Immobilized Receptors In Drug Discovery

开发了一种新的高通量筛选方法，其基础是将目标受体、伴侣蛋白或核受体包含在流动相色谱系统中。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子。测试化学品通过柱，在固定化的靶上方，化合物从柱的开始到结束所需的时间与靶与化合物之间的相互作用的强度，即配体-受体复合体的结合亲和力直接相关。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目标相互作用和不相互作用的化合物之间快速分选。同时，与靶结合的化合物本身会在低亲和力、中亲和力和高亲和力结合剂之间快速分类。因此，该方法快速地提供了信息量高的海量数据。我们已经发展了包含G蛋白偶联受体(GPCR)的柱，包括阿片受体的Kappa、Mu和Delta亚型，β2-肾上腺素能受体，各种亚型的烟碱受体，以及包含多个GPCRs的柱，包括组胺-1和P2Y1受体柱，以及包含α7和αXbetaY烟碱型乙酰胆碱受体、NMDA和GABA(A)受体的多配体门控离子通道柱。基于烟碱受体的色谱柱已被用于筛选烟草烟雾冷凝物，初步结果表明已鉴定出以前未知的化合物。正在评估这些化合物作为竞争性激动剂、拮抗剂和非竞争性抑制剂的药理活性。使用非竞争性抑制剂和非线性层析进行的研究表明，该方法可以用来鉴定和表征结合在受体中央管腔和受体外位置的非竞争性抑制剂，这些非竞争性抑制剂被鉴定为奎纳克林结合部位。化学计量学分析用于建立定量构效关系(QSAR)，所得到的方程可用于预测化合物的药理活性。分子模拟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经被用作题为用于识别和表征烟碱型乙酰胆碱受体和相关配基门控离子通道受体的非竞争性抑制剂的基于计算机的模型的专利申请的基础(参考号：E-158-2003/0-US-01)。通过将热休克90(Hsp90)蛋白固定在磁珠表面，扩展了一般的方法。Hsp90-珠子用于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形成络合物的蛋白质。这些珠子还被用来从细胞提取物中提取与Hsp90结合的蛋白质，并表明该方法可用于研究细胞内蛋白质之间的相互作用，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的呼叫线表达ERRA和ERRG，并且这些核受体是治疗脑癌的关键靶点。建立了LC-ERRA和LC-ERRG柱并对其进行了表征，并将用于筛选化学混合物和生物基质以寻找新的候选先导药物。

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.