Caliper Labchip Technology in

the Parallel Development of

Small Molecule Inhibitors of
Tyro3, Axl and Mer (TAM)
Kinases
Amy Van Deusen, Catherine Simpson, Bill Janzen, Xiaodong Wang,
Hari Patel, Jing Liu, Dmitri Kireev & Stephen V. Frye
Center for Integrative Chemical Biology & Drug Discovery
Eshelman School of Pharmacy
University of North Carolina at Chapel Hill
September 24, 2009

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 Center for Integrative Chemical Biology & Drug Discovery

• National Cancer Institute (NCI) Chemical Biology Consortium -

North Carolina Comprehensive Chemical Biology Screening Center
– School of Pharmacy - CICBDD QuickTime™ and a
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– Lineberger Comprehensive Cancer Center QuickTime™ and a

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– Center for Nanotechnology in Drug Delivery QuickTime™ and a

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– BRITE – NC Central University

– The Hamner Institute for Health Sciences

• Mission
– Dynamic interface between biology,
assay development & medicinal chemistry
– ‘Bridge the gap between basic scientific investigation & clinical research
supported by NCI to reinforce therapeutics discovery.’
• James Doroshow, MD (National Cancer Institute - CBC)

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Visit us at www.pharmacy.unc.edu/cicbdd to learn more!
 Acute Lymphoblastic Leukemia - Background

• Acute Lymphoblastic Leukemia (ALL) is the most

common malignancy in children, representing nearly one
third of pediatric cancers (Graham, 2006. Clinical Cancer
Research.)

• 10-15% of patients are resistant to current chemotherapy

• Exact genetic changes underlying causation and

resistance in ALL remain unclear

• Several single nucleotide polymorphisms (SNPs) that

distinguish ALL patients and their response to treatments
such as methotrexate were recently reported in Nature
Genetics (Trevino, 2009.) 3
 Role of Mer Tyrosine Kinase in ALL

• Mer kinase is normally expressed in monocytes

– Checks immune response to apoptotic ‘clean-up’ operations
– May be important target in context of tumor vaccine sensitization

• Mer kinase is ectopically expressed in 33% of

childhood acute lymphoblastic leukemias
– Correlates with poor prognosis in clinical settings
– Ectopic expression associated with development of various cancers
– Mer-expressing lymphocytes show statistically significant survival
advantages over wild-type cells when treated with dexamethasone

• Transgenic mice over-expressing Mer develop

histopathology of T-cell lymphoblastic leukemia
– Suggests a cooperative role for Mer in leukemogenesis
– Keating, 2006. Oncogene.
4
 TAM Kinases – Involvement in Cancer

Cancer AXL* MER Tyro3

Myeloid (AML,CML) + +
Lymphoid Leukemia's (ALL) Ect
Gastric Cancer + +
Colon Cancer +
Prostrate Cancer + +
Lung Cancer +
Breast Cancer + +
Liver Cancer +

+ Overexpression Advances in Cancer Research 2008, 100, 35-83.

Ect Ectopic expression

*Axl also shown to be involved in metastasis. (Li, 2009. Oncogene.)

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 Mer Structure & Small Molecule Binding

• Protein structure with small molecule bound recently

published by the Structural Genomics Consortium
– Huang, 2009. Journal of Structural Biology.

„Graham, 2006. Clinical Cancer Research. „Huang, 2009. Journal of Structural Biology. 6
 Project Goals

• To develop orally active • Key is selectivity of

inhibitors of Mer kinase compound for TAM over
signaling with efficacy in other tyrosine kinases
treatment of ALL &
adequate therapeutic index

7
Lemke & Rothlin, 2008. Nature Reviews: Immunology. Ashley, et al. 2002. Cell Signaling Technology, Inc.
 Project Strategy

