Design, synthesis and biological evaluation of glycolamide, glycinamide, and ß-amino carbonyl 1,2,4-triazole derivatives as DPP-4 inhibitors.

Through modification of the skeleton of Sitagliptin and Vildagliptin, we successfully synthesized and built-up four series of 1,2,4-triazole derivatives, containing N,O-disubstituted glycolamide, N,N'-disubstituted glycinamide, ß-amino ester, and ß-amino amide as linkers, for the development of new dipeptidyl peptidase 4 (DPP-4) inhibitors. The synthetic strategy for glycolamides or glycinamides involved convenient two-steps reaction: functionalized transformation of 2-chloro-N-(2,4,5-triflurophenyl)acetamide 9 (hydroxylation or amination) and esterification or amidation of 1,2,4-triazole-3-carboxylic acid. On the other hand, the one-pot synthesis procedure, including substitution and deprotection, was developed for the preparation of ß-amino carbonyl 1,2,4-triazoles from (1H-1,2,4-triazol-3-yl)methanol 12 or (1H-1,2,4-triazol-3-yl)methanamine 13 and Boc-(R)-3-amino-4-(2,4,5-trifluoro-phenyl)-butyric acid 14. All of glycolamides, glycinamides, and ß-amino carbonyl 1,2,4-triazoles were also evaluated against DPP-4 inhibitory activity. Based on the SAR study of DPP-4 inhibitory capacity, ß-amino ester 5n and ß-amino amide 1,2,4-triazoles 6d and 6p possessed the significant inhibition of DPP-4 (IC50 < 51.0 nM), particularly for compound 6d (IC50 = 34.4 nM). The selectivity evaluation indicated compound 5n and 6p had excellent selectivity over QPP, DPP-8, and DPP-9. In addition, the docking results revealed compounds 5n and 6p provided stronger π-π stacking interaction with residue Phe357 than 1,5-disubstituted 1,2,4-triazole 6d and Sitagliptin 1. In summary, compounds 5n and 6p could be promising lead compounds for further development of DPP-4 inhibitor.

Association between the growth rate of subependymal giant cell astrocytoma and age in patients with tuberous sclerosis complex.

PURPOSE: The most common neurological complications associated with tuberous sclerosis complex (TSC) include intractable seizures that begin in infancy and subependymal giant cell astrocytoma (SEGA) complicated by hydrocephalus with increasing age. Information on SEGA growth of TSC patients is limited. This study aimed to examine the TSC-SEGA growth rates by periodic neuroimaging. METHODS: This study evaluated the TSC-SEGA growth rates by serial neuroimaging. Fifty-eight patients with TSC underwent systematic evaluation, including a review of medical history and serial brain neuroimaging. RESULTS: While magnetic resonance imaging was more sensitive in detecting cortical tubers than computed tomography (73.1 vs. 0 %, p < 0.001), its efficacy in identifying intracranial lesions was comparable to that of computed tomography (96.2 vs. 100 %, p = 0.658). Significant tumor growth was observed in children (p = 0.012) and adults (p = 0.028) during follow-up periods, respectively (median for children 23.5 months, interquartile range 18-40 months and median for adults 23 months, interquartile range 12-34 months). Further, the SEGA growth rate in children was significantly higher than that in adults (75.6 vs. 16.5 %, p = 0.03). CONCLUSIONS: The results of the study show that SEGA has a significantly higher growth rate in children using serial follow-up brain imaging, suggesting the importance of performing follow-up neuroimaging at yearly intervals in childhood to identify and prevent potential comorbidities.

Monoclonal antibodies targeting the synthetic peptide corresponding to the polybasic cleavage site on H5N1 influenza hemagglutinin.

BACKGROUND: Avian influenza H5N1 virus is highly pathogenic partially because its H5 hemagglutinin contains a polybasic cleavage site that can be processed by proteases in multiple organs. METHODS: Monoclonal antibodies (mAb) specific to the synthetic peptide of hemagglutinin polybasic cleavage site of H5N1 virus were raised and tested for their neutralizing potential. RESULTS: Purified mAb showed suppression of H5N1 pseudovirus infection on Madin-Darby Canine Kidney (MDCK) cells but the efficacy was less than 50%. Since those mAb are specific to the intact uncut polybasic cleavage site of hemagglutinin, their efficacy depends on the extent of hemagglutinin cleavage on the viral surface. CONCLUSIONS: Proteolytic analysis suggests the low efficacy associated with those mAb may be due to proteolytic cleavage already present on the majority of hemagglutinin prior to the infection of virus.

Synthesis of ß-Amino Carbonyl 6-(Aminomethyl)- and 6-(Hydroxymethyl)pyrazolopyrimidines for DPP-4 Inhibition Study.

