Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, acts as a nucleotidyl transferase that catalyzes ATP and GTP to form cyclic GMP-AMP (cGAMP) and plays a critical role in innate immunity. Hyperactivation of cGAS-STING signaling contributes to hyperinflammatory responses. Therefore, cGAS is considered a promising target for the treatment of inflammatory diseases. Herein, we report the discovery and identification of several novel types of cGAS inhibitors by pyrophosphatase (PP_iase)-coupled activity assays. Among these inhibitors, 1-(1-phenyl-3,4-dihydro-1H-pyrrolo[1,2-a]pyrazin-2-yl)prop-2-yn-1-one (compound 3) displayed the highest potency and selectivity at the cellular level. Compound 3 exhibited better inhibitory activity and pathway selectivity than RU.521, which is a selective cGAS inhibitor with anti-inflammatory effects in vitro and in vivo. Thermostability analysis, nuclear magnetic resonance and isothermal titration calorimetry assays confirmed that compound 3 directly binds to the cGAS protein. Mass spectrometry and mutation analysis revealed that compound 3 covalently binds to Cys419 of cGAS. Notably, compound 3 demonstrated promising therapeutic efficacy in a dextran sulfate sodium (DSS)-induced mouse colitis model. These results collectively suggest that compound 3 will be useful for understanding the biological function of cGAS and has the potential to be further developed for inflammatory disease therapies.

环 GMP-AMP 合酶 (cGAS) 是一种胞质 DNA 传感器，充当核苷酸转移酶，催化 ATP 和 GTP 形成环 GMP-AMP (cGAMP)，在先天免疫中发挥关键作用。 cGAS-STING 信号的过度激活会导致过度炎症反应。因此，cGAS被认为是治疗炎症性疾病的一个有前景的靶点。在此，我们报告了通过焦磷酸酶（PP _i ase）偶联活性测定发现和鉴定了几种新型 cGAS 抑制剂。在这些抑制剂中，1-(1-苯基-3,4-二氢-1 H-吡咯并[1,2-a]吡嗪-2-基)丙-2-yn-1-酮(化合物3 )表现出最高的细胞水平的效力和选择性。化合物3比RU.521表现出更好的抑制活性和途径选择性，RU.521是一种选择性cGAS抑制剂，在体外和体内具有抗炎作用。热稳定性分析、核磁共振和等温滴定量热测定证实化合物3直接与 cGAS 蛋白结合。质谱和突变分析表明化合物3与 cGAS 的 Cys419 共价结合。值得注意的是，化合物3在葡聚糖硫酸钠 (DSS) 诱导的小鼠结肠炎模型中表现出良好的治疗效果。这些结果共同表明化合物3将有助于理解 cGAS 的生物学功能，并有可能进一步开发用于炎症性疾病治疗。