pshapiro kinase regulation 08

8/6/2019 PShapiro Kinase Regulation 08

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PHAR 751: Drug DesignSpring 2008

Kinase Regulation and targeting Objective: Describe the basic regulatory mechanismsof protein kinases and applications for inhibitor development.

Discussion paper: Martin et al. (2008) The DockingInteractions of Caspase-9 with ERK2. J. Biological Chemistry , 283:3854-3865.

Paul ShapiroOffice 536PH, Lab 547, 551, 555PHPhone: [email protected]


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Why study phosphorylation and kinases?

Role in disease:

1. Information transfer / signal transduction

2. Genetic mutations

3. Constitutive activation


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Phosphorylation:The balance between kinases and phosphatases.


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Why phosphorylation?

1. Causes allostericchanges in protein.2. Two negative charges.3. Attracts positive side

chains (Lys, Arg).4. Occurs on Serine,

threonine, andtyrosine.

Structure / function relationship


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Overview of protein kinases and signaling:MAP kinases

EC SignalAdaptor proteinsExchange factors

Receptor

G-proteins

MAPKs (eg. ERK,JNK, p38)

Nuclear Txn factors

Other

kinases

Other signalingproteins

Structural

proteins

MKK

MKKK (K=kinase)


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Extracellular signal-regulated kinase (ERK) signaling


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Ras

MEK1/2

ERK1/2

Raf

Mitogen Activated Protein (MAP) kinases

3. MAP kinase

2. MAPKK(MEK, MKK)

1. MAPKKK(MEKK)

MAP kinase module

TXY motif

Receptor tyrosine kinase (RTK)

G-protein (GTP)

Regulation of cell proliferation and survival


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-Oncogenic Ras mutations in ~30% of all cancers.

-Oncogenic Raf mutations in ~70% of malignant

melanomas.

-Over-expression / activation of RTK in many cancers.

Mitogen activated protein (MAP) kinases

and disease.

(The extracellular signal regulated kinases (ERK)are a subfamily of MAP kinases)


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General kinase structure

1. Often N and C-terminallobes.

2. N-terminal ATP binding site.

3. Activating / catalytic loop

(phosphorylated).4. Short sequences (red

arrowheads) are unique tospecific kinase and may

determine substratespecificity.

Cyclic AMP-dependent kinase (PKA)


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PKA ERK2

PKA comparison with ERK2


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Structural SimilaritiesBetween Protein Kinases.

Structure / FunctionRelationship

But, kinases maintainsubstrate selectivity.


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Wang et al. (1997) Proc. Natl. Acad. Sci. 94, 2327-2332

Comparison of MAP kinase members.(p38 MAPK and ERK2 structures)

p38 MAP kinase p38 MAP kinase and ERK2


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JNK1 (another MAP kinase) and JIP1 interactions

From: Heo et. al. (2004) The Embo J. 23:2185-2195.


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MAP Kinase interactions with substrates.

1. Proline directed kinases:-consensus PXS/TP site on substrates.-minimum S/TP.

1. Substrate domains:

a. FXFP motif.b. D-domain - basic residues followed by an LXL motif.c. Kinase interaction motif (KIM) on phosphatases.

3. Docking domains on MAP kinases (ERK and p38).

Tanoue et al. (2001) EMBO J. 20:466-479.


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Regulating MAP kinase function:

a. MAP kinases involved in cell proliferation and manyother physiological responses.

b. Constitutive activation is involved in disease.

c. Few MAP kinase inhibitors exist.d. Most kinase inhibitors target ATP binding, thus maylack selectivity. Why?

e. Is it advantageous to selectively inhibit some but notall MAP kinase substrates?


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Problem:-MAP kinase (ERK) proteins phosphorylate and regulate

dozens of substrates (~ 70).How can one inhibit ERK substrates involved in diseasebut not normal metabolic processes?

