Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, USA.
J Pharm Biomed Anal. 2012 Dec;71:157-61. doi: 10.1016/j.jpba.2012.07.023. Epub 2012 Aug 9.
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 (σ(1) and σ(2)) 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 K(i) values for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ(1) receptors, equivalent affinity values were observed for both methods/tissues. For σ(2) receptors, 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 σ(1) versus σ(2) receptors.
σ 受体代表了许多治疗适应症的潜在药物靶点，包括癌症、抑郁症、精神兴奋剂滥用和中风。大多数用于 σ 受体的已发表放射性配体结合研究都采用低通量方法，使用“细胞采集器”。需要高通量方法来促进大量新型化合物的有效筛选。在这项研究中，一系列参考化合物采用新的中等通量 96 孔过滤方法进行分析，并将结果与使用传统基于细胞采集器的方法获得的结果进行比较。96 孔测定法利用大鼠肝膜来确定两种已知的 σ 受体亚型（σ(1)和 σ(2)），因为这种组织含有两种亚型的高密度，并且满足与 96 孔格式一起使用的可靠要求。对于在大鼠脑膜中制备并在传统细胞采集器上处理的平行样品，新方法给出了参考配体的可比 K(i)值。对于 σ(1)受体，两种方法/组织均观察到等效的亲和力值。对于 σ(2)受体，与脑膜相比，大多数化合物在肝中的亲和力高 2 倍左右，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，它被用于筛选一系列新型 2(3H)-苯并噻唑酮化合物，结果鉴定出几种具有纳摩尔亲和力和对 σ(1)受体与 σ(2)受体大于 50 倍特异性的类似物。
