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«Abstract Title of Dissertation: Novel Extracellular Signal-Regulated Kinase (ERK) Targeted Inhibitors Effects on Apoptotic Signaling: A Potential ...»

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Abstract

Title of Dissertation: Novel Extracellular Signal-Regulated Kinase (ERK) Targeted

Inhibitors Effects on Apoptotic Signaling: A Potential Cancer Therapy

Sarice R. Boston, Doctor of Philosophy, 2011

Dissertation directed by: Paul Shapiro, Ph.D.

Associate Professor

Associate Dean for Research and Graduate Education

Department of Pharmaceutical Sciences

University of Maryland, Baltimore

The extracellular signal-regulated kinase 1 and 2 (ERK 1/2) proteins potently

mediate cell proliferation and survival signals via substrate phosphorylation events.

Aberrant activation of ERK due to mutations in upstream activators such as B-Raf is a hallmark of a variety of cancers. Thus, inhibition of substrates involved in these mechanisms provides a promising target for anti-cancer therapies.

Small molecules designed to interfere with five putative binding sites on ERK2, which are potentially involved in substrate interactions, were developed using computeraided drug design (CADD) and assessed for their ability to inhibit ERK-mediated signaling events. The inhibitors identified successfully inhibited cell proliferation and survival signals as demonstrated by inhibition of cell cycle progression and/or the induction of apoptosis in cell based assays. Evaluation of the mechanism of action of these ERK-targeted inhibitors revealed inhibitory effects on ERK mediated phosphorylation of p90Rsk-1, which plays a role in cell growth and survival as well as caspase-9, which is involved in the activation of the intrinsic apoptotic pathway resulting in the induction of apoptosis.

Additionally, the ERK-targeted inhibitors appeared to enhance chemotherapeutic drugs by inducing a cytostatic effect through selective inhibition of p90Rsk-1 phosphorylation and cyclin D1 expression indicative of a G1-phase arrest in a melanoma model harboring a B-Raf mutation. These studies suggest that selective inhibition of ERK functions is an alternative approach to complete ERK pathway inhibition and can be used to enhance the sensitivity of melanoma cells to chemotherapeutics drugs potentially reducing toxicity to normal cells and the development of drug resistance.

Novel Extracellular Signal-Regulated Kinase (ERK) Targeted Inhibitors Effects on Apoptotic Signaling: A Potential Cancer Therapy By Sarice R. Boston Dissertation submitted to the faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy © 2011 by Sarice R. Boston

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I dedicate this dissertation to my maternal grandmother Bernice Gwendolyn Hall and paternal grandfather Jack Smith Sr., both of whom lost their lives to the devastating disease of cancer. This work was completed with you mind and it was your untimely passing that motivated me through to the end. I wish you were here to celebrate this

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First I would like to give honor and glory to my Lord and Savior Jesus Christ.

Thank you for allowing me to have this opportunity, I know that it was you that got me through.

I want to thank my advisor, Dr. Paul Shapiro, for allowing me to pursue my interest under his guidance and support. Your even temperament was definitely needed at times when I thought I was going to pull my hair out. Also, I want to thank my committee members Dr. Yuan Luo, Dr. Stuart Martin, Dr. Alexander MacKerell Jr, and Dr. Wanli Smith for their helpful insights, comments, and suggestions over the course of my studies.

I would like to also acknowledge my former lab members Dr. Fengming Chen, Rahul Deshmukh, and Dr. Kim Still. You guys made lab fun even after doing a bazillion western blots…LOL. To my dear PROMISE family Antonia Tolson Baaqee, Dr.

Marishka Brown, Dr. Dawn Holt, Christina Silver Marshall, Dr. Jocelyn Reader, Dr.

Trudy Smith, and Dr. Stacey Williams, thank you for listening to all my complaining and providing constructive feedback. That was free therapy and on a grad student budget…it was needed. To the friends I’ve made throughout this process: Kerrick Nevels, Ekemini Udofa, Brandiese Beverly, Holly Porter, Tatiana Claro da Silva, Xiao Xhu, and Feiyan Jin thank you for making graduate school bearable.