Hypothesis:-Low MW compounds that bind unique MAP kinase

docking domains can selectively inhibit interactions

between the kinase and a specific substrate protein.

Thus, selective inhibition of phosphorylation andprotein function.


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ERK

S3 S4

S1 S2

ATP ADP

pS1

pS4pS3

pS2

Cell proliferation

Substrate selective inhibition of ERK functionsusing small molecules.

Test compound

ERK ERKS3 S4

S1 S2


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2. Screen compounds based on ability to inhibitERK-specific phosphorylation of substrateproteins and determine ERK binding interactions.

3. Evaluate the effectiveness of biologicallyactive compounds in inhibiting ERK-mediatedcell proliferation.

1. CADD database search of compounds that

target ERK2 docking domains (need 3D structure)

Future. Optimize lead compounds for selectiveinhibition of ERK substrates and in vivo studies.

General CADD Research Design(for ERK2 inhibitors)


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Example: ERK2 CD and ED docking domainsand computer aided drug design (CADD).

N-terminal

C-terminal

Molecular model of ERK2

ATP

substrate

Blue: D316 and D319 (common docking; CD domain).Green: T157 and T158(ED domain)


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EGFR

Ras

Raf-1

MKK1/2

ERK1/2

ELK-1

pElk-1

ppERK1/2

-tubulin

0 0 10 25 50 75 M Compound #76- + + + + + EGF

Biological testing of test compoundsidentified by CADD

Immunoblot analysis withphospho-specific antibodies.


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CADD identification of active compounds usingthe 3D structure of active ERK2

0

20

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60

80

100

pELK-1

-tubulin

ppERK1/2

- 86 87 88 89 90 91 92 93 94 95 96 97 98 Compound (100 M)

- - 86 87 88 89 90 91 92 93 94 95 96 97 98 Compound- + + + + + + + + + + + + + + EGF

pELK(% EGF)

EGFR

Ras

Raf-1

MKK1/2

ERK1/2

ELK-1


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0

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300 350 400 450 500

0

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300 350 400 450 500

nm

Fluorescence

Does compound 76 interact with ERK2?

nm

Fluorescence

DMSO final concentration =2.5%

ERK2 only+0.1 M+0.5 M+1.0 M

+2.5 M+5.0 M+10.0 M+15.0 M

+50.0 M+100.0 M

+20.0 M

ERK2 onlyERK2+DMSODMSOcontrol

Fluorescencespectroscopy.


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0

1 0 0

2 0 0

3 0 0

4 0 0

Log[M]

Fluorescence

Testcompounds

DMSO816776

36

-6. 5 -6 -5.5 -5 -4.5 -4 -3.5

Fluorescence quenching: analysis of testcompounds binding to ERK2


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Progress towards better ERK inhibitors.

Similarity searches and ERK2 binding

10 fold improvement in ERK2 binding as comparedto #76

Compound #101(similar to #76)

Kd ~ 400 nM

Conclusions: multiple docking domains


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Conclusions: multiple docking domainsregulate substrate interactions.

N-terminal

C-terminal

ATP

substrate


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Blue (Site 1, CD / ED domain)

D316, 319T157, 158

ERK2 docking domainsDocking site 1

ATP


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ERK2Docking sites 2 and 3

Blue (Site 1)

Yellow (Site 2): L114, S151,W190, Y191, E218, N222, P224

Green (Site 3): S221, R223, H237,R275

Cyan (other docking residues):L198, H230, Y231, L232, L235,

Y261

Red (activation site):T183 and Y185


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ppERK/tubulin

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pELK /tubulin

(-) EGF 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10

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pRSK /tubulin

Can substrateselectivity beachieved with

small molecules?Elk-1 vs. Rsk-1

EGFR

Ras

Raf

MKK1/2

ERK1/2

p90Rsk1 (site 1)Elk-1 (sites 1, 2 and 3)

Elk-1

Rsk-1

ERK2

pshapiro kinase regulation 08

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