Production and inhibition assay for bile salt export protein
胆汁盐输出蛋白的产生和抑制测定
基本信息
- 批准号:8827883
- 负责人:
- 金额:$ 24.33万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2015
- 资助国家:美国
- 起止时间:2015-01-01 至 2016-12-31
- 项目状态:已结题
- 来源:
- 关键词:ATP phosphohydrolaseATP-Binding Cassette TransportersBile fluidBiological AssayCell membraneClinicalDetectionDevelopmentDrug InteractionsDrug TransportEuropeanFailureFluorescenceGenesGoalsHepatocyteHousingHumanIn VitroInjury to LiverLeadLiposomesLiverMarketingMaterials TestingMeasurableMeasurementMediatingMedicineMembraneMethodsMutationNoisePermeabilityPharmaceutical PreparationsProductionProtein Export PathwayProtocols documentationPumpRadioactivityReaderReagentRiskSeriesServicesSignal TransductionSystemTechniquesTestingTimeVesicleVinblastineXenobioticsaqueousbile formationbile saltscytotoxicdesign and constructiondrug candidatedrug developmentdrug discoverydrug withdrawalhigh throughput screeninginhibitor/antagonistliver injurymembernoveloverexpressionpost-marketpre-clinicalprogramspublic health relevancereconstitutionresearch studysensortool
项目摘要
DESCRIPTION (provided by applicant): The objective of the proposed project is to design and construct a highly-specific and high throughput in vitro biological assay system to characterize the interaction of drugs and drug candidates with human BSEP. Bile Salt Export Protein (BSEP/ABCB11) is an active, ATP-dependent transporter localized in the liver canicular membrane. It mediates efflux of bile salts from hepatocytes into bile and is essential for normal bile formation and flow. Inhibition of BSEP activity due to an administered xenobiotic or a mutation in the ABCB11 gene can lead to liver injury caused by intracellular accumulation of cytotoxic bile constituents. Given that drug- induced liver injury (DILI) is a major cause of serious illness in humans, drug withdrawal, post marketing, regulatory failure, and attrition during preclinical and clinical development, regulatory agencies recommend in vitro measurement of the BSEP inhibitory potential of drug candidates. The assay system will consist of a purified and functionally-competent BSEP integrated into the novel assay platform, the Fluorosome(r) Platform. This assay system, to be referred to as Fluorosome-trans-bsep, will be marketed as a superior substitute for the only existing method which uses inverted vesicles generated from membranes of cells overexpressing BSEP. The specific aims meant to achieve these goals include Production, purification and reconstitution of active human BSEP [these steps will be accomplished in the lab of subcontract P.I. Dr. Balazs Sarkadi, a known expert in the field of ABC transporters], identification of an optimal test substrate, tentatively vinblastin, i.e. one that will be actively transported by the transporter and show significant quenching of the
sensor employed in Fluorosome-trans, determination of reconstituted BSEP ATPase activities for demonstrated BSEP substrates and inhibitors, and formation of Fluorosome-trans-bsep from reconstituted BSEP, measurement of drug transport and its inhibition, accompanied by parallel ATPase activity measurements. The result of this project will be a simple to use commercial assay that is unambiguously specific for BSEP. It will be rapid with a high signal to noise ratio, provide real time results, and will be applicable to a wide range of compounds at very small amounts. It will only require a fluorescence plate reader rather than LC-MS or radioactivity as is required for an existing assay. The new assay will be amenable to moderate and high throughput screening. It will be offered on the market both as a reagent and as an in-house service.
描述(由申请人提供):本项目的目的是设计和构建一个高特异性和高通量的体外生物测定系统,以表征药物和候选药物与人BSEP的相互作用。胆盐输出蛋白(BSEP/ABCB 11)是一种位于肝小管膜上的活性ATP依赖性转运蛋白。它介导胆汁盐从肝细胞流出到胆汁中,是正常胆汁形成和流动所必需的。由于给予外源性物质或ABCB 11基因突变导致的BSEP活性抑制可导致细胞毒性胆汁成分细胞内蓄积引起的肝损伤。鉴于药物诱导的肝损伤(DILI)是人类严重疾病、停药、上市后、监管失败以及临床前和临床开发期间的损耗的主要原因,监管机构建议体外测量候选药物的BSEP抑制潜力。 试验系统将由整合到新型试验平台Fluorosome(r)平台中的纯化和功能活性BSEP组成。该测定系统(称为荧光体-反式-bsep)将作为唯一现有方法的上级替代品上市,该方法使用过表达BSEP的细胞膜产生的倒置囊泡。旨在实现这些目标的具体目标包括活性人BSEP的生产、纯化和重建[这些步骤将在CUPPI实验室完成。Balazs Sarkadi博士,ABC转运蛋白领域的已知专家],鉴定了最佳测试底物,试验性长春碱,即将由转运蛋白主动转运并显示显著淬灭的底物。
荧光体-反式中采用的传感器,测定所证明的BSEP底物和抑制剂的重建BSEP ATP酶活性,以及由重建BSEP形成荧光体-反式-bsep,测量药物转运及其抑制,伴随平行ATP酶活性测量。 本项目的结果将是一种简单易用的商业测定方法,对BSEP具有明确的特异性。它将是快速的,具有高信噪比,提供真实的时间结果,并且将以非常少量适用于广泛的化合物。它只需要荧光板读数器,而不是现有检测所需的LC-MS或放射性。新的检测方法将适用于中等和高通量筛选。它将作为试剂和内部服务在市场上提供。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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DONALD Lee MELCHIOR其他文献
DONALD Lee MELCHIOR的其他文献
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{{ truncateString('DONALD Lee MELCHIOR', 18)}}的其他基金
Development of a fluorescence liposomal ABCG2 Multidrug Transporter assay
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- 批准号:
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Rapid in vitro substrate assay for the multi-drug resistance p-glycoprotein
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In vitro substrate assay for multi-drug resistance
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- 批准号:
6991096 - 财政年份:2005
- 资助金额:
$ 24.33万 - 项目类别:
Rapid in vitro substrate assay for the multi-drug resistance p-glycoprotein
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- 批准号:
7404207 - 财政年份:2005
- 资助金额:
$ 24.33万 - 项目类别:
ASIP-UNIVERSITY OF MASSACHUSETTES MEDICAL SCHOOL
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$ 24.33万 - 项目类别:
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