patrick an introduction to medicinal chemistry 3/e chapter 6 proteins as drug targets:

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Patrick Patrick An Introduction to Medicinal An Introduction to Medicinal

ChemistryChemistry 3/e 3/e

Chapter 6 Chapter 6

PROTEINS AS DRUG PROTEINS AS DRUG TARGETS:TARGETS:

RECEPTOR STRUCTURE & RECEPTOR STRUCTURE & SIGNAL TRANSDUCTIONSIGNAL TRANSDUCTION

Part 5: Case StudyPart 5: Case Study

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ContentsContents

Part 5: Case Study6. Case Study - Inhibitors of EGF Receptor Kinase

6.1. The target (4 slides)6.2. Testing procedures

- In vitro tests (3 slides)- In vivo tests (2 slides)- Selectivity tests

6.3. Lead compound – Staurosporine6.4. Simplification of lead compound (2 slides)6.5. X-Ray crystallographic studies (2 slides)6.6. Synthesis of analogues6.7. Structure Activity Relationships (SAR)6.8. Drug metabolism (2 slides)6.9. Further modifications (3 slides)6.10.Modelling studies on ATP binding (4 slides)6.11.Model binding studies on Dianilinophthalimides (4 slides)6.12.Selectivity of action (3 slides)6.13.Pharmacophore for EGF-receptor kinase inhibitors 6.14.Phenylaminopyrrolopyrimidines (3 slides)6.15.Pyrazolopyrimidines

[43 slides]

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6. Case Study - Inhibitors of EGF Receptor Kinase6. Case Study - Inhibitors of EGF Receptor Kinase6.1 The target6.1 The target - Epidermal growth factor receptor- Epidermal growth factor receptor

- Dual receptor / kinase enzyme role - Dual receptor / kinase enzyme role

Receptor

cell membrane

Extracellularspace

Cell

Binding site

Kinase active site(closed)

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Overexpression Overexpression of erbB1 geneof erbB1 gene

ExcessExcessreceptorreceptor

Excess sensitivityExcess sensitivityto EGFto EGF

Excess signalExcess signalfrom receptorfrom receptor

Excess cell growthExcess cell growthand division and division

Tumours Tumours

KINASE INHIBITORKINASE INHIBITOR-

6.1 The target6.1 The target

Potential Potential anticanceranticancer

agentagent

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MgTyrosine kinase

N

N N

N

N

O

OH OH

H H

H H

O P O

O

O

P O

O

O

P

O

O

O

H H

HO

HN Protein

O

Protein

Tyrosineresidue

ATP

N

N N

N

N

O

OH OH

H H

H H

O P O

O

O

P O

O

O

H H

O

HN Protein

O

Protein

PO

OO

ADPPhosphorylatedtyrosine residue

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Inhibitor DesignInhibitor Design

Possible versus binding site for tyrosine regionPossible versus binding site for tyrosine regionPossible versus binding site for ATP Possible versus binding site for ATP

Inhibitors of the ATP binding siteInhibitors of the ATP binding site

Aims: Aims: To design a potent but selective inhibitor versus EGF receptorTo design a potent but selective inhibitor versus EGF receptorkinase and not other protein kinases.kinase and not other protein kinases.


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In vitroIn vitro tests tests Enzyme assay Enzyme assay

using kinase portion of the EGF receptor produced by recombinant using kinase portion of the EGF receptor produced by recombinant DNAtechnology. Allows enzyme studies in solution.DNAtechnology. Allows enzyme studies in solution.

