PROTEIN TYROSINE KINASES

CANCER DRUG DISCOVERY AND DEVELOPMENT

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PROTF~ T~osI~ Ke,~ES FROM INHIBITORS TO USEFUL DRUGS

Edited by

DORIANO FABBRO, PhD

Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

and

FRANK NIC~ORMICK~ MD, PhD

UCSF Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA

_• HUMANA PRESS TOTOWA, NEW JERSEY

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2005006248

PREFACE

Protein kinases function as components of signal transduction pathways, play- ing a central role in diverse biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases with abnormalities in these signaling pathways is considered a promising approach for drug development. The function of many protein kinases is deregulated in human cancers. Deregu- lation, whether as a result of deletion, mutation, or amplification, is manifested as aberrant activation, prime examples of which are kinases including Bcr-Abl, EGFR family members, Flt-3, Met, etc., as well as kinases involved in the neovascularization of tumors like KDR. A decade ago, these protein kinases were considered prime targets for the development of selective inhibitors. Cur- rently, over 20 different kinases--the majority being receptor protein tyrosine kinases (RPTKs)--are being considered as potential therapeutic targets in oncol- ogy.

Although the success of agents such as Glivec ® (Imatinib mesylate, Glivec/ Gleevec ®) and Iressa TM (Gefitinib) has provided a proof of concept that such agents can be therapeutically effective and retain an acceptable safety profile, the clinical experience with other tyrosine kinase inhibitors is still limited.

A comprehensive overview of the drug discovery processes aimed at gener- ating inhibitors for the treatment of malignancies believed to be dependent on the gain of function of protein tyrosine kinases (PTKs) has to contain a sum- mary of those drug discovery programs that have devoted their efforts to gener- ating low molecular-weight (LMW) inhibitors directed against either the adenosine triphosphatase (ATP)-binding site (summarized in Chapter 1) or the Src homology 2 (SH2) domain, an important noncatalytic module that recog- nizes a short phosphotyrosine-containing sequence in other proteins. A review on the advances made targeting this critical SH2-binding event, which would result in the inactivation of undesirable signal transduction networks, is found in Chapter 2.

Epistatically, PTKs are located either upstream and/or downstream of tumor suppressor genes or oncogenes and have been demonstrated to play central roles in apoptosis, proliferation, invasion, and differentiation. The signal transduction pathways of PTKs, in particular receptor PTKs, is intimately linked to the phosphoinositide 3-kinase (PI3-K) pathway as activation of cells by a wide variety of stimuli leads to rapid changes in 3-phosphorylated inositol lipids through the action of a family of enzymes known as PI3-Ks. The dissection

vi Preface

of PI3-K signaling pathways has been greatly aided by genetic approaches and by the availability of two pharmacological tools, wortmannin and LY294002. In Chapter 3, a comprehensive summary is given to explain why the PI3-Ks represent a reasonable target for pharmaceutical intervention. All the reasoning for the activation of PI3-K as target is central to the coordinated control of multiple cell-signaling pathways leading to cell growth, cell proliferation, cell survival, and cell migration.

Aberrant activation of tyrosine kinases, owing to mutation or overexpression, is sufficient for them to become transforming in cellular and animal models. The majority of targets are RPTKs. Deregulating mutations of over half of the known RPTKs have been associated with different human malignancies. To illustrate the rationale and the progress made towards generating "selective" LMW kinase inhibitors, a few selected examples have been chosen that include the targets of Glivec (platelet-derived growth factor receptor, Kit, Bcr-Abl), FLT-3, JAKs, as well as Src. A special chapter has also been devoted to the normal function, role in disease, and application of platelet-derived growth factor antagonists. All of these efforts illustrate the tremendous biological complexity that is encoun- tered by targeting these kinases and render a conclusion about the actual level of understanding of the molecular epidemiology and pathophysiology, as well as disease relevance of these kinases. In particular, the success story of Glivec has taught the academic, as well as pharmaceutical fields, some lessons regarding the inhibition of these kinases from the points of view of therapeutics and biology (Chapters 4-8).

A successful development of protein kinase inhibitors is based primarily on solid epidemiology allowing the identification and validation of the target along with the knowledge of the structure of the kinase. The structural under- standing of protein kinases has significantly progressed as structures of kinases both in phosphorylated or nonphosphorylated forms, active or inactive states, unliganded or complexed to substrate analogs or inhibitors, and with only the catalytic domain present or in a multidomain construct including SH3 and SH2 domains have become available. All of this knowledge is being used for struc- ture-based design and has been summarized in Chapter 9.

