Immobilized Receptors In Drug Discovery

一种基于在流动色谱系统中包含靶受体、伴侣蛋白或核受体的高通量筛选新方法已经被开发出来。在这种方法中，目标蛋白被固定在固体载体上，载体被包装成一个小柱。测试化学物质通过柱，通过固定的靶标，化合物从柱的开始到柱的结束所花费的时间与靶标和化合物之间相互作用的强度直接相关，即配体-受体复合物的结合亲和力。使用这种方法，复杂的化学和生物混合物可以在与疾病相关靶标相互作用和不相互作用的化合物之间快速分类。与此同时，与靶标结合的化合物本身在低、中、高亲和力的结合剂之间迅速分类。因此，该方法可以快速提供信息量大、信息量高的数据。我们已经开发了含有g蛋白偶联受体（GPCR）的色谱柱，包括阿片受体的kappa， mu和delta亚型，β -肾上腺素能受体，烟碱受体的各种亚型，含有多种GPCR的色谱柱，包括组胺-1和P2Y1受体柱，以及含有α 7和α xbetay烟碱乙酰胆碱受体，NMDA和GABA(a)受体的多配体门控离子通道柱。基于尼古丁受体的色谱柱已被用于筛选烟草烟雾凝聚物，初步结果表明，以前未知的化合物已被确定。这些化合物正在评估其作为竞争性激动剂、拮抗剂和非竞争性抑制剂的药理活性。一项使用非竞争性抑制剂和非线性色谱的研究表明，该方法可以用于识别和表征非竞争性抑制剂，这些抑制剂结合在受体的中央管腔以及被识别为醌结合位点的受体外位点。利用化学计量学分析建立定量构效关系（QSAR），所得方程可用于预测化合物的药理活性。分子模型研究被用来描述和预测烟碱受体和非竞争性抑制剂之间的相互作用。基于计算机的研究已被用作一项专利申请的基础，该专利申请名为基于计算机的模型，用于鉴定和表征烟碱乙酰胆碱受体和相关配体门控离子通道受体的非竞争性抑制剂（参考编号：E-158-2003/0-US-01）。通过在磁珠表面固定热休克90 （Hsp90）蛋白，扩大了一般的方法。Hsp90珠被用来从化学和植物混合物中提取与Hsp90结合的小分子，并从蛋白质混合物中提取与Hsp90形成复合物的蛋白质。这些微珠还被用于从细胞提取物中提取与Hsp90结合的蛋白质，这表明该方法可用于研究细胞内蛋白质-蛋白质相互作用，并确定与Hsp90相关的新途径。我们最近已经证明星形细胞瘤和胶质母细胞瘤细胞系表达ERRa和ERRg，这些核受体是脑癌治疗的关键靶点。LC-ERRa和LC-ERRg色谱柱被创建和表征，并将用于筛选新的先导药物候选的化学混合物和生物基质。

A new method for high throughput screening has been developed based upon the inclusion of the target receptor, chaperon protein, or nuclear receptor in a flowing chromatographic system. In this approach, the target protein is immobilized on a solid support and the support packed into a small column. The test chemicals are passed through the column, over the immobilized target, and the time that it takes for the compounds to pass from the beginning of the column to its end is directly related to the strength of interaction between the target and the compound, i.e. the binding affinity of the ligand-receptor complex. Using this method, complex chemical and biological mixtures can be rapidly sorted between compounds that interact and do not interact with the disease-related target. At the same time, the compounds that bind to the target are themselves rapidly sorted between low, medium and high affinity binders. Thus, the method quickly provides a large amount of data with high information content. We have developed columns containing G-protein coupled receptors (GPCR) including the kappa, mu and delta subtypes of the opioid recpetor, beta2-adrenergic receptors, various subtypes of the nicotinic receptor and columns containing multiple GPCRs including a histamine-1 and P2Y1 receptor column and a multiple ligand-gated ion channel column containing the alpha 7 and alphaXbetaY nicotinic acetylcholine receptors, NMDA and GABA(A)receptors. The nicotinic receptor-based columns have been used to screen tobacco smoke condensates and initial results indicate that previously unknown compounds have been identified. These compounds are being assessed for their pharmacological activity as competitive agonists and antagonists and non-competitive inhibitors. A study using non-competitive inhibitors and non-linear chromatography was conducted and demonstrated that the method can be used to identify and characterize the non-competitive inhibitors which bind in the central lumen of the receptor as well as at an extra-receptor site identified as the quinacrine binding site. Chemometric analysis was used to develop quantitative structure-activity relationships (QSAR) and the resulting equations can be used to predict the pharmacological activity of a compound. Molecular modeling studies were used to describe and predict the interactions between the nicotinic receptors and non-competitive inhibitors. The computer-based studies have been used as the basis for a patent application entitled Computer-based model for identification and characterization for non-competitive inhibitors of nicotinic acetylcholine receptors and related ligand-gated ion channel receptors (ref number: E-158-2003/0-US-01). The general approach has been expanded through the immobilization of heat shock 90 (Hsp90) protein in the surface of magnetic beads. The Hsp90-beads were used to extract small molecules that bind to Hsp90 from chemical and botanical mixtures and to extract proteins that form complexes with Hsp90 from protein mixtures. The beads were also used to extract proteins that bind to Hsp90 from cellular extracts and indicate that the approach can be used to study intra-cellular protein-protein interactions and to identify new pathways associated with Hsp90. We have recently demonstrated that astrocytoma and glioblastoma call lines express the ERRa and ERRg and that these nuclear receptors are key targets in the treatment of brain cancer. The LC-ERRa and LC-ERRg columns were created and characterized and will be used to screen chemical mixtures and biological matrices for new lead drug candidates.