Faculty for Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, 90220 Oulu, Finland.
Cell Rep Methods. 2021 Dec 20;1(8):100121. doi: 10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11.
Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a G protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in and will help in ADP-ribosylation research and drug discovery.
与 ADP-核糖基相互作用的蛋白质通常参与与疾病相关的途径或病毒感染，使其成为有吸引力的药物靶点。我们提出了一种稳健且易于使用的测定法，适用于单聚和多聚 ADP-核糖基的水解或非水解结合物。该技术依赖于基于 G 蛋白α亚基肽（GAP）的 C 末端标签，允许在特定位置引入半胱氨酸连接的单聚和多聚 ADP-核糖基或类似物。通过将 GAP 标签和 ADP-核糖基结合物融合到荧光蛋白中，我们生成了强大的 FRET 供体，并证实了与 22 种已知的 ADP-核糖基结合物的相互作用。我们用 SARS-CoV-2 nsp3 结构域证明了这种测定法在高通量筛选抑制剂方面的适用性，发现苏拉明是一种中等亲和力但非特异性的抑制剂。与纳米荧光素酶融合的高亲和力 ADP-核糖基结合物补充了这项技术，使 ADP-核糖基化蛋白的简单印迹检测成为可能。所有这些工具都可以在 中生产，并将有助于 ADP-核糖基化研究和药物发现。
