Stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that serves as a molecular hub for activation of interferon and inflammatory cytokine response by multiple cellular DNA sensors. Not surprisingly, STING has been demonstrated to play an important role in host defense against microorganisms and pharmacologic activation of STING is considered as an attractive strategy to treat viral diseases and boost antitumor immunity. In light of this we established a HepAD38-derived reporter cell line that expresses firefly luciferase in response to the activation of cyclic GMP-AMP synthase (cGAS)-STING pathway for high throughput screening (HTS) of small molecular human STING agonists. This cell-based reporter assay required only 4 h treatment with a reference STING agonist to induce a robust luciferase signal and was demonstrated to have an excellent performance in HTS format. By screening 16,000 compounds, a dispiro diketopiperzine (DSDP) compound was identified to induce cytokine response in a manner dependent on the expression of functional human STING, but not mouse STING. Moreover, we showed that DSDP induced an interferon-dominant cytokine response in human skin fibroblasts and peripheral blood mononuclear cells, which in turn potently suppressed the replication of yellow fever virus, dengue virus and Zika virus. We have thus established a robust cell-based assay system suitable for rapid discovery and mechanistic analyses of cGAS-STING pathway agonists. Identification of DSDP as a human STING agonist enriches the pipelines of STING-targeting drug development for treatment of viral infections and cancers.

干扰素基因刺激物（STING）是一种内质网跨膜蛋白，可作为分子枢纽，通过多个细胞DNA传感器激活干扰素和炎症细胞因子。毫不奇怪，已证明STING在宿主对抗微生物的防御中起重要作用，并且STING的药理活化被认为是治疗病毒性疾病和增强抗肿瘤免疫力的有吸引力的策略。有鉴于此，我们建立了一个HepAD38衍生的报告细胞系，该表达细胞表达萤火虫荧光素酶，以响应环状GMP-AMP合酶（cGAS）-STING途径的激活，用于小分子人STING激动剂的高通量筛选（HTS）。这种基于细胞的报告基因检测仅需用参考STING激动剂处理4小时即可诱导出强大的荧光素酶信号，并被证明在HTS格式下具有出色的性能。通过筛选16,000种化合物，确定了双螺二酮哌嗪（DSDP）化合物以依赖功能性人STING而非小鼠STING的表达的方式诱导细胞因子应答。此外，我们表明DSDP诱导人皮肤成纤维细胞和外周血单核细胞中干扰素占主导的细胞因子反应，进而有效地抑制了黄热病病毒，登革热病毒和寨卡病毒的复制。因此，我们已经建立了一个健壮的基于细胞的分析系统，适用于cGAS-STING途径激动剂的快速发现和机理分析。