Discovery of small molecule FLT3 inhibitors that are able to overcome drug-resistant mutations

Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3 inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L, respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC₅₀s (half maximal inhibitory concentrations) of 0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively. Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good lead compound and deserves further in-depth studies.     

Differential effects of central administration of relaxin‐3 on food intake and hypothalamic neuropeptides in male and female rats

Relaxin‐3 (RLN3) is an orexigenic neuropeptide that produces sex‐specific effects on food intake by stronger stimulation of feeding in female compared with male rats. This study determined which hypothalamic nuclei and associated neuropeptides may be involved in the sex‐specific orexigenic effects of RLN3. Relaxin‐3 (800 pmol) or vehicle was injected into the lateral ventricle of female and male rats. Food and water intake were measured after the first injection, and rats were euthanized after the second injection to determine the mRNA expression of the hypothalamic neuropeptides. Food but not water intake showed sex‐specific effects of RLN3. Stimulation of food intake by RLN3 was significantly higher in female than in male rats. No effect of RLN3 injection was found on c‐fos mRNA expression in the arcuate, dorsomedial and ventromedial hypothalamic nuclei. Increased c‐fos mRNA expression was observed in the paraventricular hypothalamic nucleus (PVN) in both sexes and in the lateral hypothalamic area (LHA) in female rats. Relaxin‐3 injections led to a sex‐nonspecific increase in the expression of oxytocin mRNA in the magnocellular PVN. Conversely, RLN3‐induced expression of anorexigenic neuropeptide arginine vasopressin (AVP) was significantly higher in the parvocellular PVN in male compared with female rats. Finally, RLN3 administration significantly increased the expression of orexin (ORX) mRNA in the LHA in female but not in male rats. Stronger expression of anorexigenic AVP in the PVN in male rats and increased expression of ORX in the LHA in female rats may contribute to stronger orexigenic effects of RLN3 in female rats compared with male rats.     

Effect of palmitoylated prolactin-releasing peptide on food intake and neural activation after different routes of peripheral administration in rats

Obesity is an escalating epidemic, but an effective non-invasive therapy is still scarce. For obesity treatment, anorexigenic neuropeptides are promising tools, but their delivery from the periphery to the brain is complicated by their peptide character. In order to overcome this unfavorable fact, we have applied the lipidization of neuropeptide prolactin-releasing peptide (PrRP), whose strong anorexigenic effect was demonstrated. A palmitoylated analog of human PrRP (h palm-PrRP31) was injected in free-fed Wistar rats by three routes: subcutaneous (s.c.), intraperitoneal (i.p) (both 5 mg/kg) and intravenous (i.v.) (from 0.01 to 0.5 mg/kg). We found a circulating compound in the blood after all three applications with the highest concentration after i.v. administration. This corresponds to the effect on food intake, which was also strongest after i.v. injection. Moreover, this is in agreement with the fact that the expression of c-Fos in specific brain regions involved in food intake regulation was also highest after intravenous application. Pharmacokinetic data are further supported by results obtained from dynamic light scattering and CD spectroscopy. Human palm-PrRP31 analog showed a strong tendency to micellize, and formation of aggregates suggested lower availability after i.p. or s.c. application. We have demonstrated that palm-PrRP influenced food intake even in free fed rats. Not surprisingly, the maximal effect was achieved after the intravenous application even though two orders of magnitude lower dose was used compared to both two other applications. We believe that palm-PrRP could have a potential as an antiobesity drug when its s.c. application would be improved.     

Central administration of the RFamide peptides, QRFP-26 and QRFP-43, increases high fat food intake in rats

