Discovery of pyrazolo[3,4-d]pyrimidine derivatives as GPR119 agonists

We designed and synthesized a novel series of phenylamino- and phenoxy-substituted pyrazolo[3,4-d]pyrimidine derivatives as GPR119 agonists. SAR studies indicated that electron-withdrawing substituents on the phenyl ring are important for potency and full efficacy. Compound 26 combined good potency with a promising pharmacokinetic profile in mice, and lowered the glucose excursion in mice in an oral glucose-tolerance test.     

Discovery of 3-aryl-3-ethoxypropanoic acids as orally active GPR40 agonists

The G protein-coupled receptor 40 (GPR40) mediates enhancement of glucose-stimulated insulin secretion in pancreatic β cells. The GPR40 agonist has been attracting attention as a novel insulin secretagogue with glucose dependency for the treatment of type 2 diabetes. The optimization study of compound 1 led to a potent and bioavailable GPR40 agonist 24, which showed insulin secretion and glucose lowering effects in rat OGTT. Compound 24 is a potential lead compound for a novel insulin secretagogue with a low risk of hypoglycemia.     

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

1. Download : Download high-res image (148KB)
2. Download : Download full-size image

     

Mechano- and pH-sensing convergence on Ca2+-mobilising proteins – A recipe for cancer?

Alterations in the (bio)chemical and physical microenvironment of cells accompany and often promote disease formation and progression. This is particularly well established for solid cancers, which are typically stiffer than the healthy tissue in which they arise, and often display profound acidification of their interstitial fluid. Cell surface receptors can sense changes in the mechanical and (bio)chemical properties of the surrounding extracellular matrix and fluid, and signalling through these receptors is thought to play a key role in disease development and advancement. This review will look at ion channels and G protein coupled receptors that are activated by mechanical cues and extracellular acidosis, and stimulation of which results in increases in intracellular Ca²⁺ concentrations. Cellular Ca²⁺ levels are dysregulated in cancer as well as cancer-associated cells, and mechano- and proton-sensing proteins likely contribute to these aberrant intracellular Ca²⁺ signals, making them attractive targets for therapeutic intervention.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

人GPR81-Gi1α融合蛋白在杆状病毒系统中的表达及优化

旨在建立获得融合蛋白GPR81-Gi1α的方法和最优条件。采用RT-PCR从人胚胎肾及大脑组织总RNA中分别扩增孤儿G蛋白偶联受体GPR81和Gi1α的完整表达序列(分别为1 041bp和1 065bp),并构建各自的重组质粒pcDNA3.1(+)-GPR81及pcDNA3.1(+)-Gi1α,再以重组质粒为模板,运用重叠延伸PCR法扩增得到融合基因GPR81-Gi1α,测序无误后将融合基因与pFASTBac1重组得重组质粒pFASTBac1-GPR81-Gi1α,而后转化DH10Bac,使该融合基因重组质粒发生特异性的转座和病毒重组,获得杆状病毒表达穿梭质粒pBacmid-GPR81- Ci1α,再将该重组杆粒转染昆虫sf9细胞,获得含杆状病毒的细胞分泌上清,以上清在不同条件下(包括不同感染时间、滴度等)感染sf9细胞以优化融合蛋白在昆虫sf9细胞的表达条件。结果表明,感染72h且感染强度moi为5时是融合蛋白在sf9细胞中高效表达的理想条件。该表达体系的建立及蛋白表达条件的优化,保证了足量GPR81- Gi1α融合蛋白的制备。     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Low-conductance chloride channel activated by cAMP in the epithelial cell line T84

We have studied the modulation and pharmacological properties of two anion channels in T₈₄ cells by recording single channel and transepithelial currents. One channel had an outwardly rectifying current-voltage I/V curve, was rarely active in cell-attached patches, and was unaffected by cAMP. The other channel had lower conductance (8.7 pS at 37°C) and a more ohmic I/V relationship. Exposure to cAMP increased the probability of observing low-conductance channel activity in cell-attached patches> 6-fold. Extracellular DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or IAA-94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not affect the low-conductance channel or cAMP-stimulated transepithelial current. These results suggest the low-conductance Cl channel may contribute to apical membrane conductance during cAMP-stimulated secretion.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.