UNDERSTANDING ACTIVE ABL KINASE CONFORMATIONS

179 Pages·2015·3.78 MB·English

UNDERSTANDING ACTIVE ABL KINASE CONFORMATIONS:
APPLICATION TO DISCOVERY OF SMALL MOLECULE
ALLOSTERIC MODULATORS

by
Prerna Grover
Bachelor of Engineering in Biotechnology, Panjab University, 2009

Submitted to the Graduate Faculty of
Medicine in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy

University of Pittsburgh
2015
UNIVERSITY OF PITTSBURGH
SCHOOL OF MEDICINE

This dissertation was presented

by

Prerna Grover

It was defended on
April 13, 2015
and approved by
Jeffrey L. Brodsky, Ph.D., Dissertation Committee Chair, Biological Sciences
J. Richard Chaillet, M.D., Ph.D., Microbiology and Molecular Genetics
Qiming Jane Wang, Ph.D., Pharmacology and Chemical Biology
Ora A. Weisz, Ph.D., Cell Biology
Thomas E. Smithgall, Ph.D., Dissertation Advisor, Microbiology and Molecular Genetics

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Copyright © by Prerna Grover
2015
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UNDERSTANDING ACTIVE ABL KINASE CONFORMATIONS:
APPLICATION TO DISCOVERY OF SMALL MOLECULE
ALLOSTERIC MODULATORS
Prerna Grover, Ph.D.
University of Pittsburgh, 2015

The c-Abl protein-tyrosine kinase regulates diverse cellular signaling pathways involved in cell
growth, adhesion, and responses to genotoxic stress. Abl is well known in the context of Bcr-
Abl, the active fusion tyrosine kinase, which causes chronic myelogenous leukemia (CML) and
other leukemias. The tyrosine kinase activity of Abl is tightly regulated by auto-inhibitory
interactions involving its non-catalytic SH3 and SH2 domains. Mutations that perturb these
intramolecular interactions result in kinase activation. This study examined the effect of
activating mutations on the biochemistry and solution structure of Abl core proteins. In an active
myristic acid-binding pocket mutant (A356N), the relative positions of the regulatory N-cap,
SH3 and SH2 domains were virtually identical to those of the assembled wild-type core despite
differences in catalytic activity and thermal stability. In contrast, a dramatic structural
rearrangement in an active gatekeeper mutant (T315I) was observed with the positions of the
SH2 and SH3 domains reversed relative to wild-type. These results show that Abl kinases can
adopt multiple conformations in solution and kinase activation does not necessarily require
destabilization of the assembled core structure.
Small molecules that allosterically regulate Abl kinase activity through its non-catalytic
domains may represent selective probes of Abl function. I developed a screening assay to
identify chemical modulators of Abl kinase activity that either disrupt or stabilize the regulatory
interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP)
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assay is based on a recombinant Abl protein containing the regulatory domains (Ncap-SH3-SH2-
linker, N32L) and a short fluorescein-labeled probe peptide that binds the SH3 domain. The
probe peptide binds the recombinant Abl N32L protein in vitro producing a robust FP signal.
Mutation of the SH3 binding site (W118A) or introduction of a high-affinity linker both resulted
in loss of the FP signal. Pilot screens were performed with two chemical libraries (2800
compounds total), and thirteen compounds were found to specifically inhibit the FP signal.
Secondary assays showed that one of these hit compounds enhances Abl kinase activity in vitro.
These results show that screening assays based on the regulatory domains of Abl can identify
allosteric modulators of kinase function.