To my parents, Jack and Benita Smith, thank you for your countless prayers, love and support. The sacrifices you have made for me over the years are not forgotten and have allowed me to accomplish my goals. I can’t say thank you enough for that; I hope I made you proud. To my siblings Tanisha Laidler and Jack Smith III, thank you for your

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extended family I felt your love and prayers from afar, thank you. I also extend thanks to my in-laws Derick and Beverly Boston for their kind words of encouragement, prayers, and support throughout this journey. Marcus and Renee Boston, thank you for listening to me on good and bad days and offering words of wisdom. Andrew and Leah, just remember that anything is possible so pursue your goals.

Last and certainly not least, to my best friend, confidant, and husband I can’t say thank you enough for always being there for me. I know that you have patience and love for me because you are still here through it all. I know I was not the easiest to deal with at times while completing this degree but you remained patient and loving for the most part and I thank you tremendously for that. Seemed like forever ago that we both started our Ph.D. journeys and finally our life post-school together can begin. I can’t wait to experience the rest of my life with you.





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Chapter 1: Background into ERK Signaling and its Role in Cellular Functions

1.1 The Mitogen Activated Protein Kinase (MAPK) Family…………………………….2

1.2 ERK Activation, Signaling, and Specificity………………………………………….5

1.3 ERK and Cancer……………………………………………………………………...9

1.4 ERK’s Role in the Cell Cycle……………………………………………………….12

1.5 ERK and Apoptosis………………………………………………………………….14

1.6 Inhibitors of the ERK Pathway……………………………………………………...18 1.6.1 Epidermal Growth Factor Receptor (EGFR) Inhibitors…...……………...….19 1.6.2 Ras Inhibitors………………………………………………...……………….20 1.6.3 Raf Inhibitors…………………………………………………..……………. 21 1.6.4 MEK Inhibitors…………………………………………………..…………...24 1.6.5 ERK Inhibitors……………………………………………………...………...26

1.7 References…………………………………………………………………………...31 Chapter 2: Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

2.1 Introduction…………………………………………………………………….........48

2.2 Materials and Methods...…………………………………………………………….50 2.2.1 Cells and Reagents……………………………………………………………50 2.2.2 Protein Expression……………………………………………………………52 2.2.3 Immunoblotting………………………………………………………………52 2.2.4 Cell Proliferation Assays……………………………………………………..53 2.2.5 Kinase Assays………………………………………………………………...53 2.2.6 Caspase Activity Assays……………………………………………………...54 2.2.7 Fluorescent Activated Cell Sorting (FACS) Analysis………………………..54 2.2.8 Statistical Analysis……………………………………………………………55

2.3 Results………………………………………………………………………………..56 2.3.1 ERK-targeted Inhibitors Inhibit Cell Proliferation…………………………...56 2.3.2 ERK-targeted Inhibitors Delay Cell Cycle Progression………...……………58 2.3.3 Activation of the Intrinsic Apoptotic Pathway by ERK-targeted Inhibitors....60 2.3.4 Inhibition of ERK-mediated Phosphorylation of Pro-apoptotic Proteins..…...62 2.3.5 ERK-targeted Inhibitors Effects Pro-and Anti-apoptotic Protein Expression..64 2.3.6 ERK-targeted Inhibitors Inhibit Caspase-9 Phosphorylation………………...68 2.3.7 ERK-targeted Inhibitors are Not General Inhibitors of Kinase Signaling……68

2.4 Discussion…………………………………………………………………………...70

2.5 References…………………………………………………………………………...74

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3.1 Introduction………………………………………………………………………….81

3.2 Materials and Methods………………………………………………………………84 3.2.1 Cells and Reagents……………………………………………………………84 3.2.2 Immunoblotting………………………………………………………………85 3.2.3 Cell Proliferation Assays……………………………………………………..85 3.2.4 Caspase Activity Assays……………………………………………………...85 3.2.5 In vivo Analysis of A375 Melanoma Xenograft Model……………………...86 3.2.6 Statistical Analysis……………………………………………………………86

