Emese Kis, Zsuzsanna Rajnai, Eniko Ioja, Krisztina Herédi Szabó, Tünde Nagy, Dóra Méhn, Péter Krajcsi
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引用次数: 19
摘要
在杆状病毒感染的昆虫细胞(Sf9)系统中表达人胆汁盐输出泵(BSEP)转运体的小鼠同源物，研究膜胆固醇含量对转运体功能的影响。用已知的BSEP底物牛磺酸脱氧胆酸盐(TCDC)进行囊泡运输试验，测定了装载胆固醇的小鼠BSEP - ham - sf9囊泡的运输活性。小鼠Bsep以高速率运输TCDC，可以在atp酶试验中敏感地检测到。Sf9膜的胆固醇上传增强了TCDC运输和TCDC刺激的atp酶活性。利用载胆固醇的膜囊泡，研究了BSEP相互作用物对探针底物运输的抑制作用。在tcd刺激的mBsep atp酶实验中测量的IC(50)值与以牛磺胆酸盐(TC)为探针底物的人BSEP囊泡运输实验中测量的IC(50)值之间存在良好的等级顺序相关性。这种升级版的小鼠Bsep-HAM atp酶检测是一种用户友好、敏感、无放射性的方法，可用于早期高通量筛选具有bsep相关胆固醇抑制电位的药物。它可以补充人bsep介导的牛磺胆酸囊泡运输抑制试验。
Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs.
The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC(50) values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.
期刊介绍：
Advancing the Science of Drug Discovery: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease.
SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success.
SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies.
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