Robust predictive preclinical in vitro and, in particular, in vivo screening model systems that allow rapid optimization of lead compounds are key to a successful drug discovery effort as they are crucial for determining the safety of kinase inhibitors. Animal models as used in cancer can be divided conve- niently into models designed to understand the natural history of cancer and models that are useful for the testing, selection, and profiling of new anticancer treatment modalities. The advantages and disadvantages of in vivo preclinical models for testing protein kinase inhibitors with antitumor activity have been summarized in Chaoter 10.

vii Preface

Finally, one of the most important steps in the drug discovery process leading to kinase inhibitors is to determine "on-target" vs "off-target" effects by demonstrat- ing that the protein kinase inhibitor downregulates the function of the target in vitro and in vivo with all the expected consequences (downregulation of given pathway[s] and growth arrest). Therefore, phosphoprofiling or phosphoproteomics that include the large-scale determination of protein phosphorylation in cells and tissue is one approach that can be used to characterize biological states, including therapeutic responses to provide a comprehensive picture of cellular states. The utility of these methods in drug discovery and development is discussed in Chapter 11.

Understanding the role of a potential target in cancer development and pro- gression is as relevant as the efficient optimization of an inhibitory compound's potency, toxicity, and pharmacokinetic profile. To be a valid target, a kinase should play a fundamental role in the pathogenesis of a disease and the rationally designed LMW compounds, which are almost exclusively directed against the ATP-binding site of the kinase, should be able to revert the effects of the disease- causing kinase in preclinical models, and should be translatable to clinical settings.

Doriano Fabbro, VhD Frank McCormick, MD, PhD

CONTENTS

Preface ............................................................................................................... v Contributors ...................................................................................................... xi

1. Protein Tyrosine Kinases as Targets for Cancer and Other Indications ........................................................................... 1

Mark Pearson, Carlos Garcia-Echeverria, and Doriano Fabbro

2. Inhibitors of Signaling Interfaces: Targeting Src Homology 2 Domains in Drug Discovery .............................................................. 31

Carlos Garefa-Echeverria

3. PI3-Kinase Inhibition: A Target for Therapeutic Intervention ............ 53 Peter M. Finan and Stephen G. Ward

4. Src as a Target for Pharmaceutical Intervention: Potential and Limitations .................................................................. 71

Mira Su~a, Martin Missbach, Rainer Gamse, Michaela Kneissel, Thomas Buhl, Jiirg A. Gasser, Markus Glatt, Terence O'Reilly, Anna Teti, and Jonathan Green

5. Activated FLT3 Receptor Tyrosine Kinase as a Therapeutic Target in Leukemia ....................................................................................... 93

Blanca Scheijen and James D. Griffin

6. JAK Kinases in Leukemias, Lymphomas, and Multiple Myeloma ... 115 Renate Burger and Martin Gramatzki

7. Glivec® (Gleevec®, Imatinib, STI571): A Targeted Therapy for Chronic Myelogenous Leukemia .............. 145

Elisabeth Buchdunger and Renaud Capedeville

8. Platelet-Derived Growth Factor: Normal Function, Role in Disease, and Application of PDGF Antagonists ............................................ 161

Tobias Sj6blom, Kristian Pietras, Arne Ostman, and Carl-Henrik Heldin

9. Structural Biology of Protein Tyrosine Kinases ................................. 187 Sandra W. Cowan-Jacob, Paul Ramage, Wilhelm Stark,

Gabriele Fendrich, and Wolfgang Jahnke

10. Testing of Signal Transduction Inhibitors in Animal Models of Cancer ........................................................... 231

Terence O'Reilly and Robert Cozens

ix

x Contents

11. Phosphoproteomics in Drug Discovery and Development ................ 265 Michel F. Moran, Jarrod A. Marto, Cynthia J. Brame,

Olga Ornatsky, Mark M. Ross, Leticia M. Toledo-Sherman, Alfredo C. Castro, Brett Larsen, Henry Duewel, Christopher Hosfield, Christopher Orsi, Thodoros Topaloglou, Daniel Figeys, Jennifer A. Caldwell-Busby, and David R. Stover

Index .............................................................................................................. 279

CONTRIBUTORS

CYNTHIA J. BRAME, PhD • MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

ELISABETH BUCHDUNGER, PhD ° Senior Research Investigator I, Oncology Research, Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel, Switzerland

THOMAS BUHL, PhD ° Senior Research Investigator I, Musculoskeletal Diseases (MSD), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

RENATE BURGER, PhD ° Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School Boston, MA

JENNIFER A. CALDWELL-BUSBY, PhD ° MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

RENAUD CAPDEVILLE, MD ° Group Leader, Clinical Research, Novartis Oncology, Novartis Pharma A G, Basel Switzerland

