Development Of Small Molecule Chemical Probes For Brd4

Abstract

Bromodomain-containing proteins are epigenetic proteins that can specifically recognize acetylated histone proteins and transcription factors. There are 61 human bromodomains classified into eight subgroups based on structural and sequence similarities. The bromodomain and extra terminal domain (BET) family proteins, including BRD2, BRD3, BRD4, all contain two tandem bromodomains (D1 and D2) and an extra terminal domain. BET proteins are one of the most heavily studied bromodomain-containing proteins due to their functions in various diseases, including cancer and inflammatory diseases. Selective inhibition of BET bromodomains is challenging due to the high sequence similarity in the acetylated lysine residue binding pocket. Thus, pan-BET inhibitors, binding to eight bromodomains non-selectively, have been widely used to investigate the functions of BET proteins. Although efficacious, the use of pan-BET inhibitors obscures the function of individual bromodomains, and in some cases has led to incorrect attribution of biological activity to a specific BET bromodomain, in many cases BRD4. Clinical studies have identified dose-limiting toxicities and side effects associated with pan-BET inhibitors. Compounds with selectivity toward D1s or D2s of BET bromodomains have also entered clinical trials and show efficacy toward BET-related diseases. A D2-selective inhibitor, RVX-208, showed efficacy in decreasing the major adverse cardiovascular event in patient with high-risk type 2 diabetes mellitus in clinic trial phase III and received Breakthrough Therapy Designation from U.S. Food & Drug Administration (FDA). Despite the importance of BET proteins for regulating transcription, the individual contributions of each of their two bromodomains remains unclear. Better understanding of individual BET bromodomains and cooperative functions would facilitate therapeutic application of BET inhibitors. Small molecule inhibitors of individual BET bromodomains will improve our understanding of the molecular mechanisms underlying epigenetic regulation of disease. In this dissertation, we describe new approaches to develop potent BET bromodomains inhibitors, especially pan-D1 inhibitors and selective BRD4 D1 inhibitors. The affinity and selectivity are evaluated by biophysical assays, including fluorescent anisotropy, and their cellular activity are characterized relative to pan-BET inhibitors in different disease models. In chapter 1, the biological function of BET-proteins and effects from chemical inhibition will be described. In chapter 2, a systematic approach to remove kinase activity on dual kinase-bromodomain inhibitors based on a 1,2,3-triazole scaffold we developed will be discussed. Selective inhibition is ideal for understanding the physiological and pharmacological effect of BET inhibition as well as minimizing potential side effects although dual kinase-bromodomain inhibitors may produce synergistic effects in some cases. By modifying the hinge binding motif and removing a hydrophobic 4-fluorophenyl group, the kinase affinity was removed. In chapter 3, structure-activity-relationship will be described to further improve the affinity and D2/D1 selectivity of this 1,2,3-triazole scaffold. we describe a structure−activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes are accomplished by targeting a non-conserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, respectively. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. In chapter 4, the development of a BRD4 D1 selective inhibitor based on a reported 1,4,5-trisubstituted imidazole scaffold will be discussed. We rationalize this high level of selectivity to arise from at least two mechanisms: flexibility in a D1-conserved YNKP motif and displacement of structured-waters in the acetyl-lysine binding site of BRD4 D1, where an additional water can be displaced relative to our previous reports. Chapter 5 will describe the unpublished work on the structure-activity-relationship study based on the inhibitor in Chapter 4. We describe the development of BRD4 D1 selective inhibitors with nanomolar affinity through a structure-activity-relationship study. The unique selectivity and strong affinity are obtained from halogen bonding interaction, flexible backbone conformational effects in D1, displacement of low-energy structured waters, and targeting a D1 conserved Asp residue. Lastly, the synthesis and preliminary cellular data of bivalent BET inhibitors will be discussed in the appendix.