Development and Evaluation of Chemical Probes for the Study of Histone Demethylases

The expression of only certain particular genes out of a complete genome is what allows a cell to have a unique phenotype. Epigenetic control of gene regulation is exerted through a variety of mechanisms including DNA methylation, chromatin remodeling, and covalent histone modifications. One such modification, the methylation of histone lysine residues, is able to be ‘erased’ by several families of proteins termed histone lysine demethylases (HDMs). These proteins have been shown to be vital in many natural cellular processes, and their aberrant activity has been associated with certain disease states. A summary of these topics will be introduced along with a brief overview of relevant past work in the literature. The sequential chapters will detail the original research that encompasses this dissertation. First, the key problem of specificity was explored through the synthesis and evaluation of a peptidic probe targeting only those HDMs which contain a JmjC domain and which modify dimethylated lysines at the thirty-sixth position on histone three (H3K36me2). This probe was employed in mass spectrometry-based affinity proteomics experiments to evaluate its ability to pull out these HDMs and their associated proteins from a complex nuclear lysate. Next, techniques by which to assess in vitro binding affinity to two epigenetic proteins, JMJD2A and NgTET1, were developed via fluorescence polarization (FP). Fluorescent tracers which bind to each active site were discovered and used in the development of FP-based competition assays which allow the quantitative measurement of the dissociation constants (Ki) of non-fluorescent competitive inhibitors. These assays were applied in a high-throughput screen of a commercial library (Maybridge HitFinder v.11) to identify unique small molecule inhibitors of JHDMs with distinct carbon scaffolds. Finally, the discovery of a pentacyclic indoline inhibitor of JHDMs is reported via a screening of an in-house generated, diversity-oriented, polycyclic indoline library. A structure-activity relationship study was undertaken and a series of analogs were evaluated for binding affinity to JMJD2A, JHDM1A and NgTet1 with the FP competition assays. Ongoing investigations of the selectivity, enzymatic inhibition, and cellular activity are presented