Structure-Activity Relationship Studies and Optimization of 4-Hydroxypyridones as GPR84 Agonists.

GPR84 is a putative medium-chain fatty acid receptor that is implicated in regulation of inflammation and fibrogenesis. Studies have indicated that GPR84 agonists may have therapeutic potential in diseases such as Alzheimer's disease, atherosclerosis, and cancer, but there is a lack of quality tool compounds to explore this potential. The fatty acid analogue LY237 (4a) is the most potent GPR84 agonist disclosed to date but has unfavorable physicochemical properties. We here present a SAR study of 4a. Several highly potent agonists were identified with EC50 down to 28 pM, and with SAR generally in excellent agreement with structure-based modeling. Proper incorporation of rings and polar groups resulted in the identification of TUG-2099 (4s) and TUG-2208 (42a), both highly potent GPR84 agonists with lowered lipophilicity and good to excellent solubility, in vitro permeability, and microsomal stability, which will be valuable tools for exploring the pharmacology and therapeutic prospects of GPR84.

Hypothalamic free fatty acid receptor-1 regulates whole-body energy balance.

OBJECTIVE: Free fatty acid receptor-1 (FFAR1) is a medium- and long-chain fatty acid sensing G protein-coupled receptor that is highly expressed in the hypothalamus. Here, we investigated the central role of FFAR1 on energy balance. METHODS: Central FFAR1 agonism and virogenic knockdown were performed in mice. Energy balance studies, infrared thermographic analysis of brown adipose tissue (BAT) and molecular analysis of the hypothalamus, BAT, white adipose tissue (WAT) and liver were carried out. RESULTS: Pharmacological stimulation of FFAR1, using central administration of its agonist TUG-905 in diet-induced obese mice, decreases body weight and is associated with increased energy expenditure, BAT thermogenesis and browning of subcutaneous WAT (sWAT), as well as reduced AMP-activated protein kinase (AMPK) levels, reduced inflammation, and decreased endoplasmic reticulum (ER) stress in the hypothalamus. As FFAR1 is expressed in distinct hypothalamic neuronal subpopulations, we used an AAV vector expressing a shRNA to specifically knockdown Ffar1 in proopiomelanocortin (POMC) neurons of the arcuate nucleus of the hypothalamus (ARC) of obese mice. Our data showed that knockdown of Ffar1 in POMC neurons promoted hyperphagia and body weight gain. In parallel, these mice developed hepatic insulin resistance and steatosis. CONCLUSIONS: FFAR1 emerges as a new hypothalamic nutrient sensor regulating whole body energy balance. Moreover, pharmacological activation of FFAR1 could provide a therapeutic advance in the management of obesity and its associated metabolic disorders.

Ligand entry pathways control the chemical space recognized by GPR183.

The G protein-coupled receptor GPR183 is a chemotactic receptor with an important function in the immune system and association with a variety of diseases. It recognizes ligands with diverse physicochemical properties as both the endogenous oxysterol ligand 7α,25-OHC and synthetic molecules can activate the G protein pathway of the receptor. To better understand the ligand promiscuity of GPR183, we utilized both molecular dynamics simulations and cell-based validation experiments. Our work reveals that the receptor possesses two ligand entry channels: one lateral between transmembrane helices 4 and 5 facing the membrane, and one facing the extracellular environment. Using enhanced sampling, we provide a detailed structural model of 7α,25-OHC entry through the lateral membrane channel. Importantly, the first ligand recognition point at the receptor surface has been captured in diverse experimentally solved structures of different GPCRs. The proposed ligand binding pathway is supported by in vitro data employing GPR183 mutants with a sterically blocked lateral entrance, which display diminished binding and signaling. In addition, computer simulations and experimental validation confirm the existence of a polar water channel which might serve as an alternative entrance gate for less lipophilic ligands from the extracellular milieu. Our study reveals knowledge to understand GPR183 functionality and ligand recognition with implications for the development of drugs for this receptor. Beyond, our work provides insights into a general mechanism GPCRs may use to respond to chemically diverse ligands.

Development and Characterization of Potent Succinate Receptor Fluorescent Tracers.

