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    • 5. 发明申请
    • IMPROVED ELECTROPHYSIOLOGICAL ASSAYS USING OOCYTES THAT EXPRESS HUMAN ENaC AND THE USE OF PHENAMIL TO IMPROVE THE EFFECT OF ENaC ENHANCERS IN ASSAYS USING MEMBRANE POTENTIAL REPORTING DYES
    • 采用膜表达人类表达和使用苯胺类改善ENaC增强剂在测定中使用膜电位报告染料的效果的改进的电生理学测定
    • WO2005014848A3
    • 2005-05-06
    • PCT/US2004021853
    • 2004-07-09
    • SENOMYX INCSERVANT GUYCHANG HONGREDCROW CYRILRAY SUMITACLARK IMRAN
    • SERVANT GUYCHANG HONGREDCROW CYRILRAY SUMITACLARK IMRAN
    • C07H21/04C07K1/00C12N1/20C12P21/06C12Q20060101G01N33/50G01N33/53G01N33/566G01N33/567G01N33/68
    • C12Q1/025C12N15/85C12N2320/10C12N2503/02G01N33/5044G01N33/6872G01N2500/10
    • In one aspect, the present invention relates to a mammalian cell-based high-throughput assay for the profiling and screening of human epithelial sodium channel (hENaC) cloned from a human kidney c-DNA library and is also expressed in other tissues including human taste tissue. The present invention further relates to amphibian oocyte-based medium-throughput electrophysiological assays for identifying human ENaC modulators, preferably ENaC enhancers. Compounds that modulate ENaC function in a cell-based ENaC assay are expected to affect salty taste in humans. The assays described herein have advantages over existing cellular expression systems. In the case of mammalian cells, such assays can be run in standard 96 or 384 well culture plates in high-throughput mode with enhanced assay results being achieved by the use of a compound that inhibits ENaC function, preferably an amiloride derivative such as Phenamil. In the case of the inventive oocyte electrophysiological assays (two-electrode voltage-clamp technique), these assays facilitate the identification of compounds which specifically modulate human ENaC. The assays of the invention provide a robust screen useful to detect compounds that facilitate (enhance) or inhibit hENaC function. Compounds that enhance or block human ENaC channel activity should thereby modulate salty taste in humans.
    • 一方面,本发明涉及用于分析和筛选从人肾c-DNA文库克隆的人上皮钠通道(hENaC)的基于哺乳动物细胞的高通量测定法,并且还在包括人味觉的其他组织中表达 组织。 本发明进一步涉及用于鉴定人ENaC调节剂,优选ENaC增强子的基于两栖动物卵母细胞的中等通量电生理测定。 预期在基于细胞的ENaC测定中调节ENaC功能的化合物会影响人类的咸味。 本文所述的测定相对于现有的细胞表达系统具有优势。 在哺乳动物细胞的情况下,这种测定可以在高通量模式下在标准96或384孔培养板中进行,通过使用抑制ENaC功能的化合物,优选阿米洛利衍生物如Phenamil实现增强的测定结果。 在本发明的卵母细胞电生理学测定法(双电极电压钳技术)的情况下,这些测定法有助于鉴定特异性调节人ENaC的化合物。 本发明的测定法提供了用于检测促进(增强)或抑制hENaC功能的化合物的强力筛选。 因此增强或阻断人类ENaC通道活性的化合物应该调节人类的咸味。
    • 6. 发明申请
    • IMPROVED CELL-BASED FLUORESCENT ASSAYS FOR IDENTIFYING ALPHA AND DELTA ENaC MODULATORS
    • 改进的基于细胞的荧光测定用于鉴定ALPHA和DELTA ENCC调制器
    • WO2008013969A3
    • 2008-03-20
    • PCT/US2007016963
    • 2007-07-30
    • SENOMYX INCSERVANT GUYBRUST PAULRAY SUMITAHUNG NING
    • SERVANT GUYBRUST PAULRAY SUMITAHUNG NING
    • G01N33/53
    • G01N33/6872G01N33/5008G01N33/566
    • This invention relates to improved electrophysiological assays that measure sodium conductance activity of a delta or alpha human epithelial sodium channel (ENaC) expressed in a test cell in the presence and absence of delta hENaC enhancers. The improvement comprises contacting the test cells with an amount of sodium ion, typically from at least 15 mM to 140 mM, for a time sufficient, e.g. for at least 5 minutes to an hour, prior to the test cells being screened against at least one putative enhancer. It has been surprisingly discovered that this sodium pretreatment enhances assay sensitivity, especially assays that use membrane potential or ion sensitive dyes that detect changes in conductance fluorimetrically. These enhancer compounds have potential application in modulating (enhancing) salty taste perception and for treating disorders involving aberrant hENaC function.
    • 本发明涉及改进的电生理测定法,其测量在存在和不存在δHENaC增强子的情况下在测试细胞中表达的δ或α人上皮钠通道(ENaC)的钠电导活性。 该改进包括将测试细胞与一定量的钠离子(通常为至少15mM至140mM)接触足够的时间,例如, 在至少5分钟至1小时之前,在测试细胞被针对至少一个推定的增强子进行筛选之前。 已经惊奇地发现,这种钠预处理增强了测定灵敏度,特别是使用膜电位或离子敏感染料检测荧光测量电导的变化的测定法。 这些增强剂化合物在调节(增强)咸味感觉和治疗涉及异常hENaC功能的病症中具有潜在的应用。