Pernomian Larissa, Gomes Mayara Santos, Moreira Josimar Dornelas, da Silva Carlos Henrique Tomich de Paula, Rosa Joaquin Maria Campos, Cardoso Cristina Ribeiro de Barros
Department of Biosciences Applied to Pharmacy, Faculty of Pharmaceutical Sciences from Ribeirao Preto, University of Sao Paulo, P.O. Box: 14040-903, Ribeirao Preto, SP, Brazil.
Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences from Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
Curr Radiopharm. 2017;10(1):16-20. doi: 10.2174/1874471010666170208152420.
One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol.
合理药物开发的基石之一是测量源自配体 - 受体相互作用的分子参数，这有助于定义治疗窗口。在过去几十年中，放射性配体结合作为实现此类目的的关键方法，在该领域做出了重要贡献。然而，其局限性促使人们开发更精确的技术来确定这些参数。例如，与放射性废物相关的安全风险、放射性同位素的昂贵且受控制的处置、亲和力分析对放射性示踪剂分离的依赖性以及基于单位点数学模型的数据拟合，使得放射性配体结合在提供配体与G蛋白偶联受体（GPCR）实际亲和力构象的测量方面成为一种次优方法。高通量筛选（HTS）测定的当前进展显著扩展了监测配体亲和力的灵敏方法的选择。由纤细深海虾萤光素酶改造而来的新型生物发光供体纳米荧光素酶（Nluc）的出现，使得能够采用生物发光共振能量转移（BRET）来监测配体结合。这种名为基于Nluc的BRET（NanoBRET）结合测定的新方法是一种实时均相接近度测定法，它克服了放射性配体结合的局限性，同时确保了亲和力测量的质量。在这里，我们阐述了NanoBRET方案的主要优点以及放射性配体结合作为应用于药物发现的药理学工具箱中的分子方法所存在的不良缺点。此外，我们还为NanoBRET技术在涉及寡聚化和/或功能偏向选择性的多状态结合机制的亲和力测定中的应用提供了新的视角。这个新视角是基于赋予接近度NanoBRET方案的实时均相性所需的特定生物物理标准提出的。