6.2 Testing procedures6.2 Testing procedures

EGF-Rcell membrane

Cell

RecombinantDNA

Watersolublekinase

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In vitroIn vitro tests tests Enzyme assay Enzyme assay Test inhibitors by ability to inhibit standard enzyme catalysed reactionTest inhibitors by ability to inhibit standard enzyme catalysed reaction

Angiotensin IIAngiotensin IIOHOH

InhibitorsInhibitors

Assay productto test inhibition

• Tests inhibitory activity only and not ability to cross cell membrane• Most potent inhibitor may be inactive in vivo


Angiotensin IIAngiotensin IIO PO P

ATP ADP

kinasekinase

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In vitroIn vitro tests tests

Cell assaysCell assays • Use cancerous human epithelial cells which are sensitive to EGF for growth• Measure inhibition by measuring effect on cell growth - blocking kinase

activity blocks cell growth.• Tests inhibitors for their ability to inhibit kinase and to cross cell membrane• Assumes that enzyme inhibition is responsible for inhibition of cell growth

ChecksChecks• Assay for tyrosine phosphorylation in cells - should fall with inhibition• Assay for m-RNA produced by signal transduction - should fall with

inhibition• Assay fast growing mice cells which divide rapidly in presence of EGF


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In vivoIn vivo tests tests • Use cancerous human epithelial cells grafted onto miceUse cancerous human epithelial cells grafted onto mice

• Inject inhibitor into miceInject inhibitor into mice

• Inhibition should inhibit tumour growthInhibition should inhibit tumour growth

• Tests for inhibitory activity + favourable pharmacokineticsTests for inhibitory activity + favourable pharmacokinetics


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Selectivity testsSelectivity tests

Similar Similar in vitroin vitro and and in vivoin vivo tests carried out on serine- tests carried out on serine-threonine kinases and other tyrosine kinasesthreonine kinases and other tyrosine kinases


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6.3 Lead compound - Staurosporine6.3 Lead compound - Staurosporine

• Microbial metaboliteMicrobial metabolite• Highly potent kinase inhibitor but no selectivityHighly potent kinase inhibitor but no selectivity• Competes with ATP for ATP binding siteCompetes with ATP for ATP binding site• Complex molecule with several rings and asymmetric centresComplex molecule with several rings and asymmetric centres• Difficult to synthesiseDifficult to synthesise

HN

NN

O

O

NH

O

H3C

H3C

H3C

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HN

NHN

H

OO

6.4 Simplification of lead compound6.4 Simplification of lead compound

Arcyriaflavin A• Symmetrical molecule• Active and selective vs

PKC but not EGF-R

HN

NN

O

O

NH

O

H3C

H3C

H3C**

* *

Staurosporine

SimplificationRemove asymmetricring

HN

NHN

H

O

SimplificationSymmetry

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HN

NH

NH

OO

Simplification

HN

NHN

H

OO

maleimide ring

BisindolylmaleimidesPKC selective

indole ring indole ring

Simplification

Dianilinophthalimide (CGP 52411)• Selective inhibitor for EGF

receptor and not other kinases• Reversal of selectivity

HN

NH

NH

OO

Aniline Aniline

Phthalimide

6.4 Simplification of lead compound6.4 Simplification of lead compound

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6.5 X-Ray crystallographic studies6.5 X-Ray crystallographic studies

Different shapes implicated in different selectivityDifferent shapes implicated in different selectivity

NH

NH

O OHN

PlanarPlanar

NH

NH

O OHN

Bowl shapedBowl shaped

NH HN

O OHN

Propellor shapedPropellor shapedasymmetricasymmetric

ArcyriaflavinArcyriaflavin Bisindolyl-maleimidesBisindolyl-maleimides Dianilino-phthalimidesDianilino-phthalimides

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StericStericclashclash

StericStericclashclash

HN OO

NH HN

H HH H

Propeller conformation relieves steric clashesPropeller conformation relieves steric clashes

PlanarPlanar

TwistTwist

HN OO

NH HN

H H

H H

PropellerPropellershapeshape

6.5 X-Ray crystallographic studies6.5 X-Ray crystallographic studies

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6.6 Synthesis of analogues6.6 Synthesis of analogues

