Mutational analysis of G-protein coupled receptor--FFA2.

FFA2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate, and propionate. FFA2 is predominantly expressed in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract which suggest a possible role in inflammatory and metabolic conditions. We have previously described the identification and characterization of novel phenylacetamides as allosteric agonists of FFA2. In the current study, we have investigated the molecular determinants contributing to receptor activation with the endogenous and synthetic ligands as well as allosteric interactions between these two sites. The mutational analysis revealed previously unidentified sites that may allosterically regulate orthosteric ligand's function as well as residues potentially important for the interactions between orthosteric and allosteric binding sites.

The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators.

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions.

Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.

The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism. Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations. The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/extrusion. Here we show that GPR91 (ref. 3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to alpha-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and alpha-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure.

Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2.

FFA2 (GPR43) has been identified as a receptor for short-chain fatty acids (SCFAs) that include acetate and propionate. FFA2 is highly expressed in islets, a subset of immune cells, and adipocytes. Although the potential roles of FFA2 activation in these tissues have previously been described, the physiological functions are still unclear. The potency for SCFAs on FFA2 is low, in the high micromolar to millimolar concentrations. To identify better pharmacological tools to study receptor function, we used high-throughput screening (HTS) to discover a series of small molecule phenylacetamides as novel and more potent FFA2 agonists. This series is specific for FFA2 over FFA1 (GPR40) and FFA3 (GPR41), and it is able to activate both the Galpha(q) and Galpha(i) pathways in vitro on Chinese hamster ovary cells stably expressing FFA2. Treatment of adipocytes with these compounds also resulted in Galpha(i)-dependent inhibition of lipolysis similar to that of endogenous ligands (SCFAs). It is noteworthy that these compounds not only acted as FFA2 agonists but also exhibited positive cooperativity with acetate or propionate. The observed allosteric modulation was consistent in all the functional assays that we have explored, including cAMP, calcium mobilization, guanosine 5'-[gamma-thio]triphosphate binding, and lipolysis. Molecular modeling analysis of FFA2 based on human beta(2)-adrenergic receptor structure revealed potential nonoverlapping binding sites for the endogenous and synthetic ligands, further providing insight into the binding pocket for the allosteric interactions. This is the first report describing the identification of novel allosteric modulators with agonist activity for FFA2, and these compounds may serve as tools for further unraveling the physiological functions of the receptor and its involvement in various diseases.

High throughput screening for orphan and liganded GPCRs.

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.

Discovery of potent LPA2 (EDG4) antagonists as potential anticancer agents.

The LPA(2) protein is overexpressed in many tumor cells. We report the optimization of a series of LPA(2) antagonists using calcium mobilization assay (aequorin assay) that led to the discovery of the first reported inhibitors selective for LPA(2). Key compounds were evaluated in vitro for inhibition of LPA(2) mediated Erk activation and proliferation of HCT-116 cells. These compounds could be used to evaluate the benefits of LPA(2) inhibition both in vitro and in vivo.

Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes.

Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-alpha and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.

Differing roles for short chain fatty acids and GPR43 agonism in the regulation of intestinal barrier function and immune responses.

Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.

Utilization of human nuclear receptors as an early counter screen for off-target activity: a case study with a compendium of 615 known drugs.

Off-target effects of drugs on nuclear hormone receptors (NHRs) may result in adverse effects in multiple organs/physiological processes. Reliable assessments of the NHR activities for drug candidates are therefore crucial for drug development. However, the highly permissive structures of NHRs for vastly different ligands make it challenging to predict interactions by examining the chemical structures of the ligands. Here, we report a detailed investigation on the agonistic and antagonistic activities of 615 known drugs or drug candidates against a panel of 6 NHRs: androgen, progesterone, estrogen α/ß, and thyroid hormone α/ß receptors. Our study revealed that 4.7 and 12.4% compounds have agonistic and antagonistic activities, respectively, against this panel of NHRs. Nonetheless, potent, unintended NHR hits are relatively rare among the known drugs, indicating that such interactions are perhaps not tolerated during drug development. However, we uncovered examples of compounds that unintentionally agonize or antagonize NHRs. In addition, a number of compounds showed multi-NHR activities, suggesting that the cross-talk between multiple NHRs co-operate to elicit in vivo effects. These data highlight the merits of counter screening drug candidate against NHRs during drug discovery/development.

Activation of FFA1 mediates GLP-1 secretion in mice. Evidence for allosterism at FFA1.

FFA1 (GPR40) and GPR120 are G-protein-coupled receptors activated by long-chain fatty acids. FFA1 is expressed in pancreatic ß-cells, where it regulates glucose-dependent insulin secretion, and GPR120 has been implicated in mediating GLP-1 secretion. We show here that FFA1 co-localizes with GLP-1 in enteroendocrine cells and plays a critical role in glucose management by mediating GLP-1 secretion in vivo. Corn oil induces GLP-1 secretion in wild type mice and in GPR120-/- mice, but not in FFA1-/- mice. α-Linolenic acid, an endogenous ligand of FFA1, induces GLP-1 secretion in GLUTag cells and in primary fetal mouse intestinal cells. Synthetic partial FFA1 agonists do not stimulate GLP-1 secretion in mice, but partial and full agonists combined function cooperatively to enhance receptor activation and GLP-1 secretion both in vitro and in vivo. We conclude that allosterism at FFA1 can contribute to postprandial glucose management by stimulating insulin secretion via an extrapancreatic mechanism of action, and that GPR120 in GLP-1 secretion requires further investigation.

High-throughput TR-FRET assays for identifying inhibitors of LSD1 and JMJD2C histone lysine demethylases.

Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.

STK33 kinase activity is nonessential in KRAS-dependent cancer cells.

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.

Allosteric rescuing of loss-of-function FFAR2 mutations.

FFAR2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate and propionate. In the current study, we investigate the molecular determinants contributing to receptor activation by endogenous ligands. Mutational analysis revealed several important residues located in transmembrane domains (TM) 3, 4, 5, 6, and 7 for acetate binding. Interestingly, mutations that abolished acetate activity, including the mutation in the well-conserved D(E)RY motif, could be rescued by a recently identified synthetic allosteric agonist. These findings provide additional insight into agonist binding and activation which may aid in designing allosteric ligands for targeting receptor function in various diseases.