A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.
- MeSH
- AMP cyklický metabolismus MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- inhibiční koncentrace 50 MeSH
- isoxazoly chemie MeSH
- křečci praví MeSH
- kvartérní amoniové sloučeniny chemie MeSH
- lidé MeSH
- molekulární konformace MeSH
- oxotremorin chemie MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus MeSH
- receptory muskarinové metabolismus MeSH
- rekombinantní fúzní proteiny chemie MeSH
- signální transdukce * MeSH
- simulace molekulární dynamiky MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.
- MeSH
- CD8-pozitivní T-lymfocyty cytologie MeSH
- dánio pruhované MeSH
- histondeacetylasa 6 antagonisté a inhibitory MeSH
- inhibitory histondeacetylas chemie farmakologie MeSH
- isoxazoly chemie farmakologie MeSH
- katalytická doména MeSH
- kyseliny hydroxamové chemie farmakologie MeSH
- lidé MeSH
- makrofágy cytologie MeSH
- melanom farmakoterapie MeSH
- mikrozomy chemie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- NKT buňky cytologie MeSH
- objevování léků MeSH
- transplantace izogenní MeSH
- zinek chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Centrum pro epilepsie Brno Schválené možnosti antiepileptické terapie v dětském věku jsou omezenější než v dospělosti a proces zařazení nového antiepileptika v dětství je většinou zdlouhavější. Cílem přehledného článku je podání ucelených informací o zonisamidu v nové indikaci – přídatné terapii parciálních záchvatů v dětství od 6 let věku. Souhrně jsou prezentovány výsledky multicentrické, dvojitě zaslepené placebem kontrolované studie i přehledné výsledky otevřených studií zaměřených zejména na bezpečnost a snášenlivost a tyto poznatky jsou doplněny praktickými zkušenostmi z pracoviště autorů.
In comparison with adults approved options of antiepileptic treatment in children are limited. The process of introduction of new antiepileptic drug is often longer. The aim of this review is to present comprehensive information concerning zonisamide in new indication – add-on therapy of partial seizures in children older than 6 years. The results of multicentric, double blind, placebo controled study and results of open label studies about safety and tolerability are presented. These findings are accompanied by authors´ experience.
- MeSH
- antikonvulziva MeSH
- bezpečnost MeSH
- dítě MeSH
- dospělí MeSH
- dvojitá slepá metoda MeSH
- epilepsie farmakoterapie MeSH
- horečka MeSH
- isoxazoly * aplikace a dávkování farmakokinetika chemie škodlivé účinky terapeutické užití MeSH
- klinické zkoušky jako téma MeSH
- lékové interakce MeSH
- lidé středního věku MeSH
- multicentrické studie jako téma MeSH
- placeba MeSH
- pocení MeSH
- randomizované kontrolované studie jako téma MeSH
- senioři MeSH
- výsledek terapie MeSH
- záchvaty farmakoterapie MeSH
- závrať MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- senioři MeSH
Renal ischaemia/reperfusion (I/R) injury and hypertension represent major alloantigen-independent risk factors contributing to the development of chronic allograft nephropathy. In a model of accelerated major histocompatibility complex-independent renal injury, we evaluated the effect of leflunomide derivate - FK778 - on the progression of accelerated nephropathy. Thirty-six uninephrectomized hypertensive transgenic (m-REN-2)-27 rats received a clip on renal pedicle for 45 minutes. Animals were treated with FK778 3 mg/kg/day (I/R 3 mg, N = 12), 10 mg/kg/day (I/R 10 mg, N = 12) or placebo (N = 12) via gavage for 16 weeks. Eighteen animals were sham-operated and treated with FK778 3 mg/kg/day (sham 3 mg, N = 6), 10 mg/kg/day (sham 10 mg, N = 6) or were untreated (sham, N = 6). Proteinuria and blood pressure were evaluated throughout and the kidneys were harvested for morphological and immunohistochemical analysis at the end of the experiment. At week 16, rats with I/R injury and FK778 treatment had lower proteinuria compared with placebo-treated rats (I/R 3 mg: 48.42 +/- 26.16, I/R 10 mg 27.28 +/- 21.86 vs. Placebo: 70.13 +/- 50.19 mg/day, P < 0.05). The untreated sham group exhibited lower proteinuria compared with FK778-treated sham groups (Sham 3 mg: 24.23 +/- 10.89; Sham 10 mg: 17.37 +/- 4.13; Sham: 14.23 +/- 1.18) There was no difference in glomerulosclerosis and interstitial fibrosis among the treated groups. In the untreated animals the rate of interstitial fibrosis decline reached statistical significance (Placebo vs. Sham: 1.125 +/- 0.641 % vs. 0.250 +/- 0.500 %, P < 0.05). There was higher CD5+ leukocyte infiltration in the placebotreated group. FK778-treated rats displayed amelioration of some changes induced by the I/R injury. Our observation also suggests potential nephrotoxicity of FK778.
- MeSH
- alkyny farmakologie terapeutické užití MeSH
- financování organizované MeSH
- imunosupresiva farmakologie terapeutické užití MeSH
- isoxazoly farmakologie chemie terapeutické užití MeSH
- krevní tlak účinky léků MeSH
- krysa rodu rattus MeSH
- ledviny patologie účinky léků MeSH
- modely nemocí na zvířatech MeSH
- nemoci ledvin farmakoterapie patologie MeSH
- nitrily farmakologie terapeutické užití MeSH
- placeba terapeutické užití MeSH
- potkani transgenní MeSH
- reperfuzní poškození farmakoterapie patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi response can be calibrated by the K(+)/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Deltapsi and DeltapH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.
- MeSH
- arylsulfonany chemie MeSH
- biologické modely MeSH
- buněčná membrána fyziologie chemie MeSH
- elektrochemie MeSH
- financování organizované MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční spektrometrie MeSH
- isoxazoly chemie MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály MeSH
- proteolipidy chemie MeSH
- protonové ATPasy chemie MeSH
- protony MeSH