BACKGROUND: Type-2 diabetes is a chronic progressive metabolic disease resulting in severe vascular complications and mortality risk. Recently, DPP-4 inhibitors are conceived as a favorable class of agents for the treatment of type 2 diabetes due to the minimal side effects. METHODS: Sitagliptin is the first medicine approved for DPP-4 inhibitor. Its structure involved three fragments: 2,4,5-triflorophenyl fragment pharmacophore, enantiomerically ß-amino carbonyl linker, and tetrahydrotriazolopyridine. Herein, we are drawn to the possibility of substituting tetrahydrotriazolopyridine motif present in Sitagliptin with a series of new fused pyrazolopyrimidine bicyclic fragment to investigate potency and safety. RESULTS: Two series of fused 6-(aminomethyl)pyrazolopyrimidine and 6-(hydroxymethyl)pyrazolopyrimidine derivatives containing ß-amino ester or amide as linkers were successfully designed for the new DPP-4 inhibitors. Most fused 6-methylpyrazolopyrimidines were evaluated against DPP-4 inhibition and selectivity capacity. Based on research study, ß-amino carbonyl fused 6-(hydroxymethyl)pyrazolopyrimidine possesses the significant DPP-4 inhibition (IC50 ≤ 59.8 nM) and presents similar with Sitagliptin (IC50 = 28 nM). Particularly, they had satisfactory selectivity over DPP-8 and DPP-9, except for QPP. CONCLUSION: ß-Amino esters and amides fused 6-(hydroxymethyl)pyrazolopyrimidine were developed as the new DPP-4 inhibitors. Those compounds with a methyl group or hydrogen in N-1 position and methyl substituted group in C-3 of pyrazolopyrimidine moiety showed better potent DPP-4 inhibition (IC50 = 21.4-59.8 nM). Furthermore, they had satisfactory selectivity over DPP-8 and DPP-9 Finally, the docking results revealed that compound 9n was stabilized at DPP-4 active site and would be a potential lead drug.

A novel hydroxyfuroic acid compound as an insulin receptor activator. Structure and activity relationship of a prenylindole moiety to insulin receptor activation.

BACKGROUND: Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ) B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db) mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. METHODS: A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and fibroblast growth factor (FGF) receptors were examined in this study. RESULT AND CONCLUSION: This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639), which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

Trend of subsequent epilepsy in children with recurrent febrile seizures: A retrospective matched cohort study.

PURPOSE: Trends of epilepsy in children were correlated with febrile seizure (FS) in a previous retrospective study. In the present study, the authors obtained relevant data from a nationwide cohort database to investigate trends in subsequent epilepsy in children with a history of recurrent FS. METHODS: A total of 10,210 children with FS comprised the cohort. The diagnosis date was used as the index date. A comparison cohort was randomly matched with each case based on age, sex, urbanization level, parents' occupation, and index date. Cox proportional hazard regression was performed to estimate the hazard ratio and confidence interval of FS-associated epilepsy. RESULTS: This retrospective cohort study included 7729 children with FS and a comparison cohort of 30,916 children. The incidence of epilepsy was 11.4-fold higher in the FS cohort than in the comparison cohort (5.67 vs. 0.49 per 1000 person-years, respectively). Compared with the comparison cohort, the epilepsy incidence rate ratio increased in children with admissions for FS, from 8.62 at 1 admission to 26.2 at ≥2 admissions (95% CI 6.80-10.9, and 19.78-34.8, respectively; p for trend < 0.0001). CONCLUSION: FS may increase the risk for subsequent epilepsy in children. Recurrent FS increased the cumulative incidence of epilepsy.

Trend of nocturnal enuresis in children with attention deficit/hyperactivity disorder: a nationwide population-based study in Taiwan.

Attention-deficit/hyperactivity disorder (ADHD) and nocturnal enuresis are common disorders with extensive psychosocial suffering in affected children, and healthcare burden on parents. Whether the childhood psychological disorders and nocturnal enuresis are factors contributing to ADHD have not been clearly established. This study conducted a population-based case-control study using data sets from the National Health Research Insurance database, and identified 14 900 children diagnosed with ADHD. Risk factors that have been associated with or possibly related to ADHD development were included in this study. Performance of in groups of ADHD with enuresis was compared with controls. With adjustment for potential covariates, participants with enuresis exhibited a 2.24-fold greater risk of subsequent ADHD development compared with controls (95% CI 1.84 to 2.73). Participants with enuresis and comorbidity had a significantly greater risk of ADHD than those with no enuresis and no comorbidity (adjusted OR=8.43, 95% CI 4.38 to 16.2). Children who are assessed for ADHD should be evaluated for the presence of enuresis or other neurobehavioral comorbidities. Multidisciplinary treatment may benefit children with ADHD and minimize psychological burden on parents.

Docosahexaenoic acid inhibits inflammation via free fatty acid receptor FFA4, disruption of TAB2 interaction with TAK1/TAB1 and downregulation of ERK-dependent Egr-1 expression in EA.hy926 cells.

