A 96-well filtration method for radioligand binding analysis of σ receptor ligands.
A 96-well filtration method for radioligand binding analysis of σ receptor ligands.
复制标题
用于 α 受体配体放射性配体结合分析的 96 孔过滤方法。
DOI：
10.1016/j.jpba.2012.07.023
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发表时间：
2012
影响因子：
3.4
通讯作者：
Matsumoto,RaeR
中科院分区：
文献类型：
--
作者：
Fishback,JamesA;Rosen,Abagail;Bhat,Rohit;McCurdy,ChristopherR;Matsumoto,RaeR
σ受体代表了许多治疗适应症的潜在药物靶标，包括癌症、抑郁症、精神兴奋剂滥用和中风。大多数已发表的σ受体的放射性配体结合研究采用采用“细胞收集器”的低通量方法。需要更高通量的方法来促进大量新化合物的有效筛选。在这项研究中，一系列的参考化合物进行了分析与一种新的中等通量96孔过滤方法和结果进行了比较，使用传统的细胞收获器为基础的方法。96孔试验使用大鼠肝膜测定两种已知的σ受体亚型（σ 1和σ2），因为该组织含有高密度的两种亚型，并符合96孔格式可靠使用所需的标准。新方法给出了与在大鼠脑膜中制备并在传统细胞收获器上处理的样品平行分析的参考配体的可比Ki值。对于σ 1受体，观察到两种方法/组织的亲和力值相等。对于σ 2受体，观察到肝脏中大多数化合物的亲和力比脑膜高约2倍，但与脑衍生值保持良好的相关性。为了进一步证明新方法的实用性，它被用来筛选一系列新的2（3 H）-苯并噻唑酮化合物，导致几个类似物的鉴定与纳摩尔亲和力和大于50倍的特异性σ 1与σ 2受体。
σ receptors represent a potential drug target for numerous therapeutic indications including cancer, depression, psychostimulant abuse, and stroke. Most published radioligand binding studies for σ receptors utilize a low throughput method employing a “cell harvester.” Higher throughput methods are required to facilitate efficient screening of large numbers of novel compounds. In this study, a series of reference compounds was analyzed with a new medium-throughput 96-well filtration method and the results were compared to those obtained using the conventional cell harvester-based method. The 96-well assay utilized rat liver membranes for the determination of both known σ receptor subtypes (σ1and σ2) because this tissue contains high densities of both subtypes and fulfills criteria required for reliable use with the 96-well format. The new method gave comparable Kivalues for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ1receptors, equivalent affinity values were observed for both methods/tissues. For σ2receptors, approximately 2-fold higher affinities were observed for most compounds in liver, as compared to brain membranes, but excellent correlation with brain-derived values was maintained. To further demonstrate the utility of the new method it was used to screen a novel series of 2(3H)-benzothiazolone compounds, resulting in the identification of several analogues with nanomolar affinity and greater than 50-fold specificity for σ1versus σ2receptors.