Caliper Assay Development

-Reaction Component Optimization
-Establishing +/- Controls
-Troubleshooting

In-House Chemistry
Assay
Validation -Small Molecule Synthesis
-Computational Modeling
-Analytical Chemistry
Small Molecule Screening
-UNC-CICBDD Synthesized
-Kinase-Targeted Library
-Library of Pharmaceutically Actives

Hit Profiling
Cellular / In Vivo
&
Assays 8
Confirmation
 Our Caliper LabChip Set-Up

• 12-sipper Microfluidic Chip

• 384-well plate format (Greiner Polypropylene V-bottom)
• ~20 minutes to run single-point full plate
• FL-27 Peptide Substrate for Mer & Tyro3
• FL-30 Peptide Substrate for Axl
EZReader II
System

9
 Optimization of Reaction Components - Kinase Concentration

• Enzyme titration performed in standardized buffer

– HEPES pH 7.4, Triton-X 100, MgCl2, DTT, BSA
– Initial runs performed at 100 µM ATP
– 1 µM fluorescent peptide substrate
– Commercial kinase enzymes

• Kinetic read on EZReader

– 20 cycles ≈ 1 hour

• Results
– Mer Kinase
• > 90% total conversion
• 1 nM = 30%
– Tyro3 Kinase
• > 90% total
• 625 pM = 30%
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Optimization of Reaction Components - Kinase Concentration

• Axl
– First lot showed
consistently
decreased activity
compared to Mer &
Tyro3 kinases

1 hour < 10% conversion

O/N ≈ 56% conversion
[10X] ≈ 41% conversion
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Optimization of Reaction Components - Kinase Concentration

• Axl
– First lot showed
consistently
decreased activity
compared to Mer &
Tyro3 kinases
– Second lot showed
greater activity similar
to other kinases

2 hour > 75% conversion

O/N > 95% conversion
Result
12
2.5 nM = 30% conversion
 Optimization of Reaction Components - ATP Concentration

• Mer • Tyro • Axl

1 nM = 30% conversion 625 pM = 30% conversion 2.5 nM = 30% conversion
ATP Km = 10 µM ATP Km = 20 µM ATP Km = 75 µM

Mer ATP Km Tyro ATP Km New Axl ATP Km

3 0.8 0.6
0.7
0.5
0.6
2 0.5 0.4
Velocity

Velocity
Velocity

0.4 0.3
1 0.3
0.2
0.2
0.1 0.1
0 0.0 0.0
250 500 750 1000 1250 -0.1 250 500 750 1000 1250 250 500 750 1000 1250
-0.1
[ATP] µM [ATP] µM [ATP] µM
-1
Mer ATP Km Tyro3 ATP Km New Axl ATP Km
100 50 30
40
75
% Conversion

20

% Conversion
% Conversion

30
50
20 10
25 10

0 0
0 10 20 30 40 50 60 70
10 20 30 40 50 60 70
10 20 30 40 50 60 70
-10 Time (minutes) 13
-25 Time (minutes) Time (minutes) -10
Optimization of Reaction Components – Caliper Protocol

• Standard protocol
dictated by Caliper
fluorescent peptide
substrate utilized

• Slight increase in
post-buffer sip time
resulted in better
resolution of
individual peaks

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 Optimization of Reaction Components - Troubleshooting

• Enzyme Stability on Ice • Enzyme Stability Over Time

• On ice with multiple freeze/ • Extended time-course to

thaw cycles to reproduce ensure activity for duration
assay preparation of experiment

Mer Kinase (G. Johnson) Axl Extended Time Course

80

60

% Conversion
Fresh 1X Thaw 4X Thaw R2 = .986
40

20

0
0 1 2 3 4 5
Hours 15
 Optimization of Reaction Components - Troubleshooting

• Protease Contamination

After 1h Substrate is Normal Overnight

After 3h
2hincubation
decreased
‘Shouldering’
&intensity
new
effect
marker
of dye
yielded
observed
markerseverely
& substrate
in marker
decreased
peaks
peak
marker & substrate peaks 16
 Optimization of Reaction Components - Troubleshooting