SCOPE: Inflammation is intimately associated with many cardiovascular events and docosahexaenoic acid (DHA) has been shown to protect against CVD. Egr-1 has emerged as a key regulator in the development of atherosclerosis. Free fatty acid receptor 4 (FFA4) is an n-3 FA membrane receptor. Tumor necrosis factor alpha (TNF-α) is an inflammatory mediator and transforming growth factor-ß-activated kinase 1 (TAK1) is essential in the TNF-α-mediated activation of NF-κB. We examined the mechanisms underlying DHA inhibition of inflammation in human EA.hy926 cells. METHODS AND RESULTS: TNF-α markedly induced the interaction between TAK1 binding protein (TAB) 2 and TAK1/TAB1, the phosphorylation of ERK, p38 MAPK and Akt, the expression of Egr-1 and ICAM-1, and HL-60 (monocyte-like) cell adhesion. Pretreatment with DHA attenuated TNF-α-induced phosphorylation of ERK, expression of Egr-1 and ICAM-1 and HL-60 cell adhesion. Transfection with siFFA4 reversed the DHA-mediated inhibition of TNF-α-induced Egr-1 and ICAM-1 expression, HL-60 cell adhesion and NF-κB and DNA-binding activity. CONCLUSION: Our results suggest that the anti-inflammatory effect of DHA on the endothelium is at least partially linked to FFA4, disruption of TAB2 interaction with TAK1/TAB1 and downregulation of ERK-dependent Egr-1 and ICAM-1 expression, which leads to less HL-60 cell adhesion to TNF-α-stimulated EA.hy926 cells.

Comparison of the detection rates of RT-PCR and virus culture using a combination of specimens from multiple sites for enterovirus-associated encephalomyelitis during enterovirus 71 epidemic.

Enterovirus (EV) 71 may cause severe neurological illness in the pediatric population. The present study aimed to compare the detection rates of reverse transcription-polymerase chain reaction (RT-PCR) with pan-EV/EV71 type- specific primers and virus culture (VC) for the identification of EV and EV71 using specimens from multiple sites. In total, specimens from throat/rectal swabs, cerebrospinal fluid (CSF), and blood from 66 patients diagnosed with EV encephalomyelitis were subjected to both RT-PCR and VC for detecting the presence of pan-EV and EV71. The results revealed that the positive RT-PCR rate was higher in throat swabs (60.6%) and rectum swabs (50.0%) than in CSF (16.7%) and blood (15.6%). The same trend was also observed in case of VC: throat swabs (22.7%), rectum swabs (10.6%), and blood (3.0%). The detection rate of EV encephalomyelitis by RT-PCR was 90.9% for all samples, 63.6% of which were subtyped as EV71. The detection rates of RT-PCR were superior to those of VC, and identification using specimens from throat/rectal swabs yielded higher positive results. These findings may help physicians to identify the etiologies at an early stage during EV71 epidemics and to make emergent medical decisions for minimizing patient morbidity and mortality.

Rhubarb inhibits hepatocellular carcinoma cell metastasis via GSK-3-ß activation to enhance protein degradation and attenuate nuclear translocation of ß-catenin.

The aim of our study was to investigate the mechanisms by which rhubarb regulates ß-catenin as well as metastasis of hepatocellular carcinomas. Our results revealed that rhubarb extract inhibited HA22T cell migration ability in wound healing, migration and invasion assays in a dose-dependent manner. Rhubarb also reduced ß-catenin protein level, downregulated its downstream proteins, cyclin D, Tbx3 and c-Myc, and attenuated the expression of MMP9 and contactin-1 metastatic factors. Additionally, rhubarb inhibited ß-catenin nuclear accumulation and induced its degradation via proteasome-mediated pathway. Furthermore, we found that rhubarb suppressed the p-ser(9) GSK-3-ß protein level to inactivate Wnt signalling and reduce ß-catenin protein level. Taken together; we found that rhubarb blocked the metastatic process of HA22T hepatocellular carcinoma cells mediated through GSK-3-ß activation, and enhancement of protein degradation as well as reduction of the nuclear accumulation of ß-catenin.

Function of interdomain alpha-helix in human brain hexokinase: covalent linkage and catalytic regulation between N- and C-terminal halves.

Human brain relies on a steady supply of glucose as the source of fuel, and type I hexokinase is the major isozyme governing the introduction of glucose to glycolysis in the brain. One unique regulatory property associated with type I isozyme is the alleviation of product inhibition by inorganic phosphate which binds to the N-terminal half, and the conformational change induced by inorganic phosphate must be propagated to the active site in the C-terminal half. With a single interdomain alpha-helix as the only covalent connection between the N- and C-terminal halves, the question arises as what role the interdomain alpha-helix plays at the interdomain signal transduction. Two mutants were constructed in an attempt to answer this question. The first mutant, A464P/E465G, with a helix breaker embedded in the interdomain alpha-helix had a smaller magnitude of phosphate alleviation than the wild type. The second mutant, with an insertion of seven additional residues between Gln 466 and His 467, had this phosphate relief property further diminished. Neither mutant showed dramatic changes nor the other kinetic properties. It is speculated that the interdomain alpha-helix is important for keeping the proper non-covalent contact so that transmission of the conformational changes across the N- and C-terminal half boundary can be achieved.

Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria.

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.