Pyrogultamylated arginine-phenylalanineamide peptide (QRFP) is strongly conserved across species and is a member of the family of RFamide-related peptides, with the motif Arg-Phe-NH(2) at the C-terminal end. The precursor peptide for QRFP generates a 26-amino acid peptide (QRFP-26) and a 43-amino acid peptide (QRFP-43), both of which bind to the G protein-coupled receptor, GPR103. Recently, QRFP has been characterized in rats, mice and humans and has been reported to have orexigenic properties. In rodents, prepro-QRFP mRNA is expressed in localized regions of the mediobasal hypothalamus, a region implicated in feeding behavior. Increased intake of a high fat diet contributes to increased weight gain and obesity. Therefore, the current experiments investigated the effects of QRFP administration in rats and the effects of a high fat diet on prepro-QRFP mRNA and GPR103 receptor mRNA levels. Intracerebroventricular administration of QRFP-26 (3.0nM, 5.0nM) and QRFP-43 (1.0nM, 3.0nM) dose-dependently increased 1h, 2h, and 4h cumulative intake of high fat (55% fat), but not low fat (10% fat) diet. In Experiment 2, hypothalamic prepro-QRFP mRNA levels and GPR103 receptor mRNA levels were measured in rats fed a high fat or a low fat diet for 21 days. Prepro-QRFP mRNA was significantly increased in the ventromedial nucleus/arcuate nucleus of the hypothalamus of rats fed a high fat diet compared to those fed a low fat diet, while GPR103 mRNA levels were unchanged. These findings suggest that QRFP is a regulator of dietary fat intake and is influenced by the intake of a high fat diet.     

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.     

Intraportal administration of DPP-IV inhibitor regulates insulin secretion and food intake mediated by the hepatic vagal afferent nerve in rats

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and suppresses food intake. Recent studies indicate that the hepatic vagal afferent nerve is involved in this response. Dipeptidyl peptidase-IV (DPP-IV) inhibitor extends the half-life of endogenous GLP-1 by preventing its degradation. This study aimed to determine whether DPP-IV inhibitor-induced elevation of portal GLP-1 levels affect insulin secretion and feeding behavior via the vagal afferent nerve and hypothalamus. The effect of DPP-IV inhibitor infusion into the portal vein or peritoneum on portal and peripheral GLP-1 levels, food intake, and plasma insulin and glucose was examined in sham-operated and vagotomized male Sprague-Dawley rats. Analyses of neuronal histamine turnover and immunohistochemistry were used to identify the CNS pathway that mediated the response. Intraportal administration of the DPP-IV inhibitor significantly increased portal (but not peripheral) GLP-1 levels, increased insulin levels, and decreased glucose levels. The DPP-IV inhibitor suppressed 1- and 12- but not 24-h cumulative food intake. Intraportal infusion of the DPP-IV inhibitor increased hypothalamic neuronal histamine turnover and increased c-fos expression in several areas of the brain. These responses were blocked by vagotomy. Our results indicate that DPP-IV inhibitor-induced changes in portal but not systemic GLP-1 levels affect insulin secretion and food intake. Furthermore, our findings suggest that a neuronal pathway that includes the hepatic vagal afferent nerve and hypothalamic neuronal histamine plays an important role in the pharmacological actions of DPP-IV inhibitor.     

Discovery and pharmacological characterization of a novel small molecule inhibitor of phosphatidylinositol-5-phosphate 4-kinase,type II,beta

Phosphatidylinositol-5-phosphate 4-kinase, type II, beta (PIP5K2B) is linked to the pathogenesis of obesity, insulin resistance and diabetes. Here, we describe the identification of a novel pyrimidine-2,4-diamine PIP5K2B inhibitor, designated SAR088. The compound was identified by high-throughput screening and subsequently characterized in vitro and in vivo. SAR088 showed reasonable potency, selectivity and physicochemical properties in enzymatic and cellular assays. In vivo, SAR088 lowered blood glucose levels of obese and hyperglycemic male Zucker diabetic fatty rats treated for 3 weeks. Thus, SAR088 represents the first orally available and in vivo active PIP5K2B inhibitor and provides an excellent starting point for the development of potent and selective PIP5K2B inhibitors for the treatment of insulin resistance and diabetes.     

