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GPR40/FFAR1 is a G-protein-coupled receptor expressed in pancreatic β-cells and enteroendocrine cells. GPR40 activation stimulates secretions of insulin and incretin, both of which are the pivotal regulators of glycemic control. Therefore, a GPR40 agonist is an attractive target for the treatment of type 2 diabetes mellitus. Using the reported biaryl derivative 1, we shifted the hydrophobic moiety to the terminal aryl ring and replaced the central aryl ring with piperidine, generating 2-(4,4-dimethylpentyl)phenyl piperidine 4a, which had improved potency for GPR40 and high lipophilicity. We replaced the hydrophobic moiety with N-alkyl-N-aryl benzamides to lower the lipophilicity and restrict the N-alkyl moieties to the presumed lipophilic pocket using the intramolecular π-π stacking of cis-preferential N-alkyl-N-aryl benzamide. Among these, orally available (3S)-3-cyclopropyl-3-(2-((1-(2-((2,2-dimethylpropyl)(6-methylpyridin-2-yl)carbamoyl)-5-methoxyphenyl)piperidin-4-yl)methoxy)pyridin-4-yl)propanoic acid (SCO-267) effectively stimulated insulin secretion and GLP-1 release and ameliorated glucose tolerance in diabetic rats via GPR40 full agonism.

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood&ndash, brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson&rsquo, s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

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.