FROM CELEBREX TO A NOVEL CLASS OF PHOSPHOINOSITIDE-DEPENDENT KINASE 1 (PDK-1) INHIBITORS FOR ANDROGEN-INDEPENDENT PROSTATE CANER DISSERTATION Presented in Partial Fulfillment of the requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jiuxiang Zhu, M.S. * * * * * The Ohio State University 2005 Dissertation Committee: Approved by Professor Ching-Shih Chen, Advisor Professor Robert Brueggemeier Advisor Professor Pui-Kai (Tom) Li Graduate Program in Pharmacy Professor Matthew D. Ringel ABSTRACT Celebrex, a nonsteroidal anti-inflammatory drug (NSAID, cyclooxygenase-2 inhibitor), was reported to induce apoptosis in the prostate cancer cell line PC-3 at 50µM. Early research from our laboratory demonstrated that this apoptotic inducing effect was independent of its COX-2 inhibitory activity. Further investigation showed that PDK-1 was a major protein targeted by celebrex in PC-3 cells to induce apoptosis. However, celebrex was very weak in inhibiting PDK-1 with IC50 of 48µM. To improve its potency, two series of analogs were designed and synthesized. In the first series of 24 compounds, the 5-position methylphenyl moiety of celebrex was replaced by various aromatic ring systems to explore the optimal hydrophobic group. OSU02067 (IC50=9µM) with phenanthrene at the 5-position was the best inhibitor among this series and was selected as the lead compound for further modification. Enzyme kinetics study of PDK-1 inhibition by celebrex indicated that it competed with ATP for binding. Docking of OSU02067 into the ATP binding domain of PDK-1 showed that the sulfonamide moiety hydrogen bonded to hinge region residue Ala162. Considering the importance of H-bonding, the sulfonamide moiety was substituted with various heteroatom-rich functional groups in the 2nd series of 12 compounds. OSU03012 and OSU03013 stood out as the most potent analogs with IC50s ii of 5µM and 2µM, respectively. Such improvement was partly attributed to an additional H-bond formed with hinge region residue Ser160. Exposure of PC-3 cells to these two agents (5µM or higher) led to significant decreases in Akt and p70s6k kinase activity (both are downstream substrates of PDK-1) and an increase in apoptosis evidenced by nucleosome formation and PARP cleavage. As OSU03012 was tolerated well by nude mice at a dose of 200mg/kg, it was chosen for further study. In primary chronic lymphocytic leukemia cells and the breast cancer cell line, MDAMB453, the compound induced apoptosis. In addition, Tseng et. al. showed that OSU03012 was equally potent in imatinib-resistant and -sensitive CML cells with IC50 of 6µM. OSU03012 was able to sensitize mutant resistant cells (Ba/F3p210E255K and Ba/F3p210T315I) to imatinib partly because of the concerted effect on phospho-Akt. Currently, OSU03012 is being studied in lung, thyroid, ovarian and bladder cancers and preclinical studies are underway (including toxicology, pharmacokinetics and pharmacodynamics). Future structure modification of OSU03012 is expected to be pursued in the following three aspects. Hydrophobic pockets behind the adenine binding domain and at the C-terminal lobe below the ribose binding pocket will be explored. Crystal structures of PDK-1 with this class of inhibitors are needed to provide insights into structure-based design. Adsorption, distribution, metabolism and execretion will be integrated into inhibitor design to achieve clinically applicable drug candidates. iii Dedicated to my grandparents, parents and brother iv ACKNOWLEDGMENTS • I would like to acknowledge Dr. Chen for his guidance, constant encouragement, support and for providing excellent working environment for his students • I would also like to express my sincere appreciation for the faculty of the division, especially Drs. Brueggemeier and Li, for all their help and advice • I want to thank Kathy Brooks and Kelli Ballouz for being able to solve any problem and make graduate studies going smoothly • Colleagues and friends: Drs. Kulp, Song, Johnson, Lin and Wang, Ho-Pi, Yvette, Ping-Hui, Ya-Ting, Joe, Chung-Wai, Yeng-Jeng, Kuen-Feng, Leo, Qiang, Jim, Chang-Shi, Nicole and Jack for help in all kinds of experimental details, invaluable discussion about a variety of scientific topics and proofreading thesis draft. • Xiaohui and Yan for having lots of funs together and making graduate studies memorable. v VITA 1993-1997 B.S. Pharmaceutical Science Beijing University, China 1997-2000 M.S. Natural Product Chemistry Peking Union Medical College, China 2000-Present Graduate Teaching and Research Associate College of Pharmacy, Ohio State University PUBLICATIONS 1. Jiuxiang Zhu, Jui-Wen Huang, Joseph Fowble, Ping-Hui Tseng, Chung-Wai Shiau, Yeng-Jeng Shaw, Samuel K. Kulp, and Ching-Shih Chen From the Cyclooxygenase-2 Inhibitor Celecoxib to a Novel Class of Potent Akt-Targeted Antitumor Agents. Cancer Research, 2004;64(12):4309-18 2. Jiuxiang Zhu, Xueqing Song, Ho-pi Lin, Donn C. Young, Shunqi Yan, Victor E. Marquez, Ching-shih Chen Using Cyclooxygenase-2 Inhibitors as Molecular Platforms to Develop a New Class of Apoptosi s-inducing Agents. Journal of the National Cancer Institute 2002, 94(23), 1745-17 3. Amy J. Johnson, Lisa L. Smith, Jiuxiang Zhu, Nyla A. Heerema, Sara Jefferson, Michael Grever, Ching-shih Chen, John C. Byrd. A Novel Celecoxib Derivative Induces Apoptosis in Primary CLL Cells and Transformed B-cell Lymphoma via a Caspase and Bcl-2 Independent Mechanism. Blood, 2004, Sep 28. 