A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.     

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC₅₀ values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.     

新型冠状病毒抗体和小分子抑制剂的研究现状

世界卫生组织已宣布新型冠状病毒感染（coronavirus disease 2019,COVID-19）的爆发为全球大流行。中和抗体和小分子抑制剂在预防及治疗COVID-19中发挥重要作用。尽管已开发出了多种中和抗体以及疫苗,但是随着病原体严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的不断变异,现有的抗体及疫苗面临巨大的挑战。小分子抑制剂主要通过干扰病毒与宿主的结合以及病毒自身的复制达到消灭病毒以及抑制病毒感染的作用,并且对SARS-CoV-2突变株具有广谱抑制作用,是当前研究的热点。近年来国内外学者对SARS-CoV-2的小分子抑制剂做了大量的研究工作,本文根据中和抗体识别的抗原表位以及小分子抑制剂的作用机制分别对用于预防及治疗COVID-19的中和抗体和小分子抑制剂进行综述,讨论其研究现状,并展望小分子抑制剂的应用前景,以期为该领域的进一步研究提供参考。     

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.     

Recombinant TLR5 Agonist CBLB502 Promotes NK Cell-Mediated Anti-CMV Immunity in Mice

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.     

Small Molecule Agonist of Very Late Antigen-4 (VLA-4) Integrin Induces Progenitor Cell Adhesion

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4β1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4β1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4β1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/β subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4β7 as well as α5β1 and αLβ2. When cross-linked to αLβ2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/β subunit interface and not at the ligand-binding site in the inserted (“I”) domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.     

Mechanistic Evaluation of a Novel Small Molecule Targeting Mitochondria in Pancreatic Cancer Cells

Background

Pancreatic cancer is one of the deadliest cancers with a 5-year survival rate of 6%. Therapeutic options are very limited and there is an unmet medical need for safe and efficacious treatments. Cancer cell metabolism and mitochondria provide unexplored targets for this disease. We recently identified a novel class of triphenylphosphonium salts, TP compounds, with broad- spectrum anticancer properties. We examined the ability of our prototypical compound TP421– chosen for its fluorescent properties – to inhibit the growth of pancreatic cancer cells and further investigated the molecular mechanisms by which it exerts its anticancer effects.

Methodology/Principal Findings

TP421 exhibited sub-micromolar IC₅₀ values in all the pancreatic cancer cell lines tested using MTT and colony formation assays. TP421 localized predominantly to mitochondria and induced G₀/G₁ arrest, ROS accumulation, and activation of several stress-regulated kinases. Caspase and PARP-1 cleavage were observed indicating an apoptotic response while LC3B-II and p62 were accumulated indicating inhibition of autophagy. Furthermore, TP421 induced de-phosphorylation of key signaling molecules involved in FAK mediated adhesion that correlated with inhibition of cell migration.

Conclusions/Significance

TP421 is a representative compound of a new promising class of mitochondrial-targeted agents useful for pancreatic cancer treatment. Because of their unique mechanism of action and efficacy further development is warranted.     

Cooperative Anti-Diabetic Effects of Deoxynojirimycin-Polysaccharide by Inhibiting Glucose Absorption and Modulating Glucose Metabolism in Streptozotocin-Induced Diabetic Mice

We had previously shown that deoxynojirimycin-polysaccharide mixture (DPM) not only decreased blood glucose but also reversed the damage to pancreatic β-cells in diabetic mice, and that the anti-hyperglycemic efficacy of this combination was better than that of 1-deoxynojirimycin (DNJ) or polysachharide alone. However, the mechanisms behind these effects were not fully understood. The present study aimed to evaluate the therapeutic effects of DPM on streptozotocin (STZ)-induced diabetic symptoms and their potential mechanisms. Diabetic mice were treated with DPM (150 mg/kg body weight) for 90 days and continued to be fed without DPM for an additional 30 days. Strikingly, decrease of blood glucose levels was observed in all DPM treated diabetic mice, which persisted 30 days after cessation of DPM administration. Significant decrease of glycosylated hemoglobin and hepatic pyruvate concentrations, along with marked increase of serum insulin and hepatic glycogen levels were detected in DPM treated diabetic mice. Results of a labeled ¹³C₆-glucose uptake assay indicated that DPM can restrain glucose absorption. Additionally, DPM down-regulated the mRNA and protein expression of jejunal Na⁺/glucose cotransporter, Na⁺/K⁺-ATPase and glucose transporter 2, and enhanced the activities as well as mRNA and protein levels of hepatic glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylas E1). Activity and expression of hepatic gluconeogenesis enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) were also found to be attenuated in diabetic mice treated with DPM. Purified enzyme activity assays verified that the increased activities of glucose glycolysis enzymes resulted not from their direct activation, but from the relative increase in protein expression. Importantly, our histopathological observations support the results of our biochemical analyses and validate the protective effects of DPM on STZ-induced damage to the pancreas. Thus, DPM has significant potential as a therapeutic agent against diabetes.     

Anti-Tumor Activity of a Novel HS-Mimetic-Vascular Endothelial Growth Factor Binding Small Molecule

The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF) pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl)-3H-imidazole-4-carbaldehyde) was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS), which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7) which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor.     

