contacts to the hinge and lipophilic specificity pockets. 9 was found to show good selectivity profile and cellular potency.

PK Selectivity

Indole Fragment
The indole fragment 2 also binds to the hinge region of the ATP binding site forming an H-bond with the main chain amide nitrogen of Met109, with the 4-pyridyl and 3-indolyl moieties having a gauche conformation about the central ethylene spacer. The indolyl moiety is buried deep within the hydrophobic cleft of the specificity pocket. Both rings make substantial contacts including the side chains of Lys53. The indolyl NH H-bonds to the backbone carbonyl of Ala51. A number of analogues were made; the less basic pyrimidine was less active than 2. Substituted pyrimidines containing groups that may bind to Met109 showed improved potency. To target the DFG activation loop, an aromatic amide-indole hybrid 22 was made. X-ray showed that the indole ring again targeted the hydrophobic specificity pocket. 22 makes no contact with Met109 and instead forms two hydrogen bonds from the amide carbonyl and NH to the NH of Asp168 and a side chain oxygen of a salt bridging Glu171. Significant conformational rearrangement of DFG is induced, with the aromatic side chain of Phe169 shielding the hydrophobic regions of 22 from solvent; the morpholine ring of 22 is located in the pocket vacated by Phe169.

X-ray of 2

22 Bound to p38alpha

Hybrid Analogs

To further gain in activity, is became obvious to use 2 and 18 and to add the functionality present in 22. From overlays of the x-rays, 3,5- and 1,6-disubstituted indoles were likely to be more active. 23 and 24 were prepared and these found to have IC50s of 630 and 340 nM. X-ray shows these compounds to have similar binding modes.

Append Fxn. To 18

Similar Binding Modes

Discovery of Glivec Capdeville, R., Buchdunger, E., Zimmermann, J., Matter, A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov, 1, 493-502, (2002)

Until 1980s, drug discovery programs were focused almost exclusively on DNA synthesis and cell division resulted in agents such as antimetabolites, alkylating agents and microtuble destabilizers. These drugs generally showed tox. Due to lack of selectivity. Next genes were discovered that were cancer causing, these were later called oncogenes. One interesting cancer is chronic myelogenous leukaemia (CML)- a haematological stem-cell disorder that is characterized by excessive proliferation of cells of the myeloid lineage.

Lead from PKC Screen

BCR-ABL was established as the molecular pathogenic event in CML. The tyrosine-kinase activity of BCR-ABL is crucial for its transforming activity; the enzymatic activity of this deregulated gene was thus deemed an attractive target for addressing BCR-ABL positive leukaemias. The Bcr-Abl fusion proteins have constitutive activity. In the case of Glivec, a lead compound was identified in a screen for inhibitors of PKC. A phenylaminopyridine had potential lead like properties, high potential for diversity, allowing simple chemistry to be used to alter potency and selectivity. Strong PKC inhibition was obtained with derivatives bearing a 3-pyridyl group at the 3-position of the pyrimidine. Presence of an amide group on the phenyl ring gave inhibitory activity against tyrosine kinases, such as the BCR-ABL kinase. Next, it was found that substitution at position 6 of the diaminophenyl ring abolished PKC inhibitory activity, while activity against tyrosine kinases was retained or even enhanced. By attaching a highly polar side chain (N-methylpiperazine), solubility was improved and oral bioavailability.

Glivec binds to inactive form of kinase.

Docking studies and x-ray showed that binding of Glivec occurs at the ATP-binding site. Anaylsis of the crystal structure showed that Glivec inhibits the ABL kinase by binding with high specificity to the inactive form of the kinase. The need for the kinase to adopt this unusual conformation which favors binding may contribute to the high selectivity of the compound. In broader screen of kinases, Glivec was found to inhibit c-KIT, PDGF, and ARG kinase. Glivec selectively induced apoptosis in BCR-ABL positive cell lines, and induced cell killing in primary leukaemia cells from patients with Philadelphiachromosome-positive CML and acute lymphoblastic leukaemia.

Chemical optimization

X-ray structure
In the x-ray structure of a variant of STI-571, with Abl kinase (2), STI extends much further into the catalytic domain, and its pyridinyl group is inserted underneath helix alphaC in the NH2-terminal lobe of the kinase. The compound is kinked at the secondary amino group and it straddles the highly conserved NH2-terminal region of the activation loop. Tyr393 in the activation loop is the major site of phosphorylation in Abl, but the form that was crystallized is not phosphorylated. The activation loop is folded into the active site of the kinase, and Tyr393 forms a H-bond with Asp363, a side chain crucial for catalysis. The activation loop mimics the binding mode of substrates. There are a number of H-bond and van der Waals interactions that stabilize STI in complex with Abl, but it does not directly contact the activation loop except for N-terminal anchor region.

Dual Src-Abl kinase inhibitors

By targeting the tyrosine kinase activity of Bcr-Abl, Glivec normalizes peripheral white blood cell counts and reduces the Philadelphia chromosome positive clone of stem cells in bone marrow. The c-Src proto-oncogene also plays a major role in the development, growth (3), progression, and metastasis of a wide variety of human cancers. Src activation in terms of elevated kinase activity and protein expression has been demonstrated in several cancer type including colon, breast, lung, and brain carcinomas. Lck kinase activity of 2-acylamino-5-carboxamidothiazoles has been described, and improvement in activity found by replacing the acyl group with heterocycles. Compound 1 was found to be inhibitor of Lck, Fyn, Src, and Hck. The compound also was found to block BcrAbl kinases with IcC50 of < 1 nM.

SAR
Compound 2 was potent against 4 tumor cell lines when tested in cellular assays, but produced low circulating plasma levels. Eventually compound 13 was found to have broad spectrum antiproliferative activity and oral exposure in the mouse screening assay. X-ray structure of 13 with Abl was determined. Placement of the activation loop in an active conformation and 3 H-bonds were found. 2-Amino hydrogen of 13 and carbonyl oxygen of Met318 and between the 3-nitrogen of the thiazole ring of 13 and the amide nitrogen of Met 318. The hydroxyl oxygen of Thr315 and the amide nitrogen of 13 also make H-bond. Ki against Src and Bcr-Abl are 16 and 30 pM respectively.

SAR Table

Synthesis of 13

X-ray

In vivo activity against CML

The in vivo activity of 13 was evaluated in a K562 xenograft assay in nude mice (Figure 3). Tumor cells were implanted subcutaneously and staged to approximately 300 mg prior to two cycles of oral drug administration on a 5 day on and 2 day off schedule. Following once daily doses of either 5 or 50 mg/kg, compound 13 showed partial tumor regressions after one treatment cycle and complete disappearance of tumor mass at the end of the drug treatment.