Wang Xingqi, Gou Lingshan, Gao Yuzhi, Huang Yuqing, Kuai Rui, Li Yu, Wang Yujing, Chen Yanhong, Li Jun, Cheng Chao, Feng Zhaojun, Wu Xuefeng, Yao Ruiqin
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, 101 Shanghai Road, Xuzhou 221116, Jiangsu, China.
Center for Genetic Medicine, Xuzhou Maternity and Child Health Care Hospital, Xuzhou 221009, Jiangsu, China.
Int Immunopharmacol. 2020 Nov;88:106898. doi: 10.1016/j.intimp.2020.106898. Epub 2020 Aug 28.
The NLRP3 inflammasome is an important mediator of inflammatory responses and its regulation is an active area of research. RalA is a Ras-like GTPase, which play pivotal roles in the biology of cells. So far, there have been very few studies on RalA regulating inflammatory responses. Bioinformatics analysis predicted that RalA might participate in the regulatory network of NLRP3 inflammasome, which has been confirmed in THP-1 macrophages. After virtual screening of compounds, it was found that levonidazole selected from our virtual small molecule compound library has the potential to bind to RalA. Of note, the interaction of RalA/levornidazole was verified by Surface Plasmon Resonance-Biacore T200, LC/MS analysis and Western blotting analysis. Molecular dynamics simulations revealed that the conformational changes of RalA might be regulated by levornidazole. Additionally, IL-1β/IL-18 secretion from ATP + LPS stimulated THP-1-derived macrophages was RalA-dependently suppressed by levornidazole, suggesting that RalA might have an inhibitory effect on NLRP3 inflammasome activation. The results of co-immunoprecipitation and RalA depletion experiments showed that levornidazole could induce RalA to block the assembly of NLRP3/ASC/pro-caspase-1 complex, thereby reducing the levels of cleaved-caspase-1 and IL-1β/IL-18 secretion. Our study has suggested an anti-inflammatory function of RalA and identified its targeting chemical compound. Overall, this study clarifies a novel pharmacological mechanism by which RalA/levornidazole inhibits NLRP3 inflammasome activation and IL-1β/IL-18 secretion.
NLRP3炎性小体是炎症反应的重要介质，对其调控是一个活跃的研究领域。RalA是一种类Ras GTP酶，在细胞生物学中发挥关键作用。到目前为止，关于RalA调节炎症反应的研究非常少。生物信息学分析预测RalA可能参与NLRP3炎性小体的调控网络，这在THP-1巨噬细胞中得到了证实。在对化合物进行虚拟筛选后，发现从我们的虚拟小分子化合物库中选出的左硝唑有可能与RalA结合。值得注意的是，通过表面等离子体共振-Biacore T200、液相色谱/质谱分析和蛋白质免疫印迹分析验证了RalA/左硝唑的相互作用。分子动力学模拟显示，左硝唑可能调节RalA的构象变化。此外，左硝唑以RalA依赖的方式抑制ATP + LPS刺激的THP-1来源巨噬细胞分泌IL-1β/IL-18，这表明RalA可能对NLRP3炎性小体的激活具有抑制作用。免疫共沉淀和RalA缺失实验结果表明，左硝唑可诱导RalA阻断NLRP3/ASC/前半胱天冬酶-1复合物的组装，从而降低裂解的半胱天冬酶-1水平和IL-1β/IL-18的分泌。我们的研究表明了RalA的抗炎功能并鉴定了其靶向化合物。总体而言，本研究阐明了一种新的药理学机制，即RalA/左硝唑抑制NLRP3炎性小体激活和IL-1β/IL-18分泌。
