Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly's drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC50=55nM) was reached. Four lead compounds (EC50 range 55-410nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F=10.3%) and plasma levels achieved on oral administration.

Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric Modulator

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu(173) or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [S-35] guanosine 5'-(3-O-thio) triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized alpha-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized alpha-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.

Prediction of adipose tissue composition using Raman spectroscopy: average properties and individual fatty acids.

Abstract: Raman spectroscopy has been used for the first time to predict the FA composition of unextracted adipose tissue of pork, beef, lamb, and chicken. It was found that the bulk unsaturation parameters could be predicted successfully [R-2 = 0.97, root mean square error of prediction (RMSEP) = 4.6% of 4 sigma], with cis unsaturation, which accounted for the majority of the unsaturation, giving similar correlations. The combined abundance of all measured PUFA (>= 2 double bonds per chain) was also well predicted with R-2 = 0.97 and RMSEP = 4.0% of 4 sigma. Trans unsaturation was not as well modeled (R-2 = 0.52, RMSEP = 18% of 4 sigma); this reduced prediction ability can be attributed to the low levels of trans FA found in adipose tissue (0.035 times the cis unsaturation level). For the individual FA, the average partial least squares (PLS) regression coefficient of the 18 most abundant FA (relative abundances ranging from 0.1 to 38.6% of the total FA content) was R-2 = 0.73; the average RMSEP = 11.9% of 4 sigma. Regression coefficients and prediction errors for the five most abundant FA were all better than the average value (in some cases as low as RMSEP = 4.7% of 4 sigma). Cross-correlation between the abundances of the minor FA and more abundant acids could be determined by principal component analysis methods, and the resulting groups of correlated compounds were also well-predicted using PLS. The accuracy of the prediction of individual FA was at least as good as other spectroscopic methods, and the extremely straightforward sampling method meant that very rapid analysis of samples at ambient temperature was easily achieved. This work shows that Raman profiling of hundreds of samples per day is easily achievable with an automated sampling system.

Discovery of novel Agonists and antagonists of the free fatty acid receptor 1 (FFAR1) using virtual screening

The G-protein-coupled receptor free fatty acid receptor 1 (FFAR1), previously named GPR40, is a possible novel target for the treatment of type 2 diabetes. In an attempt to identify new ligands for this receptor, we performed virtual screening (VS) based on two-dimensional (2D) similarity, three-dimensional (3D) pharmacophore searches, and docking studies by using the structure of known agonists and our model of the ligand binding site, which was validated by mutagenesis. VS of a database of 2.6 million compounds followed by extraction of structural neighbors of functionally confirmed hits resulted in identification of 15 compounds active at FFAR1 either as full agonists, partial agonists, or pure antagonists. Site-directed mutagenesis and docking studies revealed different patterns of ligand-receptor interactions and provided important information on the role of specific amino acids in binding and activation of FFAR1.

Acute and chronic effects of dietary fatty acids on cholecystokinin expression, storage and secretion in enteroendocrine STC-1 cells

Cholecystokinin (CCK) is a peptide hormone secreted from the I-cells of the intestine and it has important physiological actions related to appetite regulation and satiety. In this study we used STC-1 cells to investigate the effects of common dietary-derived fatty acids (FAs) on I-cell secretory function and metabolism. We extend earlier studies by measuring the acute and chronic effects of 11 FAs on CCK secretion, cellular CCK content, CCK mRNA levels, cellular DNA synthesis, cellular viability and cytotoxicity. FAs were selected in order to assess the importance of chain length, degree of saturation, and double bond position and conformation. The results demonstrate that secretory responses elicited by dietary FAs are highly selective. For example, altering the conformation of a double bond from cis to trans (i.e. oleic acid versus elaidic acid) completely abolishes CCK secretion. Lauric acid appears to adversely affect I-cell metabolism and arachidonic acid suppresses DNA synthesis. Our studies reveal for the first time that conjugated linoleic acid isoforms are particularly potent CCK secretagogues, which also boost intracellular stores of CCK. These actions of conjugated linoleic acid may explain satiating actions observed in dietary intervention studies.

Synthesis and in Vitro and in Vivo Evaluation of a Series of Dihydroisocoumarin Derivatives Conjugated with Fatty Acids, Alcohols, and Amines as Potential Anticancer Agents

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues Conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to (he dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds Conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1.3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo ill a number of human tumor xenograft models.

Antimicrobial Efficacy of an Innovative Emulsion of Medium Chain Triglycerides against Canine and Feline Periodontopathogens

OBJECTIVES: To test the in vitro antimicrobial efficacy of a non-toxic emulsion of free fatty acids against clinically relevant canine and feline periodontopathogens 
METHODS: Antimicrobial kill kinetics were established utilising an alamarBlue® viability assay against 10 species of canine and feline periodontopathogens in the biofilm mode of growth at a concentration of 0·125% v/v medium chain triglyceride (ML:8) emulsion. The results were compared with 0·12% v/v chlorhexidine digluconate and a xylitol-containing dental formulation. Mammalian cellular cytotoxicity was also investigated for both the ML:8 emulsion and chlorhexidine digluconate (0·25 to 0·0625% v/v) using in vitro tissue culture techniques.
RESULTS: No statistically significant difference was observed in the antimicrobial activity of the ML:8 emulsion and chlorhexidine digluconate; a high percentage kill rate (>70%) was achieved within 5 minutes of exposure and was maintained at subsequent time points. A statistically significant improvement in antibiofilm activity was observed with the ML:8 emulsion compared with the xylitol-containing formulation. The ML:8 emulsion possessed a significantly lower (P<0·001) toxicity profile compared with the chlorhexidine digluconate in mammalian cellular cytotoxicity assays.
CLINICAL SIGNIFICANCE: The ML:8 emulsion exhibited significant potential as a putative effective antimi-crobial alternative to chlorhexidine- and xylitol- based products for the reduction of canine and feline periodontopathogens.

A randomised interventional trial of ω-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus

Objective: To determine the clinical effect of dietary supplementation with low-dose ?-3-polyunsaturated fatty acids on disease activity and endothelial function in patients with systemic lupus erythematosus. Methods: A 24-week randomised double-blind placebo-controlled parallel trial of the effect of 3 g of ?-3-polyunsaturated fatty acids on 60 patients with systemic lupus erythematosus was performed. Serial measurements of disease activity using the revised Systemic Lupus Activity Measure (SLAM-R) and British Isles Lupus Assessment Group index of disease activity for systemic lupus erythematosus (BILAG), endothelial function using flow-mediated dilation (FMD) of the brachial artery, oxidative stress using platelet 8-isoprostanes and analysis of platelet membrane fatty acids were taken at baseline, 12 and 24 weeks. Results: In the fish oil group there was a significant improvement at 24 weeks in SLAM-R (from 9.4 (SD 3.0) to 6.3 (2.5), p

Small-angle scattering from long-chain alkylimidazolium-based ionic liquids

This paper compares the structure of 1-alkyl-3-methylim ridazolium salts using SAXS and X-ray reflectivity. A range of anions have been investigated namely chloride, bromide, trifluoromethanesulfonate (OTf), bis(trifluoromethanesulfonyl)imide (TFI) and tetrachloropalladate(II) with cation alkyl chains ranging from n = 12-20. In general, the salts show liquid crystalline behaviour whose structure is still observed on melting into an isotropic liquid.