胆汁酸G-蛋白偶联受体激动剂齐墩果酸对高脂饮食小鼠体内糖脂代谢的影响

目的观察胆汁酸G-蛋白偶联受体（Gprotein—coupled receptor for bile acids,TGR5）激动剂齐墩果酸（oleanolic acid,OA）对肥胖小鼠体重及糖、脂代谢的影响,探讨齐墩果酸减轻肥胖小鼠体重的机制。方法建立高脂饮食诱导的肥胖小鼠动物模型,并喂食OA进行干预。动态测定体重及第17周后内脏脂肪、肝脏重量,并进行葡萄糖耐量实验（glucose tolerence test,GTT）;肝脏组织石蜡切片HE染色,光镜观察病理变化;Realtime PCR检测肝脏组织糖代谢相关基因的表达及白色脂肪组织脂肪合成酶（fatty acid synthase,FAS）的表达。结果OA减轻肥胖小鼠的体重、内脏脂肪及肝脏的重量;改善肝脏脂质沉积;增强胰岛素敏感性。OA抑制肝脏内葡萄糖-6-磷酸酶（glucose-6-phosphatase,G6Pc）的表达,并下调肥胖小鼠脂肪组织FAS的mRNA水平的表达。结论TGR5激动剂OA能减少高脂诱导的肥胖小鼠的脂肪堆积,其机制可能与OA能改善肥胖小鼠胰岛素抵抗,减少脂质合成有关。     

Dual Activation of the Bile Acid Nuclear Receptor FXR and G-Protein-Coupled Receptor TGR5 Protects Mice against Atherosclerosis

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE^−/− mice and LDLR^−/− mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE^−/− and LDLR^−/− mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE^−/− mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.     

Blockade but Not Overexpression of the Junctional Adhesion Molecule C Influences Virus-Induced Type 1 Diabetes in Mice

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.     

一种新型PPARr激动剂对人肾癌细胞增殖抑制的实验研究

目的：探讨新型过氧化物酶体增殖激活物受体(PPARr)激动剂DH9 对人肾癌细胞OS-RC-2 的增殖抑制作用。方法：予以不 同浓度的DH9 及罗格列酮作用OS-RC-2 细胞12 h、24 h和48 h,荧光素酶活性检测比较两种药物的PPARr激动效应;MTT 法 检测细胞增殖情况;流式细胞术观察细胞周期;AnnexinV-FITC/PI双染色流式细胞术测定细胞凋亡率;Western blot 检测细胞内 Bax 及Bcl-2等蛋白的变化。结果：不同浓度的DH9 与罗格列酮相比,对PPARr的激动效应DH9明显低于罗格列酮,增殖抑制 作用优于罗格列酮(P＜0.05),并呈现明显的浓度、时间依赖性;加入PPARr抑制剂GW9662 前后DH9 的增殖抑制作用差异无统 计学意义(P＞0.05);DH9 作用细胞48小时后,G0/G1 期细胞比例明显增加(P＜0.05),S期细胞明显减少(P＜0.05)。DH9可诱导细 胞凋亡,伴随Bcl-2 表达的减少以及Bax表达的增加。结论：OS-RC-2 细胞中,DH9 的增殖作用明显优于罗格列酮,且是通过 PPARr非依赖途径实现;DH9 能将OS-RC-2 细胞阻滞在G0/G1 期,并通过影响Bcl-2 和Bax 蛋白表达促进细胞凋亡。     

Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability

NADH cytochrome b₅ reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC₅₀ = ∼275 μm), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC₅₀ = 10.81 μm) and ZINC39395747 (IC₅₀ = 9.14 μm), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development.     

Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism

ATP-sensitive potassium (K_ATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant K_ATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct K_ATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited K_ATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the K_ATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant K_ATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore K_ATP channel expression and function in a subset of congenital hyperinsulinism patients.     

Effects of Small Molecule Modulators on ATP Binding to Skeletal Ryanodine Receptor

Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca²⁺-channel and is strongly affected by intrinsic modulators like Ca²⁺, Mg²⁺ and ATP. We showed differential effects on ATP binding in the presence of Ca²⁺ or Mg²⁺ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408–9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca²⁺, suggestive of a stable “open” channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca²⁺, while in the presence of inhibitory Ca²⁺, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.     

Indirect Effects of Glucagon-Like Peptide-1 Receptor Agonist Exendin-4 on the Peripheral Circadian Clocks in Mice

Circadian clocks in peripheral tissues are powerfully entrained by feeding. The mechanisms underlying this food entrainment remain unclear, although various humoral and neural factors have been reported to affect peripheral clocks. Because glucagon-like peptide-1 (GLP-1), which is rapidly secreted in response to food ingestion, influences multiple humoral and neural signaling pathways, we suggest that GLP-1 plays a role in the food entrainment of peripheral clocks. To test this, we compared the effects of exendin-4, a GLP-1 receptor agonist, on mRNA expression of the clock genes (Clock, Bmal1, Nr1d1, Per1, Per2, and Cry1) with those of refeeding. In addition, we investigated whether exendin-4 could affect the rhythms of the peripheral clocks. In male C57BL/6J mice, although refeeding rapidly (within 2 h) altered mRNA levels of Per1 and Per2 in the liver and that of Per1 in adipose tissue, a single i.p. injection of exendin-4 did not cause such changes. However, unlike the GLP-1 receptor antagonist exendin-(9–39), exendin-4 significantly influenced Per1 mRNA levels in the liver at 12 h after injection. Moreover, pretreatment with exendin-4 affected the rapid-feeding-induced change in Per1 not only in the liver, but also in adipose tissue, without effect on food intake. Furthermore, during light-phase restricted feeding, repeated dosing of exendin-4 at the beginning of the dark phase profoundly influenced both the food intake and daily rhythms of clock gene expression in peripheral tissues. Thus, these results suggest that exendin-4 modulates peripheral clocks via multiple mechanisms different from those of refeeding.