4. Haiming Ding, Chunhua Han, Jiuxiang Zhu, Ching-Shih Chen, Steven M.D’Ambrosio. Celecoxib Derivatives Induce Apoptosis via Disruption of Mitochondrial Membrane Potential and Activation of Caspase 9. Int. J Cancer., 2004 Oct 21. FIIELDS OF STUDY Major Field: Pharmacy vi TABLE OF CONTENTS Page Abstract……………………………………………………………………………........ii Dedication……………………………………………………………………………...iv Acknowledgments..…………………………………………………………………......v Vita……………………………………………………………………………………..vi List of Tables…………………………………………………………………………...ix List of Figures………………………………………………………………………......x Abbreviations…………………………………………………………………………xiv Chapter 1 Introduction………………………………………………………………….1 1.1 Hormone-Refractory Prostate Cancer (HRPC) and Therapies……………..1 1.2 PI3K/PDK-1/Akt Signaling Pathway ……………………………………...5 1.3 Protein Kinases (PKs) and Their Inhibitors………………………………...9 1.3.1 Kinases and Cancer…………………………………………………...9 1.3.2 Protein Kinase Inhibitors (PKIs)…………………………………….12 1.4 Cyclooxygenase-2 (COX-2) Inhibitors as Anti-cancer Agents…………....15 Chapter 2 Molecular Target(s) for Celebrex and Project Design……………………...27 2.1 Molecular Target of Celebrex…………………………………………….27 2.1.1 COX-2 Independent Mechanism…………………………………….27 2.1.2 PDK-1, a Major COX-2 Independent Target for Celebrex………….29 2.2 Aims and Project Design………………………………………………….30 Chapter 3 Design, Synthesis and Biological Activities of the1st Series of Celebrex Derivatives…………………………………………..……………………...39 3.1 Design of the 1st Series of Derivatives……………………………..……...39 3.2 Synthesis of the 1st Series of Derivatives…...……………………………..40 3.3 Structures of the Analogs and Their Biological Activities…….…..……...41 3.4 OSU02067………………………………………………………………....42 3.4.1 Effect on Downstream Protein Akt…………………………………...42 3.4.2 Cellular Effect of OSU02067…………………………………………42 3.4.3 In vivo Study of OSU02067………………………………………......43 vii Chapter 4 Structure-Based Design of PDK-1 Inhibitors………………………………49 4.1 Crystal Structure of PDK-1 Catalyic Domain in Complex with ATP…….49 4.2 Docking of OSU02067 to ATP Binding Domain and Design of the 2nd Series Analogs….……………………………………………..……………….50 4.3 Synthesis of the 2nd Series of Compounds……………………………..….52 4.4 Optimal Compounds-OSU03012 and OSU03013………………………...54 4.5 In vitro Effect of OSU03012 and OSU03013 in PC-3……………………55 4.5.1 Effects on Downstream Proteins Akt and p70S6K…………………..55 4.5.2 Cellular Effects of OSU03012 and OSU03013…………………….55 Chapter 5 Application of OSU03012 to Other Cancers……………………………….71 5.1 In Primary Chronic Lymphocytic Leukemia (CLL)………………………71 5.2 In Breast Cancer cells MDAMB453…..………………………..…………71 5.3 In Gleevec Resitant Chronic Myelogenous Leukemia (CML) Cells……...72 Chapter 6 Conclusions and Future Directions………………………………………...73 6.1 Conclusions……………………………………………………………...73 6.2 Future Directions………………………………………………………..73 6.3 Concerns about Anticancer Drug Development………………………...77 6.3.1 Specific PKIs versus Non-Specific (or Broad Spectrum) PKIs…...78 6.3.2 Targeted Cancer Therapy-Lessons from Gefitinib Clinical Trial…79 Chapter 7 Experimental Methods and Material……………………………………….83 7.1 Synthesis of the 1st Series of Compounds……………………………..…..83 7.1.1 Preparation of Starting Material for Synthesis of Compound 1-4….83 7.1.2 Preparation of Starting Material for Synthesis of Compound 10-20.84 7.1.3 General Procedure…………………………………………………..85 7.2 Synthesis of the 2nd Series of Compounds………………………………...87 7.3 Nomenclatures, 1H NMR (proton nuclear magnetic resonance), and HRMS (high resolution mass spectrometry) Characterizations of Compounds 1 – 36………………………..………………..………………….93 7.4 PDK-1 Kinase Assay……………………………………………………...93 7.5 Cell Viability Assay……………………………………………………….94 7.5.1 Cell culture………...………………………………………………..94 7.5.2 MTT Assay………………………………………………………….94 7.6 Immunoblotting……………………………………………………………95 7.7 Immunoprecipitated Akt Kinase Assay…………………………………....96 7.8 Immunoprecipitated p70S6K Kinase Assay………………………………...96 7.9 Cell Death Detection ELISA………………………………………………97 7.10 Molecular Modeling……………………………………………………..98 Bibliography………………………………………………………………………….108 viii