Examination of the Role of Lysine Specific Demethylase 1 (LSD1) and Associated Proteins in Breast Cancer Proliferation using 2‐Phenylcyclopropylamine Inhibitors by Julie Ann Pollock Department of Chemistry Duke University Date:_______________________ Approved: ___________________________ Dewey G. McCafferty, Supervisor ___________________________ Donald P. McDonnell ___________________________ Jiyong Hong ___________________________ Michael Fitzgerald Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the Graduate School of Duke University 2011 ABSTRACT Examination of the Role of Lysine Specific Demethylase 1 (LSD1) and Associated Proteins in Breast Cancer Proliferation using 2‐Phenylcyclopropylamine Inhibitors by Julie Ann Pollock Department of Chemistry Duke University Date:_______________________ Approved: ___________________________ Dewey G. McCafferty, Supervisor ___________________________ Donald P. McDonnell ___________________________ Jiyong Hong ___________________________ Michael Fitzgerald An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the Graduate School of Duke University 2011 Copyright by Julie Ann Pollock 2011 Abstract Lysine specific demethylase 1 (LSD1) is a FAD‐dependent amine oxidase enzyme responsible for removing methyl groups from the side chain nitrogen of lysine within histones in order to regulate gene transcription. By its interaction with various transcriptional complexes, including those containing estrogen receptor α (ERα), LSD1 mediates expression of many genes important in cancer proliferation and progression. Herein, we report our efforts towards understanding the function of LSD1 in breast cancer. We have developed a straightforward method for the syntheses of 2‐ arylcyclopropylamines as irreversible mechanism‐based inactivators of LSD1. We employed these small molecules as probes of LSD1 activity, and together with experiments involving the knockout of LSD1 by small interfering RNA (siRNA), we have shown that LSD1 activity is essential for both ERα‐postive and ERα‐negative breast cancer proliferation. LSD1 inhibitors induce a dramatic cell cycle arrest without causing apoptosis. Furthermore, we observe that LSD1 and ERα work cooperatively to express certain estrogen‐target genes through simultaneous recruitment to promoters; LSD1 inhibition diminishes ERα recruitment. Similarly, knockdown of CoREST, a binding partner of LSD1, results in comparable changes in gene expression. Although, we have not observed a direct interaction between LSD1 and ERα, we believe that CoREST may iv be facilitating this interaction. We have made efforts to inhibit the interaction between LSD1 and CoREST in vitro in hopes of targeting this interface in breast cancer cells in order to disrupt the necessary functional complex and prevent LSD1 activity. v To my parents who have always supported my dreams vi Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................xiv List of Schemes ........................................................................................................................... xxi List of Abbreviations ................................................................................................................ xxii Acknowledgements .................................................................................................................. xxv Acknowledgements .................................................................................................................. xxv 1. Introduction ............................................................................................................................... 1 1.1 Eukaryotic gene regulation ............................................................................................. 1 1.1.1 The nucleosome ........................................................................................................... 1 1.1.2 Post‐translational modifications of histones............................................................ 6 1.2 Histone lysine methylation and demethylation ......................................................... 10 1.3 LSD1 ................................................................................................................................. 16 1.3.1 LSD1 mechanism and function ............................................................................... 16 1.3.2 LSD1 structure ........................................................................................................... 17 1.3.3 LSD1 inhibition .......................................................................................................... 22 1.4 Thesis objectives ............................................................................................................. 24 2. Synthesis of small molecule compounds: 2‐arylcyclopropylamines and propargylamines ......................................................................................................................... 25 2.1 Background information ............................................................................................... 25 2.2 Synthesis of 2‐arylcyclopropylamine derivatives ...................................................... 31 vii 2.2.1. Design of synthetic plan .......................................................................................... 31 2.2.2 Results ......................................................................................................................... 32 2.3 Synthesis of propargylamine derivatives ................................................................... 50 2.4 Methylation of 2‐PCPA .................................................................................................. 51 2.5 Mechanistic evaluation of Corey‐Chaykovsky cyclopropanation ........................... 53 2.6 Conclusions and future directions ............................................................................... 55 2.7 Experimental section ...................................................................................................... 57 3. Inhibition of FAD‐dependent amine oxidase enzymes ................................................... 104 3.1 Background information ............................................................................................. 104 3.2 Optimization of the overexpression and purification of LSD1 .............................. 107 3.2.1 Expression tests of NΔ122 ...................................................................................... 108 3.2.2 Efforts towards refolding insoluble LSD1 from inclusion bodies .................... 109 3.2.3 Final overexpression and purification using a codon‐optimized clone .......... 112 3.3 Inhibition of LSD1 ........................................................................................................ 114 3.3.1 LSD1 activity assay ................................................................................................. 114 3.3.2 Reversibility of inhibition of LSD1 by 2‐phenylcyclopropylamines ................ 115 3.3.3 Time‐dependent inhibition of LSD1 ..................................................................... 115 3.4 Inhibition of MAO A and MAO B .............................................................................. 120 3.5 Conclusions and future directions ............................................................................. 123 3.5.1. Selectivity issues and other inhibitors ................................................................. 123 3.5.2 Lysine specific demethylase 2 (LSD2) .................................................................. 124 3.5.3 Future directions...................................................................................................... 133 viii 3.6 Experimental section .................................................................................................... 133 4. Evaluation of small molecule inhibitors as probes of LSD1 function in breast cancer cells .............................................................................................................................................. 140 4.1 Background information ............................................................................................. 140 4.1.1 Breast cancer ............................................................................................................. 140 4.1.2 Estrogen Receptor‐positive breast cancer ............................................................ 142 4.1.3 Estrogen Receptor‐negative breast cancer ........................................................... 145 4.1.4 Epigenetics as a target for cancer therapy ........................................................... 145 4.2 Small molecules as probes of LSD1 function in breast cancer. .............................. 147 4.3 LSD1 expression and importance in breast cancer cells and tumors .................... 149 4.4 LSD1 as a coactivator of estrogen‐dependent gene transcription ........................