Discovery of thiazolidin-4-one urea analogues as novel multikinase inhibitors that potently inhibit FLT3 and VEGFR2

A series of novel thiazolidine-4-one urea analogues were designed, synthesized and biologically evaluated. The structure-activity relationship (SAR) at several positions of the scaffolds was investigated and its binding mode was analyzed by molecular modeling studies. Compound 17b proved to be the most potent one, and IC₅₀ values against A549 and HT-29 cancer cell lines were 0.65?μM and 0.11?μM, respectively. The results of kinase profile demonstrated that compound 17b is a multikinase inhibitor that potently inhibits FLT3 (IC₅₀?=?8.6?nM) and VEGFR2 (IC₅₀?=?18.7?nM). The results of real-time live-cell imaging indicated that compound 17b showed excellent cytotoxicity and anti-proliferative activity against HT-29 cancer cells in a time- and dose-dependent manner, which was significantly potent than that of Cabozantinib. In addition, in vitro antitumor activity was associated with inducing cancer cell apoptosis and suppression of cancer cell migration.     

The identification of GPR3 inverse agonist AF64394; The first small molecule inhibitor of GPR3 receptor function

The identification of the novel and selective GPR3 inverse agonist AF64394, the first small molecule inhibitor of GPR3 receptor function, is described. Structure activity relationships and syntheses based around AF64394 are reported.     

急、慢性运动对2型糖尿病大鼠脂肪PI3K/AKT/GLUT4通路的影响

目的:探讨急性和慢性运动对2型糖尿病(T2DM)大鼠脂肪组织明磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/葡萄糖运载体4(GLUT4)信号通路的影响。方法:15月龄SD雄性大鼠52只随机分为正常对照组(n=13)和高脂组(n=39),分别喂养普通和高脂饲料。8周后,高脂组体重>正常对照组20%,注射小剂量STZ后,血糖>16.7 mmol/l,造模成功。将糖尿病模型组随机分为糖尿病对照组(DC,n=13),糖尿病慢性运动组(DCE,n=13),糖尿病急性运动组(DAE,n=13)。DCE组进行8周的游泳运动,DAE组进行一次性游泳运动。测定血脂,血糖和血清胰岛素,Western blot法测定脂肪PI3K、AKT和GLUT4蛋白含量。结果:糖尿病组体重、血脂、血糖、胰岛素显著高于正常对照组(P均<0.01);高密度脂蛋白胆固醇(HDL-C)水平降低(P<0.05),脂肪组织中PI3K、AKT和GLUT4蛋白表达下降(P均<0.01)。糖尿病慢性运动组体重、血脂、血糖、胰岛素均出现显著性下降(P均<0.01);HDL-C升高(P<0.05...     

Effects of oral preload, CCK or bombesin administration on short term food intake of melanocortin 4-receptor knockout (MC4RKO) mice

We investigated whether either heterozygous (HET) or homozygous (knockout, KO) disruption of the melanocortin type 4 receptor (MC4R) gene alters post ingestive responsiveness of mice. Specifically, we tested the hypothesis that hyperphagia in MC4RKO mice might be due to a deficit in processes that sustain intermeal intervals (satiety) and/or processes that terminate ongoing episodes of eating (satiation). To test satiety, mice drank an oral preload and then we monitored intake of a subsequent liquid diet test meal. To test satiation, we examined the effect of exogenous administration of cholecystokinin (CCK) and bombesin (BN) on the size of a liquid diet meal. Experiment 1 was comprised of two studies. In the first, we determined that the intake of all three genotypes following fasts of either 6, 12, or 24 h were comparable, and so chose 12 h deprivation for the subsequent studies. In the second, 12 h fasted mice were allowed to consume a fixed preload, approximately 50% of their expected mean intake and, following delays of either 30 or 60 min, were allowed to consume to satiation. Compared with no preload, the preload significantly reduced meal size comparably in all three genotypes. The reduction in intake was greater when the test meal was presented 30 compared with 60 min after the preload, again with no genotype differences in this decay of satiety. In experiment 2, we administered either CCK or BN and examined suppression of meal size after a 12 h fast. Mice were tested repeatedly with CCK-8 (2, 6, or 18 μg/kg ip) or BN (2, 4 or 8 μg/kg ip) with vehicle injection days intervening. The 30 min intakes of HET and KO mice were suppressed more than those of WT following either CCK or BN. These experiments suggest that diminished responsiveness to nutrients or gut satiety hormones is not responsible for hyperphagia in MC4RKO mice.     