• Phosphatase Contamination
– Decreased signal observed within first 3 hours of reaction

Axl Titrat
1 Hour
16

14

12

10

6 2 Hours
4

-2
0 500 1000 1500 2000 2500 3000 3500 4000 3 Hours
[Axl] u

1 Hour 2 Hours 3 Hou

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 Optimization of Reaction Components - Troubleshooting

• Protease & Phosphatase Contamination

Axl Titration + Inhibitor Cocktail

1hr

2hr

5hr

Resolution: Include protease & phosphatase

inhibitor cocktail in assay buffers! 18
 Optimization of Reaction Components – Establishing Controls

• DMSO Tolerance DMSO Tolerance

100
Mer

% Control Conversion
80 Tyro3
– Titration performed from Axl
10% → 0.01% 60

– Result – no effect observed at 40

our assay target of 1% DMSO 20

for any of TAM receptors
0

00
01
02
04
08
16
32
64
25
50

10 0
0
0
.0
• Staurosporine Inhibition

0.
0.
0.
0.
0.
0.
0.
0.
1.
2.
5.
% DMSO

– Titration performed using 2-fold dilutions from 10 µM → 10 pM

Tyro3
Mer 40 Axl
% Conversion

% Conversion
20 15
% Conversion

IC50= 3nM 30
IC50=13nM IC50=109 nM
15
10
20
10
10 5
5

-6 -4 -2 0 2 4
-6 -4 -2 2 4 -6 -4 -2 0 2 4
-5
Log[Staurosporine] Log[Staurosporine] Log[Staurosporine] 19
 Optimization of Reaction Components – Repeats for New Lots

• Mer • Tyro3 • Axl

1 nM = 30% conversion 8 nM = 30% conversion 250 nM = 30% conversion
ATP Km = 6 µM ATP Km = 42 µM ATP Km = 75 µM
Staurosporine IC50 = 3 nM Staurosporine IC50 = 13 nM Staurosporine IC50 = 109 nM

Mer Tyro3
Axl Titration
40
100 60

80
% Conversion 30
%Conversion

40

% Conversion
60 20
40
10 20
20
0
0 0
20 40 60 80 20 40 60 80
20 40 60 80
-20 Time (minutes) -10 Time (minutes)
Time (minutes)
-20

Mer ATP Km Tyro3 ATP Km Axl ATP Km

0.3 0.6
0.3

0.2
% C onversion

0.2 0.4
Velocity

Velocity
R2=.958 R2=.998 R2=.976
0.1 0.1
0.2

0.0
0.0
500 1000 1500 0.0 20
0 500 1000 1500 0 500 1000 1500
ATPµM [ATP] µM [ATP] µM
-0.1
 Optimization of Reaction Components - Troubleshooting

• Enzyme Stability
Multidrop Combi

• Protease Contamination

• Multi-Drop Technical Issues

– Distinct pattern reflects path of
Multi-Drop dispensing

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 Optimization of Reaction Components - Troubleshooting

1% BSA

• Enzyme Stability

• Protease Contamination

1% BSA + Metal Tips

• Multi-Drop Technical Issues
– Distinct pattern reflects path of
Multi-Drop dispensing
– Prime lines with 1% BSA
– Change from plastic to metal-
tipped dispensing head
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 Standard Assay Validation

• Three days with two full plates of Max Signal, Min Signal, and
Inhibitor Dose Response Curves (or Mid-Range Signal).

– "Max" signal: Measures maximum signal. For assays this is untreated

constitutively active condition in the presence of 1% DMSO.

– "Min" signal: Measures background signal. For assays this is signal when
controlled inhibitor is added to completely inactivated enzyme.

– Inhibitor Dose Response Curves: Serial dilution of known inhibitor to

yield an inhibition curve.

– "Mid" signal: Estimates signal variability at some point between the

maximum and minimum signals. Typically, mid-point is reached by adding
an IC50 concentration of a standard inhibitor to each well.

