Identification of Novel, Structurally Diverse, Small Molecule Modulators of GPR119.

GPR119 drug discovery efforts in the pharmaceutical industry for the treatment of type 2 diabetes mellitus (T2DM) and obesity, were initiated based on its restricted distribution in pancreas and GI tract, and its possible role in glucose homeostasis. While a number of lead series have emerged, the pharmacological endpoints they provide have not been clear. In particular, many lead series have demonstrated loss of efficacy and significant toxic side effects. Thus, we sought to identify novel, potent, positive modulators of GPR119. In this study, we have successfully developed and optimized a high-throughput screening strategy to identify GPR119 modulators using a live cell assay format that utilizes a cyclic nucleotide-gated channel as a biosensor for cAMP production. Our high-throughput screening (HTS) approach is unique to that of previous HTS approaches targeting this receptor, as changes in cAMP were measured both in the presence and absence of an EC10 of the endogenous ligand, oleoylethanolamide, enabling detection of both agonists and potential allosteric modulators in a single assay. From these efforts, we have identified positive modulators of GPR119 with similar as well as unique scaffolds compared to existing compounds and similar as well as unique signaling properties. Our compounds will not only serve as novel molecular probes to better understand GPR119 pleiotropic signaling and the underlying physiological consequences of receptor activation, but are also well-suited for translation as potential therapeutic agents.

Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus.

Enhancers can regulate designate promoters over long distances by forming chromatin loops. Whether chromatin loops are lost or reconfigured during gene repression is largely unexplored. We examined the chromosome conformation of the Kit gene that is expressed during early erythropoiesis but is downregulated upon cell maturation. Kit expression is controlled by sequential occupancy of two GATA family transcription factors. In immature cells, a distal enhancer bound by GATA-2 is in physical proximity with the active Kit promoter. Upon cell maturation, GATA-1 displaces GATA-2 and triggers a loss of the enhancer/promoter interaction. Moreover, GATA-1 reciprocally increases the proximity in nuclear space among distinct downstream GATA elements. GATA-1-induced transitions in chromatin conformation are not simply the consequence of transcription inhibition and require the cofactor FOG-1. This work shows that a GATA factor exchange reconfigures higher-order chromatin organization, and suggests that de novo chromatin loop formation is employed by nuclear factors to specify repressive outcomes.

Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin.

Methylation of histones modulates chromatin structure and function. Whereas methylation of histone H3 on lysines 4, 36, and 79 has been linked with gene activation, methylation of H3 on lysines 9 and 27 and histone H4 on lysine 20 is associated with heterochromatin and some repressed genes within euchromatin. Here, we show that H3K9 di- and trimethylation occur in the transcribed region of active genes in mammalian chromatin. This modification is dynamic, as it increases during activation of transcription and is rapidly removed upon gene repression. Heterochromatin Protein 1gamma (HP1gamma), a protein containing a chromo-domain that recognizes H3K9 methylation, is also present in the transcribed region of all active genes examined. Both the presence of HP1gamma and H3K9 methylation are dependent upon elongation by RNA polymerase II. These findings demonstrate novel roles for H3K9 methylation and HP1gamma in transcription activation.