Spring 2008

PHAR 751: Drug Design Kinase Regulation and targeting

Objective: Describe the basic regulatory mechanisms of protein kinases and applications for inhibitor development. Discussion paper: Martin et al. (2008) The Docking Interactions of Caspase-9 with ERK2.J. Biological Chemistry, 283:3854-3865.

Paul Shapiro Office 536PH, Lab 547, 551, 555PH Phone: 6-8522 pshapiro@rx.umaryland.edu

Why study phosphorylation and kinases? Role in disease: 1. Information transfer / signal transduction 2. Genetic mutations 3. Constitutive activation

Phosphorylation: The balance between kinases and phosphatases.

Why phosphorylation?
1. Causes allosteric changes in protein. 2. Two negative charges. 3. Attracts positive side chains (Lys, Arg). 4. Occurs on Serine, threonine, and tyrosine.

Structure / function relationship

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Overview of protein kinases and signaling: MAP kinases

Receptor EC Signal G-proteins
Adaptor proteins Exchange factors

MKKK MKK

(K=kinase)

Nuclear Txn factors

Other signaling proteins Structural proteins

MAPKs (eg. ERK, JNK, p38) Other kinases

Extracellular signal-regulated kinase (ERK) signaling

Mitogen Activated Protein (MAP) kinases

Receptor tyrosine kinase (RTK) Ras MAP kinase module 1. MAPKKK (MEKK) 2. MAPKK (MEK, MKK) 3. MAP kinase TXY motif Raf MEK1/2 G-protein (GTP)

ERK1/2 Regulation of cell proliferation and survival

Mitogen activated protein (MAP) kinases and disease.

(The extracellular signal regulated kinases (ERK) are a subfamily of MAP kinases) -Oncogenic Ras mutations in ~30% of all cancers. -Oncogenic Raf mutations in ~70% of malignant melanomas. -Over-expression / activation of RTK in many cancers.
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General kinase structure

1. Often N and C-terminal lobes. 2. N-terminal ATP binding site. 3. Activating / catalytic loop (phosphorylated). 4. Short sequences (red arrowheads) are unique to specific kinase and may determine substrate specificity. Cyclic AMP-dependent kinase (PKA)
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PKA comparison with ERK2

PKA

ERK2
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Structural Similarities Between Protein Kinases. Structure / Function Relationship

But, kinases maintain substrate selectivity.

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

p38 MAP kinase

p38 MAP kinase and ERK2

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

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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 many other physiological responses. b. Constitutive activation is involved in disease. c. Few MAP kinase inhibitors exist. d. Most kinase inhibitors target ATP binding, thus may lack selectivity. Why? e. Is it advantageous to selectively inhibit some but not all 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 disease but 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 and protein function.
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Substrate selective inhibition of ERK functions using small molecules.

Cell proliferation
S1 S2 S1 S2 pS1 pS2

ERK
S3 S4

ERK
S3 S4 pS3

ERK
pS4

ATP Test compound

ADP

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General CADD Research Design

(for ERK2 inhibitors)
1. CADD database search of compounds that target ERK2 docking domains (need 3D structure)

2. Screen compounds based on ability to inhibit ERK-specific phosphorylation of substrate proteins and determine ERK binding interactions.

3. Evaluate the effectiveness of biologically active compounds in inhibiting ERK-mediated cell proliferation.

Future. Optimize lead compounds for selective inhibition of ERK substrates and in vivo studies.
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Example: ERK2 CD and ED docking domains and computer aided drug design (CADD).

ATP

N-terminal

substrate

C-terminal

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

Molecular model of ERK2

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Biological testing of test compounds identified by CADD

EGFR Ras Raf-1
ppERK1/2 0

0 10 25 50 75 M Compound #76 pElk-1

+ +

+ EGF

MKK1/2 ERK1/2 ELK-1

-tubulin

Immunoblot analysis with phospho-specific antibodies.

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CADD identification of active compounds using the 3D structure of active ERK2

EGFR Ras Raf-1
100 80 60

+ + + + + + + + + + + + + + EGF - 86 87 88 89 90 91 92 93 94 95 96 97 98 Compound
pELK-1 -tubulin ppERK1/2

MKK1/2 ERK1/2 ELK-1

pELK (% EGF)

40 20 0

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

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Does compound 76 interact with ERK2?

450 400 350 300 250 200 150 100 50 0 300 350 400 450 500

ERK2 only +0.1 M +0.5 M +1.0 M +2.5 M +5.0 M +10.0 M +15.0 M +20.0 M +50.0 M +100.0 M

Fluorescence

Fluorescence spectroscopy.

400 350 300 250

nm

Fluorescence

200 150 100 50 0 300 350 400 450 500

DMSO control

ERK2 only ERK2+DMSO

DMSO final concentration =2.5%

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nm

Fluorescence quenching: analysis of test compounds binding to ERK2

400

Fluorescence

300 200 100 0

DMSO 81 Test 67 76 compounds 36 -6. 5 -6 -5.5 -5 -4.5 -4 -3.5

Log[M]

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Progress towards better ERK inhibitors. Similarity searches and ERK2 binding

Compound #101 (similar to #76) Kd ~ 400 nM

10 fold improvement in ERK2 binding as compared to #76

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Conclusions: multiple docking domains regulate substrate interactions.

ATP

N-terminal

substrate

C-terminal
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ERK2 docking domains

Docking site 1
Blue (Site 1, CD / ED domain) D316, 319 T157, 158

ATP

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ERK2 Docking 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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nl ubu / KL Ep i t

Can substrate selectivity be achieved with small molecules? Elk-1 vs. Rsk-1
EGFR Ras Raf

7 0 6 0 5 0 4 0 3 0 2 0 1 0 0

Elk-1

10 2

10 0

8 0

6 0

4 0

Rsk-1

2 0

nl ub t i ppERK/ tubulinu / KS Rp

MKK1/2 ERK1/2 p90Rsk1 (site 1) Elk-1 (sites 1, 2 and 3)

0.30 0.25 0.20 0.15 0.10 0.05 0.00

ERK2
(-) 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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