Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

Calcium-dependent protein kinases (CDPKs) play pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites, and comprise a CaMK-like kinase domain regulated by a calcium-binding domain in the C-terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N-terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate-binding site. This large conformational change constitutes a distinct mechanism in calcium signal transduction pathways.

SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the G9a-8 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. The co-crystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling.Multicellular organisms have evolved elaborate mechanisms to enable differential and celltype specific expression of genes. Epigenetics refers to these heritable changes in how the genome is accessed in different cell-types and during development and differentiation. This capability permits specialization of function between cells even though each cell contains the same genome. Over the last decade, the cellular machinery that creates these heritable changes has been the subject of intense scientific investigation as there is no area of biology or for that matter no area of human health, where epigenetics may not play a fundamental role. 1 The template upon which the epigenome is written is chromatin -the complex of histone proteins, RNA and DNA that efficiently package the genome in an appropriately accessible state within each cell. The state of chromatin, and therefore access to the genetic code, is mainly regulated by covalent and reversible PTMs to histone proteins and DNA, and the recognition of these marks by other proteins and protein complexes. The PTMs of histones and DNA include: histone lysine methylation, arginine methylation, lysine acetylation, sumoylation, † The coordinates and structure factors of UNC0224 co-crystallized with G9a have been deposited in the Protein Data Bank (www.pdb.org, PDB code 3K5K). ubiquitination, glycosylation and phosphorylation, and DNA methylation. 2 Given the wide-spread importance of chromatin regulation to cell biology, the enzymes that produce these modifications (the 'writers'), the proteins that recognize them (the 'readers'), and the enzymes that remove them (the 'erasers') are critical targets for manipulation in order to further understand the histone code 3, 4 and its role in human disease. Indeed, small molecule histone de-acetylase inhibitors5 and DNA methyltransferase inhibitors6 have already proven useful in the treatment of cancer.Histone lysine methylation refers to covalent methylation of histone lysine tails to produce mono-,di-, or trimethylated states. Among a myriad of PTMs, histone lysine methylation catalyzed by histone lysine methyltransferases (HMTs) has received great attention because of its essential function in many biological processes including gene expression and transcriptional regulation, heterochromatin formation, and X-chromosome inactivation. 7 It is therefore considered to be one of the most significant PTMs of histones. Since the first HMT was characterized in 20008, more than 50 human histone methyltransferases have been ...