Lysine demethylases (KDMs) catalyze the removal of methyl groups from the side-chain amino groups of tri-, di- and mono-methylysines (Kme3/2/1), notably at specific sites in the N-terminal 'tails' of histones. There are two groups of KDMs: 1) the lysine-specific demethylases (LSDs 1 and 2), flavin dependent amine oxidases that can demethylate Kme2/1 and 2) the JmjC-domain-containing oxidases, Fe2+ and ?-ketoglutarate requiring enzymes that, in many cases, can also demethylate Kme3. Histone lysine methylations are epigenetic regulatory modifications that profoundly affect gene expression and aberrant histone lysine methylation occurs in a number of diseases including cancer. Excess activity of a number of particular KDMs is implicated in a variety of cancers. Interest in the discovery of drugs that inhibit KDMs is therefore growing but, especially for the JmjC enzymes, which is the largest of the two groups of KDMs. A factor which is slowing progress toward eventual development of drugs that target the JmjC KDMs is a lack of chemical probes, with probes defined as highly potent and highly specific inhibitors of individual JmjC KDMs. One circumstance that may be contributing to the lack of specific probes is that the existing inhibitor exclusively target the highly conserved binding site of the co-substrate, 2-oxoglutarate (2-OG) and/or chelate the active site Fe2+. In order to provide tools that may help remedy this situation, we are proposing to produce a comprehensive collection of JmjC KDM proteins, which will include full-length proteins, catalytic domain (demethylase) constructs and separate individual constructs for each of the many 'reader' domains (e.g. PHD or double Tudor domains) that form part of most, but not all, full-length JmjC KDMs. Once these proteins are in hand, we will develop at least one assay (binding or demethylation) and begin by screening all of them against a collection of FDA-approved drugs and compounds from clinical trials. The chemical profiles of the demethylase and reader domains thus generated will be incorporated in a database and made publicly available as a general aid to research in this area. The proteins that are produced in the process will become available as products and the assays that are developed will become services that we offer. Finally, these proteins will be screened against a much larger chemically diverse library (>80,000 compounds). Inhibitors that are identified will also become products. In Phase II we envision that some of these inhibitors will be used as the starting point for chemistry to produce more potent probes or to produce fluorophor-labeled probes for fluorescence polarization assays.
Histones, proteins which are packaged together with DNA into chromatin, can be chemically modified in a number of ways, including methylation on specific lysines in their amino acid sequences. Such modifications can regulate gene expression and are termed 'epigenetic', because while they sometimes can be inherited, they do not alter the cell's genetics, the underlying DNA sequence. Epigenetic regulation of gene expression is essential to normal development and when dysfunctional can be involved in the development and maintenance of disease states including cancer, neurodegeneration and metabolic disorders. This has led to intense research into epigenetic modifications (e.g. NIH's Epigenomics Program) as well as efforts to discover drugs that affect the enzymes that add or remove these modifications. Two FDA approved drugs for cutaneous T-cell lymphoma (CTCL) target enzymes (histone deacetylases, HDACs) that reverse acetylation of histone lysines. A younger field of study, relative to the HDACs, are the enzymes that remove lysine methyl groups (lysine demethylases, KDMs). The largest subgroup of the KDMs, the JmjC-domain containing KDMs (JmjC KDMs) contains members whose hyperactivity is implicated in a number of cancers, making them promising targets for the development of anti-cancer therapies. A circumstance that is slowing down progress toward JmjC KDM-targeted therapies is the absence of what are termed chemical 'probes' for the individual JmjC KDMs. A 'probe' in this context means a chemical that is a powerful inhibitor of a particular protein, but does not, o only weakly inhibits related proteins. This makes such compounds useful in addressing question such as, if we were to eliminate the activity of this particular protein, and only this protein, ina certain kind of cancer cell, will that kill that kind of cell? In other words, such compounds are tools that help validate a protein as one which would be worthwhile to develop a drug against, one whose inhibition might actually benefit a patient with a particular form of cancer. In order to help remedy this lack probes for the JmjC KDMs, we are proposing to produce a comprehensive collection of the human JmjC KDM proteins, which will include not only the complete version of each protein but also the individual functional fragments of each ('domains'). Then we will test all of these proteins for inhibition by chemicals that are part of collections that we hold here at Reaction Biology Corp. (RBC). One part of our chemical collection consists of FDA-approved drugs and chemicals that have been in clinical trials. The information about which KDMs and which parts of the KDMs are inhibited by these drugs, and potential drugs. will go into a public database and will be available to any researcher to use in his or her research on the targeting of JmjC KDMs. We will also attempt to directly contribute to the discovery of probes for the KDMs by testing the effects of a much larger collection of chemicals (>80,000) on the various KDM proteins we have made. Overall this project will benefit public health by speeding up the discovery of drugs that are specific to particular KDMs and thus enable the addition of an entirely new class of anti-cancer therapeutics to the health care arsenal.