O

O

TMSCl, NEt3

DMF, 100 oC

OSi(CH3)3(H3C)3SiO

OO

OO

H3C CH3Anilines

Acetic acid, 120 oC

R2NNR2

OO

OO

H3C CH3

R1 R1

a) LiOH, MeOH

b) (Ac)2O, toluene

R2NNR2

OO O

R1 R1

NH3 or formamides

140-150 oC

RN OO

NR2 R2NR1 R1

H2C

H2C O

O

Si (CH3)3

Si (CH3)3MeO2C

C

C

CO2Me

Diels AlderToluene

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• R=H R=H Activity lost if N is substitutedActivity lost if N is substituted• Aniline aromatic rings essential (activity lost if cyclohexane)Aniline aromatic rings essential (activity lost if cyclohexane)• RR11=H or F (small groups). Activity drops for Me and lost for Et=H or F (small groups). Activity drops for Me and lost for Et• RR22=H Activity drops if N substituted=H Activity drops if N substituted• Aniline N’Aniline N’ss essential. Activity lost if replaced with S essential. Activity lost if replaced with S• Both carbonyl groups important. Activity drops for lactamBoth carbonyl groups important. Activity drops for lactam

6.7 Structure Activity Relationships (SAR)6.7 Structure Activity Relationships (SAR)RN OO

NR2 R2NR1 R1

HN O

NH HN

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6.7 Structure Activity Relationships (SAR)6.7 Structure Activity Relationships (SAR)

Parent Structure: R=RParent Structure: R=R11=R=R22=H chosen for preclinical trials=H chosen for preclinical trialsICIC5050 = 0.7 = 0.7 MM

HN OO

NH HN

CGP 52411

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6.8 Drug metabolism6.8 Drug metabolism

Excretion

Excretion

HN OO

NH HN

CGP 52411

Metabolism(man,mouse,rat, dog)

Metabolism(monkey)

HN OO

NH HNHO

HN OO

NH HNHO OH

GlucuronylationOGlucose DrugDrug

GlucuronylationOGlucose O GlucoseDrugDrug

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Metabolicblocker

Metabolicblocker

Introduce F at Introduce F at parapara position as metabolic blocker position as metabolic blocker

HN OO

NH HNF F

CGP 53353

6.8 Drug metabolism6.8 Drug metabolism

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6.9 Further modifications6.9 Further modifications

a) Chain extensiona) Chain extension

Activity dropsActivity drops

HN OO

NH HN

CGP58109

Chain extensionChain extension

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b) Ring extension / expansionb) Ring extension / expansion

CGP54690 (IC50 0.12M)Inactive in cellular assays due to polarity (unable to cross cell membrane)


extensionextension

CGP 52411 (IC50 0.7M)

HN OO

NH HN NH HN

NHHN

O O removeremovepolar groupspolar groups

ring ring expansionexpansion

CGP57198 (IC50 0.18M)Active in vitro and in vivo

NH HN

NHN

O

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CGP58522Similar activity in enzyme assayInactive in cellular assay


c) Simplificationc) Simplification

HN OO

NH HN

CGP52411

HN OO

NH OH

SimplificationSimplification

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6.10 Modelling studies on ATP binding6.10 Modelling studies on ATP binding

• No crystal structure for EGF- receptor availableNo crystal structure for EGF- receptor available

• Make a model active site based on structure of an Make a model active site based on structure of an analogous protein which has been crystallisedanalogous protein which has been crystallised

• Cyclic AMP dependant protein kinase used as templateCyclic AMP dependant protein kinase used as template

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Cyclic AMP dependant protein kinase Cyclic AMP dependant protein kinase + Mg + ATP + Inhibitor (bound at + Mg + ATP + Inhibitor (bound at substrate site)substrate site)

X-Ray CrystallographyX-Ray Crystallography

Molecular modellingMolecular modelling

CrystalsCrystals

CrystalliseCrystallise

Structure of protein /Structure of protein /inhibitor / ATP complexinhibitor / ATP complex

Identify active siteIdentify active siteand binding interactionsand binding interactionsfor ATPfor ATP


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• ATP bound into a cleft in the enzyme with adenine portion ATP bound into a cleft in the enzyme with adenine portion buried deep close to hydrophobic region. buried deep close to hydrophobic region.

