Guo Dong, van Dorp Erika J H, Mulder-Krieger Thea, van Veldhoven Jacobus P D, Brussee Johannes, Ijzerman Adriaan P, Heitman Laura H
Leiden University, Leiden, the Netherlands.
J Biomol Screen. 2013 Mar;18(3):309-20. doi: 10.1177/1087057112464776. Epub 2012 Oct 23.
The concept of ligand-receptor binding kinetics is emerging as an important parameter in the early phase of drug discovery. Since the currently used kinetic assays are laborious and low throughput, we developed a method that enables fast and large format screening. It is a so-called dual-point competition association assay, which measures radioligand binding at two different time points in the absence or presence of unlabeled competitors. Specifically, this assay yields the kinetic rate index (KRI), which is a measure for the binding kinetics of the unlabeled ligands screened. As a prototypical drug target, the adenosine A(1) receptor (A(1)R) was chosen for assay validation and optimization. A screen with 35 high-affinity A(1)R antagonists yielded seven compounds with a KRI value above 1.0, which indicated a relatively slow dissociation from the target. All other compounds had a KRI value below or equal to 1.0, predicting a relatively fast dissociation rate. Several compounds were selected for follow-up kinetic quantifications in classical kinetic assays and were shown to have kinetic rates that corresponded to their KRI values. The dual-point assay and KRI value may have general applicability at other G-protein-coupled receptors, as well as at drug targets from other protein families.
配体-受体结合动力学的概念正在成为药物发现早期阶段的一个重要参数。由于目前使用的动力学检测方法费力且通量低，我们开发了一种能够进行快速和大规模筛选的方法。这是一种所谓的双点竞争结合测定法，它在不存在或存在未标记竞争剂的情况下，于两个不同时间点测量放射性配体结合情况。具体而言，该测定法产生动力学速率指数（KRI），它是对所筛选的未标记配体结合动力学的一种度量。作为典型的药物靶点，选择腺苷A(1)受体（A(1)R）进行测定验证和优化。对35种高亲和力A(1)R拮抗剂进行的筛选产生了7种KRI值高于1.0的化合物，这表明其与靶点的解离相对较慢。所有其他化合物的KRI值低于或等于1.0，预示着解离速率相对较快。选择了几种化合物在经典动力学测定中进行后续动力学定量，结果显示其动力学速率与其KRI值相符。双点测定法和KRI值可能在其他G蛋白偶联受体以及其他蛋白质家族的药物靶点中具有普遍适用性。
