Immobilized Proteins In Drug Discovery

一种新的高通量筛选方法已经被开发出来，该方法是基于将目标受体、跨膜受体(细胞、核或线粒体)包含在流动色谱系统中。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。我们开发了包含尼古丁受体不同亚型的柱子。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。通过将SIRT6蛋白固定在开放的管状毛细管表面和磁珠表面，扩展了一般的方法。SIRT6-MBS用于从化学和植物混合物中提取与SIRT6结合的小分子。为了鉴定SIRT6激活剂和SIRT6抑制剂，还开发和优化了其他SIRT6活性测定方法。利用这种方法，已经从复杂的基质中鉴定了几个新的SIRT6激动子。已鉴定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.