• Ribose and phosphate extend outwards towards opening of Ribose and phosphate extend outwards towards opening of cleftcleft

• Identify binding interactions (measure distances between Identify binding interactions (measure distances between atoms of ATP and complementary atoms in binding site to atoms of ATP and complementary atoms in binding site to see if they are correct distance for binding)see if they are correct distance for binding)

• Construct model ATP binding site for EGF-receptor kinase Construct model ATP binding site for EGF-receptor kinase by replacing amino acid’s of cyclic AMP dependent protein by replacing amino acid’s of cyclic AMP dependent protein kinase for those present in EGF receptor kinasekinase for those present in EGF receptor kinase


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N

N N

N

N

O

OH OH

H H

H H

O P O

O

O

P O

O

O

P

O

O

O

H H

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emptyemptypocketpocket

'ribose' pocket'ribose' pocket

H-bond interactionsH-bond interactions

N

N

O

H

H

O

HN

HN

OHO

H2NOC

H3C

H3C

O

SH3C

Gln767Thr766

Met769

Leu768

1N is a H bond acceptor6-NH2 is a H-bond donorRibose forms H-bonds to Glu in ribose pocket


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6.11 Model binding studies on Dianilinophthalimides6.11 Model binding studies on Dianilinophthalimides

N

N

O

H

H

O

HN

HN

OHO

H2NOC

H3C

H3C

O

SH3C

Gln767Thr766

Met769

Leu768



N

NH

HN

O

O

H

H-bond interactionH-bond interaction

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• Both imide carbonyls act as H-bond acceptors (disrupted if Both imide carbonyls act as H-bond acceptors (disrupted if carbonyl reduced)carbonyl reduced)

• Imide NH acts as H bond donor (disrupted if N is substituted)Imide NH acts as H bond donor (disrupted if N is substituted)

• Aniline aromatic ring fits small tight ribose pocketAniline aromatic ring fits small tight ribose pocket

• Substitution on aromatic ring or chain extension prevents Substitution on aromatic ring or chain extension prevents aromatic ring fitting pocketaromatic ring fitting pocket

• Bisindolylmaleimides form H-bond interactions but cannot fit Bisindolylmaleimides form H-bond interactions but cannot fit aromatic ring into ribose pocket. aromatic ring into ribose pocket.

• Implies ribose pocket interaction is crucial for selectivityImplies ribose pocket interaction is crucial for selectivity


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6.12 Selectivity of action6.12 Selectivity of action

POSERS ?POSERS ?

• Ribose pocket normally accepts a polar ribose so why can it Ribose pocket normally accepts a polar ribose so why can it accept an aromatic ring?accept an aromatic ring?

• Why can’t other kinases bind dianilinophthalimides in the same Why can’t other kinases bind dianilinophthalimides in the same manner?manner?

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Amino Acids present in the ribose pocketAmino Acids present in the ribose pocket

Hydrophobic Hydrophilic

Protein Kinase A

EGF Receptor Kinase

Leu,Gly,Val,LeuLeu,Gly,Val,Leu

Leu,Gly,Val,Leu,CysLeu,Gly,Val,Leu,Cys

Glu,Glu,Asn,ThrGlu,Glu,Asn,Thr

Arg,Asn,ThrArg,Asn,Thr


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• Ribose pocket is more hydrophobic in EGF-receptor kinaseRibose pocket is more hydrophobic in EGF-receptor kinase• Cys can stabilise and bind to aromatic rings (S-Ar interaction)Cys can stabilise and bind to aromatic rings (S-Ar interaction)

• Stabilisation by S-Ar interaction not present in other kinasesStabilisation by S-Ar interaction not present in other kinases• Leads to selectivity of actionLeads to selectivity of action

N

N

O

H

H

O

HN

HN

OHO

H2NOC

H3C

H3C

O

SH3C

Gln767Thr766

Met769

Leu768



N

NH

HN

O

O

H

SSH


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6.13 Pharmacophore for EGF-receptor kinase inhibitors6.13 Pharmacophore for EGF-receptor kinase inhibitors

