Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1′-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

中文翻译：

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GS-441524-二磷酸核糖衍生物作为 SARS-CoV-2 和其他病毒宏结构域的纳摩尔粘合剂和荧光偏振示踪剂

可以结合或水解蛋白质腺苷二磷酸核糖基化（ADP-核糖基化）的病毒大结构域已成为抗病毒药物开发的有希望的靶点。许多抑制剂的开发工作都是针对严重急性呼吸综合征冠状病毒 2 宏结构域 1 (SARS-CoV-2 Mac1)。然而，仍然缺乏有效的病毒大结构域抑制剂，最好的抑制剂仍在微摩尔范围内。基于瑞德西韦前体GS-441524和我们之前的研究，我们设计并合成了 SARS-CoV-2 Mac1 和其他病毒大结构域的有效结合物，包括中东呼吸综合征冠状病毒 (MERS-CoV)、委内瑞拉马脑炎的病毒大结构域病毒（VEEV）和基孔肯雅病毒（CHIKV）。我们表明， GS-441524的 1'-CN 基团促进与所有四种测试的病毒大结构域的结合，同时用简单的苯环封盖GS-441524 -二磷酸核糖的 1''-OH进一步有助于结合。结合这两个结构特征，最好的结合剂对 SARS-CoV-2、MERS-CoV、VEEV 和 CHIKV 宏结构域的结合亲和力比 ADP-核糖增加 20 至 6000 倍。此外，在这些有效的结合剂的基础上，我们开发了两种高灵敏度的荧光偏振示踪剂，它们只需要纳摩尔蛋白质，并且可以有效解析纳摩尔抑制剂的结合亲和力。我们在此描述的发现和探针将有助于未来开发更有效的病毒大结构域抑制剂。