Homology modeling and explicit membrane molecular dynamics simulation to delineate the mode of binding of thiazolidinediones into FFAR1 and the mechanism of receptor activation

Free fatty acid receptor 1 (FFAR1) is a member of a previously characterized cluster of orphan G protein-coupled receptors (GPCRs). Later, this orphan receptor was identified as a target of medium- to long-chain free fatty acids in β-cells of the pancreas. Administration of FFAR1 agonists has been proved to potentiate glucose-stimulated insulin secretion from pancreatic β-cells. It was reported that some thiazolidinediones (TZDs), the best studied PPARγ agonists, are also able to stimulate FFAR1 in a dose-dependent manner. In the present study, a homology model of the human FFAR1 was constructed and inserted into a pre-equilibrated DPPC/TIP3P membrane system. This system was then simulated for 20 ns in complex with the FFAR1 agonist GW9085, as well as rosiglitazone and pioglitazone. We noticed that the salt bridge between Glu172 and Arg258 and the H bond between Glu145 and His153 could be responsible for the stabilization of the receptor in the inactive state. Moreover, we described for the first time the binding mode of TZDs in the binding site of FFAR1. The thiazolidinedione head forms a hydrogen bonding network with the critical polar residues in the binding site, Arg258 and Asn244, while the rest of the molecule is embedded into the receptor hydrophobic pocket. Based on this modeling study, we arrived at a proposal of the pharmacophore required for binding to both PPARγ and FFAR1. Insights gained from this investigation should provide future directions for the design of novel dual acting antidiabetic agents.     

Receptor-specific regulation of ERK1/2 activation by members of the “free fatty acid receptor” family

Context: The “free fatty acid receptors” (FFARs) GPR40, GPR41, and GPR43 regulate various physiological homeostases, and are all linked to activation of extracellular signal-regulated kinases (ERK)1/2.

Objective: Investigation of coupling of FFARs to two other mitogen-activated protein kinases (MAPKs) sometimes regulated by G protein-coupled receptors (GPCRs), c-Jun N-terminal kinase (JNK) and p38MAPK, and characterization of signaling proteins involved in the regulation of FFAR-mediated ERK1/2 activation.

Methods: FFARs were recombinantly expressed, cells challenged with the respective agonist, and MAPK activation quantitatively determined using an AlphaScreen SureFire assay. Inhibitors for signaling proteins were utilized to characterize ERK1/2 pathways.

Results: Propionate-stimulated GPR41 strongly coupled to ERK1/2 activation, while the coupling of linoleic acid-activated GPR40 and acetate-activated GPR43 was weaker. JNK and p38MAPK were weakly activated by FFARs. All three receptors activated ERK1/2 fully or partially via G_i/o and Rac. PI3K was relevant for GPR40- and GPR41-mediated ERK1/2 activation, and Src was essential for GPR40- and GPR43-induced activation. Raf-1 was not involved in the GPR43-triggered activation.

Conclusion: The results demonstrate a novel role of Rac in GPCR-mediated ERK1/2 signaling, and that GPCRs belonging to the same family can regulate ERK1/2 activation by different receptor-specific mechanisms.     

Identification of free fatty acid receptor 2 agonists using virtual screening

Structure- and ligand-based virtual-screening methods (docking, 2D- and 3D-similarity searching) were analyzed for their effectiveness in virtual screening against FFAR2. To evaluate the performance of these methods, retrospective virtual screening was performed. Statistical quality of the methods was evaluated by BEDROC and RIE. The results revealed that electrostatic similarity search protocol using EON (ET combo) outperformed all other protocols with outstanding enrichment of >95% in top 1% and 2% of the dataset with an AUC of 0.958. Interestingly, the hit lists that are obtained from different virtual-screening methods are generally highly complementary to hits found from electrostatic similarity searching. These results suggest that considering electrostatic similarity searching first increases the chance of identifying more (and more diverse) active compounds from a virtual-screening campaign. Accordingly, prospective virtual screening using electrostatic similarity searching was used to identify novel FFAR2 ligands. The discovered compounds provide new chemical matter starting points for the initiation of a medicinal chemistry campaign.     