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS .................................................................................................... XIV
1.0 INTRODUCTION ........................................................................................................ 1
1.1 C-ABL KINASE OVERVIEW ........................................................................... 1
1.2 STRUCTURE AND REGULATION OF C-ABL ............................................. 3
1.2.1 Regulation by intramolecular interactions.................................................... 4
1.2.1.1 N-terminal cap (N-cap) ......................................................................... 4
1.2.1.2 SH3 domain ........................................................................................... 6
1.2.1.3 SH2 domain ........................................................................................... 8
1.2.1.4 SH2-kinase linker .................................................................................. 9
1.2.1.5 Kinase domain ..................................................................................... 12
1.2.1.6 C-terminal region ................................................................................ 16
1.2.2 Conformational dynamics of Abl proteins .................................................. 16
1.2.2.1 Small Angle X-ray Scattering (SAXS) .............................................. 17
1.2.2.2 Hydrogen Exchange Mass Spectrometry (HXMS) .......................... 19
1.2.2.3 Nuclear Magnetic Resonance Spectroscopy (NMR) ........................ 22
1.2.3 Physiological regulation of Abl kinase function ......................................... 24
1.2.3.1 Phosphorylation .................................................................................. 25
1.2.3.2 Interaction with binding partners ..................................................... 26
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1.2.3.3 Growth factor induced activation ...................................................... 28
1.3 C-ABL IN DNA DAMAGE ............................................................................... 29
1.3.1 Role of Abl in the DNA damage repair pathway ........................................ 30
1.3.2 Abl signals inducing apoptosis ..................................................................... 32
1.4 BCR-ABL AND CHRONIC MYELOGENOUS LEUKEMIA ..................... 33
1.4.1 Disease overview ............................................................................................ 33
1.4.2 Bcr-Abl: origin and mechanism of activation ............................................. 34
1.4.3 Imatinib: targeted Bcr-Abl kinase inhibitor ............................................... 37
1.4.3.1 Imatinib: mechanism of action .......................................................... 38
1.4.3.2 Mechanisms of resistance to imatinib ............................................... 39
1.4.4 Second and third generation ATP-competitive inhibitors of Bcr-Abl ...... 44
1.4.5 Allosteric inhibitors of Bcr-Abl .................................................................... 46
1.5 ROLE OF C-ABL IN SOLID TUMORS ......................................................... 48
1.5.1 Abl as a promoter of tumor growth ............................................................. 49
1.5.2 Abl as a suppressor of tumor growth........................................................... 49
1.6 HYPOTHESIS AND SPECIFIC AIMS........................................................... 50
2.0 THE C-ABL KINASE ADOPTS MULTIPLE ACTIVE CONFORMATIONAL
STATES IN SOLUTION* .......................................................................................................... 52
2.1 SUMMARY ........................................................................................................ 52
2.2 INTRODUCTION ............................................................................................. 53
2.3 MATERIALS AND METHODS ...................................................................... 58
2.3.1 Recombinant protein expression and purification ..................................... 58
2.3.2 Protein kinase activity measurements ......................................................... 58
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2.3.3 Transient expression of Abl proteins in HEK 293T cells ........................... 59
2.3.4 Kinetic protein kinase assay ......................................................................... 59
2.3.5 Differential Scanning Fluorimetry (DSF) .................................................... 60
2.3.6 X-ray solution scattering data collection ..................................................... 61
2.3.7 Reconstruction of molecular envelopes ....................................................... 62
2.4 RESULTS ........................................................................................................... 63
2.4.1 Biochemical characterization of the kinase activity of Abl kinase proteins
63
2.4.2 Thermal stability of Abl proteins ................................................................. 67
2.4.3 X-ray scattering analysis ............................................................................... 72
2.4.4 Shape reconstructions from X-ray solution scattering data ...................... 73
2.4.5 Enhanced SH3-linker interaction reverses the structural changes induced
by the T315I mutation ............................................................................................... 77
2.4.6 Effect of small molecules on thermal stability of Abl kinase proteins ...... 78
2.5 DISCUSSION ..................................................................................................... 81
3.0 FLUORESCENCE POLARIZATION SCREENING ASSAYS FOR SMALL
MOLECULE ALLOSTERIC MODULATORS OF C-ABL KINASE FUNCTION* ......... 84
3.1 SUMMARY ........................................................................................................ 84
3.2 INTRODUCTION ............................................................................................. 85
3.3 MATERIALS AND METHODS ...................................................................... 90
3.3.1 Expression and purification of recombinant Abl proteins ........................ 90
3.3.2 Peptide synthesis ............................................................................................ 91
3.3.3 Fluorescence polarization assay ................................................................... 92
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3.3.4 Chemical library screening ........................................................................... 92
3.3.5 Differential Scanning Fluorimetry (DSF) .................................................... 93
3.3.6 Surface Plasmon Resonance (SPR) .............................................................. 94
3.3.7 Protein kinase assays ..................................................................................... 94
3.3.8 Molecular dynamics ...................................................................................... 96
3.3.9 Computational docking ................................................................................. 97
3.4 RESULTS AND DISCUSSION ........................................................................ 98
3.4.1 Abl fluorescence polarization (FP) assay design ......................................... 98
3.4.2 Recombinant Abl regulatory proteins for FP assay development ............ 99
3.4.3 Structural basis for high affinity probe peptide binding to the Abl SH3
domain ....................................................................................................................... 101
3.4.4 Selection of a probe peptide for the Abl N32L FP assay .......................... 103
3.4.5 Abl N32L FP assay development and optimization .................................. 105
3.4.6 Identification of inhibitors of p41 interaction with Abl N32L................. 107
3.4.7 Compounds identified in the Abl N32L FP screen interact directly with
the Abl N32L protein in orthogonal assays............................................................ 112
3.4.8 Allosteric activation of Abl kinase by compound 142 .............................. 117
3.4.9 Molecular dynamics simulations and docking studies predict binding of
compound 142 to the SH3:linker interface in the Abl kinase core ...................... 124
3.5 SUMMARY AND CONCLUSIONS .............................................................. 127
4.0 OVERALL DISCUSSION ...................................................................................... 130
4.1 EFFECT OF ACTIVATING AND STABILIZING MUTATIONS ON ABL
KINASE ACTIVITY, STABILITY, AND CONFORMATION .................................. 131
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4.2 IDENTIFICATION OF ALLOSTERIC MODULATORS OF ABL KINASE
FUNCTION ....................................................................................................................... 135
4.3 CLOSING REMARKS ................................................................................... 141
APPENDIX A ............................................................................................................................ 142
BIBLIOGRAPHY ..................................................................................................................... 144
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