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 Assay Validation Results – Mer Kinase

• Maximum • Minimum • Staurosporine IC50

– CV ≤ 3.6% – CV ≤ 1.2% – R2 = 0.826
– Z’ = 0.952 – Z’ = 0.981 – Z’ = 0.939

Tyro3

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 Targeted Library Screening

• Kinase-Targeted Library Rationale

– Broad families with related function & high sequence homology
– Inhibition site recognizable throughout this family-based design
– 3D structures with co-crystal inhibitors enable structure-based
computational approaches
– Published inhibitor series enable ligand-based approaches
• Use of Commercially Designed Kinase Libraries
– Apply filters for generic ‘drug-likeness’ & kinase-specific motifs
– Select a diverse and representative set

• Virtual Screening
– In silico screening of a large pool of commercially available 25
compounds performed by Dmitri Kireev
 In-House Compound Screening

•In-house Chemistry Compounds

• Initially based on known

Axl inhibitor MP470

MP470 Z’ = .951

• Additional compounds
generated using structure-
based & ligand-based
design Z’ =26.938
Initial Hits from Targeted Screen

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 Conclusions

• Importance of validation & necessity for thoughtful

optimization of assay protocols as well as equipment

• Utility of Caliper technology for protein kinases

– Ease of technique & simplicity of assay buffers
– Sensitive & consistent results yielding low CVs
– Ability to screen thousands of compounds a day
– Direct measurement of peptide substrate phosphorylation
instead of downstream effects
– Lack of molecular tags or fusion proteins that can generate
experimental artifacts
– Availability of Caliper ProfilerPro® products simplifies further
characterization of small molecule target specificity
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 Acknowledgements

Assay Development & Compound Profiling CICBDD Administration QuickTime™ and a

William Janzen, Director
Catherine Simpson, Senior Scientist
Tim Wigle, Postdoctoral Research Associate
Jacqueline Norris, Senior Scientist
Emily Hull-Ryde, Research Operations Manager
Adam Cheely, Research Technician
Stephen V. Frye, Director decompressor
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Barbara Dearry, Business Services
Computational Drug Discovery
Dmitri Kireev, Director
QuickTime™ and a
decompressor
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Medicinal Chemistry Collaborators

Xiaodong Wang, Assistant Director Gary Johnson, Chair UNC Pharmacology Department
Jian Jin, Assistant Director H. Shelton Earp, Director - Lineberger Cancer Center
Hari Patel, Senior Scientist Wistar Protein Purification Institute, Philadelphia, PA
Jing Liu, Senior Scientist
Sab Randhawa, Senior Scientist
George Adjabeng, Senior Scientist
Xin Chen, Scientist
Feng Liu, Postdoctoral Research Associate
Martin Herold, Postdoctoral Research Associate
Chris MacNevin, Postdoctoral Research Associate

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 References

• Graham, D.K., et al., Ectopic expression of the proto-oncogene Mer in

pediatric T-cell acute lymphoblastic leukemia. Clin Cancer Res 2006, 12,
2662-2669.

• Huang, X., et al., Structural insights into the inhibited states of the Mer
receptor tyrosine kinase. J Struct Biol, 2009. 165(2): p. 88-96.

• Keating, A.K., et al., Lymphoblastic leukemia/lymphoma in mice

overexpressing the Mer (MerTK) receptor tyrosine kinase. Oncogene 2006,
25, 6092-6100.

• Lemke, G; Rothlin C. V., Immunobiology of the TAM receptors. Nature

Reviews: Immunology 2008, 8, 327.

• Li, Y., et al., Axl as a potential therapeutic target in cancer: role of Axl in
tumor growth, metastatsis & angiogenesis. Oncogene 2009, E-Pub July 27
ahead of print.

• Trevino, L.R., et al., Germline genomic variants associated with childhood

acute lymphoblastic leukemia. Nature Genet 2009, E-Pub Aug 16 ahead of
print. 30