Synthesis,biological evaluation,and molecular docking investigation of benzhydrol- and indole-based dual PPAR-γ/FFAR1 agonists

Type-2 diabetes mellitus is a progressive cluster of metabolic disorders, representing a global public health burden affecting more than 366?million people worldwide. We recently reported the discovery of three series of novel agents showing balanced activity on two metabolic receptors, peroxisome proliferator activated receptor-γ (PPAR-γ) and free fatty acid receptor 1 (FFAR1), also known as GPCR40. Our designing strategy relied on linking the thiazolidinedione head with known GPCR privilege structures. To further investigate this concept, two new scaffolds, the benzhydrol- and indole-based chemotypes, were introduced here in. Our optimization campaign resulted in three compounds; 15a, 15c, and 15d, with affinities in the low micromolar range on both targets. In vivo study of selected test compounds, revealed that 15c possesses a significant anti-hyperglycemic and anti-hyperlipidemic activities superior to rosiglitazone in fat-fed animal models. Molecular docking analysis was conducted to explain the binding modes of both series. These compounds could lead to the development of the unique antidiabetic agent acting as insulin sensitizer as well as insulin secretagogue.     

Discovery of N-arylpyrroles as agonists of GPR120 for the treatment of type II diabetes

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.     

Production and characterization of a monoclonal antibody against GPR40 (FFAR1; free fatty acid receptor 1)

GPR40 is G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs), and it has been implicated to play an important role in FFA-mediated enhancement of glucose-stimulated insulin release. We have developed a monoclonal antibody against the extracellular domain of GPR40. Specificity of the antibody was demonstrated by immunoprecipitation and cell surface staining using GPR40-transfected cells. GPR40 immunoreactivity was highly abundant in mouse pancreatic β-cells and splenocytes, THP-1 cells, and human peripheral blood mononuclear cells. The anti-GPR40 monoclonal antibody should prove valuable for further studying the function of this nutrient sensing receptor.     

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.     

Featured Article: Deficiency of G-protein coupled receptor 40, a lipid-activated receptor,heightens in?vitro- and in?vivo-induced murine osteoarthritis

Osteoarthritis (OA) is an age-related degenerative joint disease. To date, its management is focused on symptoms (pain and inflammation). Studies suggest that fatty acids can reduce the expression of inflammatory and catalytic mediators, and improve in vivo joint function. Free fatty acid receptors (FFARs) such as G-protein coupled receptor 40 (GPR40) are proposed as attractive therapeutic targets to counteract inflammation and cartilage degradation observed in OA. This study aims to elucidate the involvement of GPR40 in OA. In this study, we used an in vitro model of OA, and surgically induced OA by ligament transection and partial meniscectomy in wild-type and GPR40 deficient mice. OA phenotype was investigated in vivo by histology and genes expression. We demonstrate that IL-1β-treated GPR40^−/− chondrocytes secret more inflammatory mediators (nitric oxide, interleukin-6, prostaglandin E2) and active catabolic enzymes (metalloproteinase-2, -9 [MMP-2, MMP-9]), and show decreased anabolism (glycoaminoglycan) compared to GPR40^+/+ cells. In accordance with these results, we show that GPR40^−/− mice exhibit an aggravated OA-induced phenotype characterized by higher tidemark exposure, frequency of osteophyte formation and subchondral bone sclerosis. Altogether our results demonstrate that GPR40 deficiency leads to an extended OA phenotype, providing evidence that increasing GPR40 activity, by natural or synthetic ligands, could be a new strategy in the management of OA.     

Generating thermostabilized agonist-bound GPR40/FFAR1 using virus-like particles and a label-free binding assay

Elucidating the detailed mechanism of activation of membrane protein receptors and their ligand binding is essential for structure-based drug design. Membrane protein crystal structure analysis successfully aids in understanding these fundamental molecular interactions. However, protein crystal structure analysis of the G-protein-coupled receptor (GPCR) remains challenging, even for the class of GPCRs which have been included in the majority of structure analysis reports among membrane proteins, due to the substantial instability of these receptors when extracted from lipid bilayer membranes. It is known that increased thermostability tends to decrease conformational flexibility, which contributes to the generation of diffraction quality crystals. However, this is still not straightforward, and significant effort is required to identify thermostabilized mutants that are optimal for crystallography. To address this issue, a versatile screening platform based on a label-free ligand binding assay combined with transient overexpression in virus-like particles was developed. This platform was used to generate thermostabilized GPR40 [also known as free fatty acid receptor 1 (FFAR1)] for fasiglifam (TAK-875). This demonstrated that the thermostabilized mutant GPR40 (L42A/F88A/G103A/Y202F) was successfully used for crystal structure analysis.     

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 [(35)S]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 α-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized α-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.     

β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins G_q/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of G_q/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of G_q activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to G_q/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.     

Effects of short chain fatty acid producing bacteria on epigenetic regulation of FFAR3 in type 2 diabetes and obesity

The human gut microbiota and microbial influences on lipid and glucose metabolism, satiety, and chronic low-grade inflammation are known to be involved in metabolic syndrome. Fermentation end products, especially short chain fatty acids, are believed to engage the epigenetic regulation of inflammatory reactions via FFARs (free fatty acid receptor) and other short chain fatty acid receptors.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。