The succinate receptor (SUCNR1) has emerged as a potential target for the treatment of various metabolic and inflammatory diseases, including hypertension, inflammatory bowel disease, and rheumatoid arthritis. While several ligands for this receptor have been reported, species differences in pharmacology between human and rodent orthologs have limited the validation of SUCNR1's therapeutic potential. Here, we describe the development of the first potent fluorescent tool compounds for SUCNR1 and use these to define key differences in ligand binding to human and mouse SUCNR1. Starting from known agonist scaffolds, we developed a potent agonist tracer, TUG-2384 (22), with affinity for both human and mouse SUCNR1. In addition, we developed a novel antagonist tracer, TUG-2465 (46), which displayed high affinity for human SUCNR1. Using 46 we demonstrate that three humanizing mutations on mouse SUCNR1, N181.31E, K2697.32N, and G84EL1W, are sufficient to restore high-affinity binding of SUCNR1 antagonists to the mouse receptor ortholog.

Discovery of Potent Tetrazole Free Fatty Acid Receptor 2 Antagonists.

The free fatty acid receptor 2 (FFA2), also known as GPR43, mediates effects of short-chain fatty acids and has attracted interest as a potential target for treatment of various metabolic and inflammatory diseases. Herein, we report the results from bioisosteric replacement of the carboxylic acid group of the established FFA2 antagonist CATPB and SAR investigations around these compounds, leading to the discovery of the first high-potency FFA2 antagonists, with the preferred compound TUG-2304 (16l) featuring IC50 values of 3-4 nM in both cAMP and GTPγS assays, favorable physicochemical and pharmacokinetic properties, and the ability to completely inhibit propionate-induced neutrophil migration and respiratory burst.

Glucose-stimulated insulin secretion depends on FFA1 and Gq in neonatal mouse islets.

AIMS/HYPOTHESIS: After birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion. This study investigates the role of FFA1 in neonatal beta cell function and in the adaptation of offspring beta cells to parental high-fat feeding. METHODS: Wild-type (WT) and Ffar1-/- mice were fed high-fat (HFD) or chow diet (CD) for 8 weeks before mating, and during gestation and lactation. Blood variables, pancreas weight and insulin content were assessed in 1-, 6-, 11- and 26-day old (P1-P26) offspring. Beta cell mass and proliferation were determined in P1-P26 pancreatic tissue sections. FFA1/Gq dependence of insulin secretion was evaluated in isolated islets and INS-1E cells using pharmacological inhibitors and siRNA strategy. Transcriptome analysis was conducted in isolated islets. RESULTS: Blood glucose levels were higher in CD-fed Ffar1-/- P6-offspring compared with CD-fed WT P6-offspring. Accordingly, glucose-stimulated insulin secretion (GSIS) and its potentiation by palmitate were impaired in CD Ffar1-/- P6-islets. In CD WT P6-islets, insulin secretion was stimulated four- to fivefold by glucose and five- and sixfold over GSIS by palmitate and exendin-4, respectively. Although parental HFD increased blood glucose in WT P6-offspring, it did not change insulin secretion from WT P6-islets. In contrast, parental HFD abolished glucose responsiveness (i.e. GSIS) in Ffar1-/- P6-islets. Inhibition of Gq by FR900359 or YM-254890 in WT P6-islets mimicked the effect of Ffar1 deletion, i.e. suppression of GSIS and of palmitate-augmented GSIS. The blockage of Gi/o by pertussis toxin (PTX) enhanced (100-fold) GSIS in WT P6-islets and rendered Ffar1-/- P6-islets glucose responsive, suggesting constitutive activation of Gi/o. In WT P6-islets, FR900359 cancelled 90% of PTX-mediated stimulation, while in Ffar1-/- P6-islets it completely abolished PTX-elevated GSIS. The secretory defect of Ffar1-/- P6-islets did not originate from insufficient beta cells, since beta cell mass increased with the offspring's age irrespective of genotype and diet. In spite of that, in the breastfed offspring (i.e. P1-P11) beta cell proliferation and pancreatic insulin content had a genotype- and diet-driven dynamic. Under CD, the highest proliferation rate was reached by the Ffar1-/- P6 offspring (3.95% vs 1.88% in WT P6), whose islets also showed increased mRNA levels of genes (e.g. Fos, Egr1, Jun) typically high in immature beta cells. Although parental HFD increased beta cell proliferation in both WT (4.48%) and Ffar1-/- (5.19%) P11 offspring, only the WT offspring significantly increased their pancreatic insulin content upon parental HFD (5.18 µg under CD to 16.93 µg under HFD). CONCLUSIONS/INTERPRETATION: FFA1 promotes glucose-responsive insulin secretion and functional maturation of newborn islets and is required for adaptive offspring insulin secretion in the face of metabolic challenge, such as parental HFD.