3.3 Results……………………………………………………………………………….87 3.3.1 ERK-targeted Inhibitors Selectively Inhibit Substrate Phosphorylation……..87 3.3.2 Effects of ERK-targeted Inhibitors on Melanoma and Normal Melanocyte Cell Proliferation………………...………………………………………...….89 3.3.3 ERK-targeted Inhibitors Enhance Melanoma Growth Inhibition of Chemotherapeutic Agents…………………………………………………….91 3.3.4 ERK-targeted Inhibitors Down Regulate Cyclin D1 Expression…………….93 3.3.5 ERK-targeted Inhibitors Enhance Down Regulation of Mcl-1 by temozolomide…………………………………………………………………95 3.3.6 ERK-targeted Inhibitor 76.3 Enhances the Efficacy of Temozolomide in vivo………………………………………………………...97

3.4 Discussion…………………………………………………………………………...99

3.5 References………………………………………………………………………….103 Chapter 4: Identification and Evaluation of Lead Compounds Targeting Potentially Novel ERK2 Substrate Interaction Sites

4.1 Introduction………………………………………………………………………...110

4.2 Materials and Methods……………………………………………………………..112 4.2.1 CADD Screening……………………………………………………………112 4.2.2 Cells and Reagents…………………………………………………………..113 4.2.3 Immunoblottiing…………………………………………………………….114 4.2.4 Cell Proliferation Assays……………………………………………………115 4.2.5 Kinase Assays……………………………………………………………….115 4.2.6 Fluorescent Activated Cell Sorting (FACS) Analysis………………………116 4.2.7 Statistical Analysis…………………………………………………………..116

4.3 Results……………………………………………………………………………...117 4.3.1 ERK-targeted Inhibitor Identification and Screening……………………….117 4.3.2 Active ERK-targeted Inhibitors Effects on Cell Proliferation………………119 4.3.3 Active ERK-targeted Inhibitors Induce Apoptosis………………………….122 4.3.4 Active ERK-targeted Inhibitors Effects on Survival Signals……………….124 4.3.5 ERK-targeted Inhibitors Effects on p38 MAPK…………………………….126

4.4 Discussion………………………………………………………………………….126

4.5 References………………………………………………………………………….132 
 vii
 Chapter 5: Future Directions

5.1 Introduction………………………………………………………………………..137

5.2 Future Studies……………………………………………………………………...138 5.2.1 Similarity Searching/Lead Optimization……………………………………138 5.2.2 Further Characterization of Identified ERK-targeted Inhibitors……………139 5.2.3 Combination Studies with Traditional Chemotherapeutics………………....140 5.2.4 Co-crystallization of ERK-inhibitor Complexes……………………………143

5.3 Conclusion…………………………………………………………………………143

5.4 References………………………………………………………………………….144 Chapter 6: Cumulative References

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Figures

1.1 Overview of the Mitogen Activated Protein Kinase (MAPK) family pathway………4

1.2 Overview of the extracellular signal-regulated kinase (ERK) pathway……………...8

1.3 An overview of the ERK pathway’s effects on apoptotic signaling.………………..15

1.4 Putative Binding Sites on the ERK2 protein as identified by CADD…….…………30

2.1 Structure of test compounds used in these studies…………………………………..57

2.2 Effects of ERK targeted inhibitors on cell proliferation……………………...……..59

2.3 ERK-targeted inhibitors effects cell cycle progression during G2/M-phase……......61

2.4 ERK-targeted inhibitors induce the intrinsic apoptosis pathway……………………63

2.5 ERK-targeted inhibitors reduce ERK-mediated phosphorylation of p90Rsk-1 and Bad………………………………………………………………...…66

2.6 ERK-targeted inhibitors effect the expression of the pro- and anti-apoptotic proteins, Bad and Mcl-1……..……………………………………....67



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