We screened a library of bioactive small molecules for activators and inhibitors of innate immune signaling through IRF3 and NFkB pathways with the goals of advancing pathway understanding and discovering probes for immunology research. We used high content screening to measure the translocation from the cytoplasm to nucleus of IRF3 and NFkB in primary human macrophages; these transcription factors play a critical role in the activation of STING and other pro-inflammatory pathways. Our pathway activator screen yielded a diverse set of hits that promoted nuclear translocation of IRF3 and/or NFkB, but the majority of these compounds did not cause activation of downstream pathways. Screening for antagonists of the STING pathway yielded multiple kinase inhibitors, some of which inhibit kinases not previously known to regulate the activity of this pathway. Structure–activity relationships (SARs) and subsequent chemical proteomics experiments suggested that MAPKAPK5 (PRAK) is a kinase that regulates IRF3 translocation in human macrophages. Our work establishes a high content screening approach for measuring pro-inflammatory pathways in human macrophages and identifies novel ways to inhibit such pathways; among the targets of the screen are several molecules that may merit further development as anti-inflammatory drugs.

中文翻译：

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巨噬细胞中的高内涵屏幕可识别STING-IRF3和NFkB信号传导的小分子调节剂。

我们筛选了一个生物活性小分子文库，用于通过IRF3和NFkB途径激活和抑制先天性免疫信号转导，目的是促进对途径的了解和发现用于免疫学研究的探针。我们使用高含量的筛选方法来检测人类原代巨噬细胞中IRF3和NFkB从细胞质到细胞核的转运；这些转录因子在STING和其他促炎途径的激活中起关键作用。我们的途径激活剂筛选产生了一系列促进IRF3和/或NFkB核易位的命中点，但这些化合物中的大多数并未引起下游途径的激活。筛选STING途径的拮抗剂可产生多种激酶抑制剂，其中一些抑制以前未知的激酶来调节该途径的活性。结构-活性关系（SARs）和随后的化学蛋白质组学实验表明，MAPKAPK5（PRAK）是一种调节人类巨噬细胞中IRF3易位的激酶。我们的工作建立了一种高含量的筛选方法来测量人类巨噬细胞中的促炎途径，并确定了抑制这种途径的新方法。在筛选目标中，有几种分子可能需要作为抗炎药进一步发展。我们的工作建立了一种高含量的筛选方法来测量人类巨噬细胞中的促炎途径，并确定了抑制这种途径的新方法。在筛选目标中，有几种分子可能需要作为抗炎药进一步发展。我们的工作建立了一种高含量的筛选方法来测量人类巨噬细胞中的促炎途径，并确定了抑制这种途径的新方法。在筛选目标中，有几种分子可能需要作为抗炎药进一步发展。