ALFREDO C. CASTRO, PhD ° Syntonix Pharmaceuticals, Waltham, MA SANDRA W. COWAN-JACOB, PhD ° Group Leader, Discovery Technologies,

Protein Structure Unit (DT/PSU), Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel Switzerland

ROBERT COZENS, PhD ° Unit Head, Drug Discovery Pharmacology (DDP), Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel Switzerland

HENRY DUEWEL, PhD ° MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

DOmANO FABBRO, PhD ° Unit Head, In Vitro Profiling (IVP), and FIP Head of Signaling Pathways/Oncology Research, Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel, Switzerland

GABRIELE FENDRICH, PhD ° Research Investigator I, Discovery Technologies, Protein Structure Unit (DT/PSU), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

DANIEL FIGEYS, PhD ° MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

PETER M. FINAN, PhD • Novartis Horsham Research Centre, Horsham, UK RAINER GAMSE, MD • DA Project Management Director, MSD Management,

Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

xi

xii Contributors

CARLOS GARdA-ECHEVERR~A, PhD ° Global Discovery Chemistry-Oncology, Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

Jt~RG A. GASSER, PhD • Senior Research Investigator I, Musculoskeletal Diseases (MSD), Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel, Switzerland

MARKUS GLATT, PhD ° Senior Research Investigator I, Musculoskeletal Diseases (MSD), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

MARTIN GRAMATZKI, MD ° Division of Stem Cell and Immunotherapy II, and Department of Medicine, University of Kiel, Kiel, Germany

JONATHAN GREEN, PhD • Senior Research Investigator II/NDS, Musculoskeletal Diseases (MSD), Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel, Switzerland

JAMES D. GRIFFIN, MD ° Department of Medical Oncology, Dana-Farber Cancer Institute; Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

CARL-HENRIK HELDIN, PhD "Ludwig Institute for Cancer Research, Uppsala, Sweden CHRISTOPHER HOSFmLD, PhD • MDS Proteomics Inc., Toronto, Canada

and Charlottesville, VA WOLFGANG JAHNKE, PhD o Senior Research Investigator I/NLS, Discovery

Technologies, Protein Structure Unit (DT/PSU), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel Switzerland

MICHAELA KNEISSEL, PhD ° Research Investigator II, Musculoskeletal Diseases (MSD), Novartis Institutes for BioMedical Research Basel Novartis Pharma AG, Basel, Switzerland

BRETT LARSEN, MSc ° MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

JARROD A. MARTO, PhD • MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

FRANK MCCORMICK, MD, PhD ° UCSF Comprehensive Cancer Center, University of California at San Francisco, San Francisco, CA

MARTIN MISSBACH, PhD ° Unit Head, Disease Area Bone, Muscle, and Gastrointestinal (BMG), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

MICHEL F. MORAN, PhD ° McLaughlin Centre for Molecular Medicine, Hospital for Sick Children, University of Toronto, Toronto, Canada

OLGA ORNATSKY, PhO " M D S Proteomics Inc., Toronto, Canada and Charlottesville. VA

Contributors xiii

TERENCE O'REILLY, PhD "Senior Research Investigator I/NLS, Oncology, Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

CHRISTOPHER ORSl, MZng " MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

ARNE OSTMAN, PhD • Cancer Center Karolinska, Karolinska Institute, Stockholm, Sweden

MARK PEARSON, PhD • Research Investigator II, Oncology In Vitro Profiling (IVP), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

KR~ST~AN PmTRAS, PhD ° Ludwig Institute for Cancer Research, Stockholm, Sweden PAUL RAMAGE, PhD • Group Leader, Discovery Technologies, Protein

Structure Unit (DT/PSU), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

MARK M. Ross, PhD • MDS Proteomics Inc., Toronto, Canada and Charlottesville, VA

BLANCA SCHEIJEN, PhD • Department of Medical Oncology, Dana-Farber Cancer Institute; Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

TOBIAS SJOBLOM, PhD ° The Sydney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD

MIRA SV~A, PhD • Senior Research Investigator I/NLS, Musculoskeletal Diseases (MSD), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

WILHELM STARK, PhD • Research Investigator I, Discovery Technologies, Protein Structure Unit (DT/PSU), Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

DAVID R. STOVER, PhD • Oncology Antibody Lab L Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland

ANNA TETI, PhD • University L'Aquilla, L'Aquilla, Italy LETICIA M . TOLEDO-SHERMAN, PhD • MDS Proteomics Inc., Toronto, Canada

and Charlottesville, VA THODOROS TOPALOGLOU, PhD • MDS Proteomics Inc., Toronto, Canada

and Charlottesville, VA STEPHEN G . WARD, PhD • Department of Pharmacy and Pharmacology,

University of Bath, Bath, UK