Immobilized Proteins In Drug Discovery

一种基于目标受体、跨膜受体（细胞、核或线粒体）在流动色谱系统中包含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。我们已经开发了包含烟碱受体的各种亚型的色谱。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。通过将SIRT6蛋白固定在开放管状毛细管表面和磁珠表面，扩展了一般方法。SIRT6- mb用于从化学和植物混合物中提取与SIRT6结合的小分子。开发并优化了其他SIRT6活性测定方法，用于鉴定SIRT6激活剂和SIRT6抑制剂。利用这种方法，从复杂基质中鉴定出了几种新的SIRT6激活剂。鉴定出的化合物将在体内进行活性测试。

A new method for high throughput screening has been developed based upon the inclusion of the target receptors, transmembrane receptors (cellular, nuclear or mitochondrial) 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 various subtypes of the nicotinic receptor. 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 general approach has been expanded through the immobilization of SIRT6 protein onto the surface of an open tubular capillary and on the surface of magnetic beads. The SIRT6-MBs were used to extract small molecules that bind to SIRT6 from chemical and botanical mixtures. Additional SIRT6 activity assays were developed and optimized for the identification of SIRT6 activators and SIRT6 inhibitors. Using this approach, several novel SIRT6 activators have been identified from complex matrixes. The identified compounds will be tested for activity in vivo.