• Pharmacophore allows identification of other potential inhibitorsPharmacophore allows identification of other potential inhibitors• Search databases for structures containing same pharmacophoreSearch databases for structures containing same pharmacophore• Can rationalise activity of different structural classes of inhibitorCan rationalise activity of different structural classes of inhibitor

N

NH

HN

O

O

HHBDHBD

HBAHBA

ArAr

HBDHBD

HBAHBA

ArArPharmacophorePharmacophore

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HBDHBD

HBAHBA

ArAr

Mode IIMode II

HBDHBD

HBAHBA

Mode IMode I

N

N

N

NH

Cl

H

6.14 Phenylaminopyrrolopyrimidines6.14 Phenylaminopyrrolopyrimidines

Only mode II tallies with pharmacophore and explains activity Only mode II tallies with pharmacophore and explains activity and selectivity and selectivity

CGP 59326 - Two possible binding modes for H-bondingCGP 59326 - Two possible binding modes for H-bonding

N

N NH

NH

Cl

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Illustrates dangers in comparing structures and assuming similar Illustrates dangers in comparing structures and assuming similar interactions (e.g. comparing CGP59326 with ATP)interactions (e.g. comparing CGP59326 with ATP)


Binding Mode I like ATP (not favoured)

N

N NH

NH

N

O

H

Cl

S

H

emptypocket

'ribose' pocket

CGP59326

Binding mode II (favoured)

N

O

H

N

N

N

N

H

Cl

H

S

H

emptypocket

'ribose' pocket

CGP59326

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• Both structures are selective EGF-receptor kinase inhibitorsBoth structures are selective EGF-receptor kinase inhibitors• Both structures belong to same class of compoundsBoth structures belong to same class of compounds• Docking experiments reveal different binding modes to obey Docking experiments reveal different binding modes to obey

pharmacophore pharmacophore

6.15 Pyrazolopyrimidines6.15 Pyrazolopyrimidines i) Lead compoundsi) Lead compounds

N

N N

N

NH2

(I) EC50 0.80M

N

N NH

N

NH2

H2N

Cl

(II) EC50 0.22M

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HBDHBD

HBAHBA

ArAr

N

N N

N

NH H

ii) Structure Iii) Structure I

6.15 Pyrazolopyrimidines6.15 Pyrazolopyrimidines

N

N N

N

NH H

N

O

H

S

H

emptypocket

'ribose' pocket

Structure I

Extra binding Extra binding interactionsinteractions

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iii) Structure IIiii) Structure II • Cannot bind in same mode since no fit to ribose pocketCannot bind in same mode since no fit to ribose pocket

N

O

HN

N

NNH

NH

Cl

H2N

S

H

emptypocket

'ribose' pocket

Structure II

• Binds in similar mode to phenylaminopyrrolopyrimidinesBinds in similar mode to phenylaminopyrrolopyrimidines


unoccupiedunoccupied

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ExtraExtraH-bondingH-bondinginteractioninteraction

HBDHBD

HBAHBA

ArAr

HBDHBD

HBAHBA

iv) Drug design on structure IIiv) Drug design on structure II

• Upper binding pocket is larger than ribose pocket allowing greater Upper binding pocket is larger than ribose pocket allowing greater variation of substituents on the ‘upper’ aromatic ringvariation of substituents on the ‘upper’ aromatic ring


SimplificationSimplification(remove extra (remove extra functional group)functional group)N

N

NNH

NH

Cl

H2N

(II) EC50 0.22M

N

N

NNH

NH

Cl

(IV) EC50 0.16M

Activity increases

ExtensionExtension(add aromatic (add aromatic ring for ribosering for ribosepocket)pocket)

N

N

NNH

NH

Cl

NH

Cl

(V) EC50 0.033M

Activity increasesAr fits ribose pocket

ExtensionExtensionN

N

NNH

NH

OH

NH

Cl

(VI) EC50 0.001MActivity increases

patrick an introduction to medicinal chemistry 3/e chapter 6 proteins as drug targets:

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