Structure-Activity Relationship Explorations and Discovery of a Potent Antagonist for the Free Fatty Acid Receptor 2.

Free fatty acid receptor 2 (FFA2) is a sensor for short-chain fatty acids that has been identified as an interesting potential drug target for treatment of metabolic and inflammatory diseases. Although several ligand series are known for the receptor, there is still a need for improved compounds. One of the most potent and frequently used antagonists is the amide-substituted phenylbutanoic acid known as CATPB (1). We here report the structure-activity relationship exploration of this compound, leading to the identification of homologues with increased potency. The preferred compound 37 (TUG-1958) was found, besides improved potency, to have high solubility and favorable pharmacokinetic properties.

FFA2-, but not FFA3-agonists inhibit GSIS of human pseudoislets: a comparative study with mouse islets and rat INS-1E cells.

The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2 > FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3 > > Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.

Discovery of a Potent Thiazolidine Free Fatty Acid Receptor 2 Agonist with Favorable Pharmacokinetic Properties.

Free fatty acid receptor 2 (FFA2/GPR43) is a receptor for short-chain fatty acids reported to be involved in regulation of metabolism, appetite, fat accumulation, and inflammatory responses and is a potential target for treatment of various inflammatory and metabolic diseases. By bioisosteric replacement of the central pyrrolidine core of a previously disclosed FFA2 agonist with a synthetically more tractable thiazolidine, we were able to rapidly synthesize and screen analogues modified at both the 2- and 3-positions on the thiazolidine core. Herein, we report SAR exploration of thiazolidine FFA2 agonists and the identification of 31 (TUG-1375), a compound with significantly increased potency (7-fold in a cAMP assay) and reduced lipophilicity (50-fold reduced clog P) relative to the pyrrolidine lead structure. The compound has high solubility, high chemical, microsomal, and hepatocyte stability, and favorable pharmacokinetic properties and was confirmed to induce human neutrophil mobilization and to inhibit lipolysis in murine adipocytes.

Dihydropyridine Fluorophores Allow for Specific Detection of Human Antibodies in Serum.

Antigen recognition by antibodies plays an important role in human biology and in the development of diseases. This interaction provides a basis for multiple diagnostic assays and is a guide for treatments. We have developed dihydropyridine-based fluorophores that form stable complexes with double-stranded DNA and upon recognition of the antibodies to DNA (anti-DNA) provide an optical response. The fluorophores described herein have advantageous optical properties compared to those of the currently available dyes making them valuable for research and clinical diagnostics. By studying a series of novel fluorophores, crucial parameters for the design were established, providing the required sensitivity and specificity in the detection of antibodies. Using these DNA-fluorophore complexes in a direct immunofluorescence assay, antibodies to DNA are specifically detected in 80 patients diagnosed with an autoimmune disease, systemic lupus erythematosus. Positivity indicated by emission change of α-(4'-O-methoxyphenyl)-2-furyl dihydropyridine strongly correlates with other disease biomarkers and autoimmune arthritis.

Structure-Activity Investigations and Optimisations of Non-metabolite Agonists for the Succinate Receptor 1.

The succinate receptor 1 (SUCNR1) is a receptor for the metabolite succinate, which functions as a metabolic stress signal in the liver, kidney, adipose tissue and the retina. However, potent non-metabolite tool compounds are needed to reveal the physiological role and pharmacological potential of SUCNR1. Recently, we published the discovery of a computationally receptor-structure derived non-metabolite SUCNR1 agonist series with high target selectivity. We here report our structure-activity exploration and optimisation that has resulted in the development of agonists with nanomolar potency and excellent solubility and stability properties in a number of in vitro assays. Ligand-guided receptor models with high discriminative power between binding of active and inactive compounds were developed for design of novel chemotypes.

Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91.

OBJECTIVE: Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. METHODS AND RESULTS: Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase. CONCLUSIONS: These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy.