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.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.chemical biology ͉ crystallography ͉ human S tructural, functional, and chemical genomics (proteomics) are disciplines that aim to determine the biochemical, cellular, and physiological functions of proteins on a genome scale. Many of the central, important experimental approaches that are involved, such as protein-based screens for small-molecule inhibitors, depend on the availability of purified and active proteins. To meet this demand, many large projects are devoted to developing methods to generate large numbers of purified proteins. However, the task is proving challenging: on average, for proteins from prokaryotes, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity (1, 2). The success rates for human proteins are predicted to be significantly lower.To improve the general rates of protein purification, efforts have focused largely on alterations of the recombinant host, the expression conditions, changes of the construct encoding the protein, and the purification conditions. It is also known that the expression and purification of a protein can be improved significantly by the addition of a specific ligand, which serves to stabilize the protein, thereby reducing its propensity to unfold, aggregate, or succumb to proteolysis. This parameter has not been studied systematically, although in individual cases the addition of a specific ligand has had dramatic effects. For example, the recombinant expression of the guinea pig and human forms of the enzyme 11␤-hydroxysteroid dehydrogenase-1 in bacteria was increased dramatically by the addition of an inhibitor of the enzyme to the growing cells (3) Wu, K. L. Kav...

The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.