闪烁接近测定(SPA)是一种放射性同位素技术格式,用于测量广泛的生物相互作用,包括药物-靶标结合亲和力研究。该测定法本质上是均匀的,因为它依赖于“混合和度量”格式。它不涉及过滤步骤以将结合的配体与游离配体分离,如在传统的受体结合测定中的情况。对于G蛋白偶联受体(GPCRs),它已经表明,最佳的结合动力学,除了配体的高亲和力之外,可以导致更理想的药理学概况。然而,传统的评估动力学参数的技术往往是繁琐和费力的。因此,我们旨在评估SPA是否可以作为GPCRs上实时受体结合动力学测量的替代平台。要做到这一点,我们首先验证了SPA技术在原型A类GPCR上的平衡结合研究,人腺苷A1受体(hA1R)。与经典动力学研究不同,SPA技术允许我们几乎实时地研究结合动力学过程,这在过滤分析中是不可能的。为了证明这种技术在动力学方面的可靠性,我们进行了所谓的竞争协会实验。可靠且快速地确定了未标记的hA1R配体的缔合和解离速率常数(kon和koff),并与同时进行的传统过滤测定的相同参数非常吻合。总之,SPA是确定药物-靶标相互作用的动力学特征的非常有前途的技术。其鲁棒性和高通量的潜力可能使该技术成为进一步动力学研究的首选。
Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a \"mix and measure\" format. It does not involve a filtration step to separate bound from free ligand as is the
case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.