Identification of a small molecule that stabilizes lipoprotein lipase in vitro and lowers triglycerides in vivo

Patients at increased cardiovascular risk commonly display high levels of plasma triglycerides (TGs), elevated LDL cholesterol, small dense LDL particles and low levels of HDL-cholesterol. Many remain at high risk even after successful statin therapy, presumably because TG levels remain high. Lipoprotein lipase (LPL) maintains TG homeostasis in blood by hydrolysis of TG-rich lipoproteins. Efficient clearance of TGs is accompanied by increased levels of HDL-cholesterol and decreased levels of small dense LDL. Given the central role of LPL in lipid metabolism we sought to find small molecules that could increase LPL activity and serve as starting points for drug development efforts against cardiovascular disease. Using a small molecule screening approach we have identified small molecules that can protect LPL from inactivation by the controller protein angiopoietin-like protein 4 during incubations in vitro. One of the selected compounds, 50F10, was directly shown to preserve the active homodimer structure of LPL, as demonstrated by heparin-Sepharose chromatography. On injection to hypertriglyceridemic apolipoprotein A-V deficient mice the compound ameliorated the postprandial response after an olive oil gavage. This is a potential lead compound for the development of drugs that could reduce the residual risk associated with elevated plasma TGs in dyslipidemia.     

Tissue-specific variation of δC in mature canopy trees in a temperate forest in central Europe

Stable carbon isotope analysis has become a key tool in functional ecology, yet considerable natural variability often limits the interpretations. In this study we document the spatial, taxonomic, temporal and tissue-specific δ¹³C variability in 10 tree species of a temperate European forest. The Swiss Canopy Crane provided access to the three dimensional space within 55 trees 30–35 m high representing the genera Acer, Carpinus, Fagus, Prunus, Quercus, Tilia, Abies, Larix, Picea and Pinus. The results from six broad-leaved and four conifer species (seven deciduous, three evergreen) documented that the species effect was not significant in contrast to tissue-specific and spatial differences in the canopy. Year-to-year differences were not large but still significant.

Our analysis confirmed a significant difference between δ¹³C of foliage collected in the upper and lower canopy, but revealed no systematic differences with respect to azimuthal directions in tree crowns of the broad-leaved trees, as opposed to the conifers, which show clear differences between the sun-exposed and the shaded side. Tissue-specific differences were significant, despite surprisingly similar mean values for most tissue types. Such tissue effect was largely due to young branch xylem, which exhibited a systematic less negative deviation from the other tissue types. These findings were consistent across the species tested and provided some guidelines towards a representative sampling strategy for ¹³C analysis.     

Vanillic acid, a metabolite of 4-hydroxy-3-methoxyphenylglycol and 4-hydroxy-3-methoxymandelic acid in man

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with ¹⁴C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 ± 1.1% (mean ± SEM) was excreted as ¹⁴C-labelled vantllic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium-labelled HMPG ([²H₃]HMPG) and HMMA ([²H₆]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [²H₃]VA from [²H₃]HMPG was 8.3 ± 2.1% and the recovery of [²H₆]VA from [²H₆]HMMA was 9.0 ± 2.1%. The ²H-labelled VAs were probably formed by a decar boxylation reaction, in the case of HMPG after previous oxidation to HMMA.     

MicroRNA 483-3p suppresses the expression of DPC4/Smad4 in pancreatic cancer

Both deregulation of tumor-suppressor genes and misexpression of microRNAs (miRNAs) have been implicated in the development of pancreatic cancer, but their relationship during this process remains less clear. Here, we report that the expression of miR-483-3p is strongly enhanced in pancreatic cancer tissues compared to side normal tissues using a miRNA-array differential analysis. Furthermore, DPC4/Smad4 is identified as a target of miR-483-3p and their expression levels are inversely correlated in human clinical specimens. Ectopic expression of miR-483-3p significantly represses DPC4/Smad4 protein levels in pancreatic cancer cell lines, and simultaneously promotes cell proliferation and colony formation in vitro. Our findings identify miR-483-3p as a potent regulator of DPC4/Smad4, which may provide a novel therapeutic strategy for the treatment of DPC4/Smad4-driven pancreatic cancer.