半胱氨酸天冬氨酸蛋白水解酶caspase-1抑制剂体外筛选模型的构建及应用

Establishment and application of screening model for caspase-1 inhibitor in vitro

半胱氨酸天冬氨酸特异性蛋白水解酶caspase-1(Cystein-containing aspartat-specific protease-1)通过介导白介素-1β和18(interleukin-1β,IL-1β;interleukin-18,IL-18)及Gasdermin D的成熟,启动炎症反应并诱导细胞焦亡,参与多种疾病的发生发展.因此构建出高效稳定的caspase-1抑制剂筛选模型对于发现新型caspase-1小分子抑制剂并深入研究caspase-1功能具有重要意义.通过应用大肠杆菌系统克隆表达可溶的人源caspase-1蛋白活性区域,以VX-765为阳性抑制剂,建立caspase-1抑制剂体外筛选模型,对实验室化合物文库中868个化合物进行筛选.结果显示,黄酮类化合物E-3-(4-(diethylamino)-benzylidene)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-methoxychroman-4-one(CIB-1710),在体外对caspase-1活性有良好的抑制作用,其IC50为36.27μmol/L.进一步研究发现CIB-1710在小鼠单核巨噬细胞系J774A.1上可抑制由脂多糖(lipopolysaccharide,LPS)及尼日利亚菌素(nigericin)刺激所诱导的IL-1β释放,且在高浓度下不影响细胞活力;分子对接显示CIB-1710可与caspase-1蛋白的催化活性结构域高亲和力结合来抑制蛋白的剪切活性;透析实验显示CIB-1710可逆抑制caspase-1剪切活性.综上所述,本研究所建立的caspase-1抑制剂体外筛选模型为发现新型高效的caspase-1小分子抑制剂提供了高效低成本的途径,所发现的活性化合物CIB-1710可作为caspase-1抑制剂开展进一步研究.(图7表1参30)

Cysteine-containing aspartate-specific protease-1(Caspase-1)plays a critical role in innate immunity,which initiates an inflammatory response and induces pyroptosis by mediating the maturation of interleukin(IL)-1β,IL-18,and gasdermin D participating in the development and progression of multiple diseases.Therefore,the establishment of an efficient and stable caspase-1 inhibitor screening model is of great significance for the discovery of novel caspase-1 small-molecule inhibitors and in-depth investigation of caspase-1 functions.In this study,the soluble human caspase-1 protein was cloned and expressed in Escherichia coli.Using VX-765 as a positive inhibitor,an in vitro screening model for caspase-1 inhibitors was established and an in-house library consisting of 868 compounds was screened using this model.The results showed that a flavonoid compound,(E)-3-(4-(diethylamino)-benzylidene)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-methoxychroman-4-one(CIB-1710),had a significant inhibitory effect on caspase-1 activity in vitro,with an IC50 of 36.27μmol/L.Further studies revealed that CIB-1710,acting on the mouse mononuclear macrophage cell line J774A.1,inhibited the release of IL-1βinduced by lipopolysaccharide and nigericin without affecting cellular viability.Molecular docking showed t hat CIB-1710 c an b ind to t he c atalytically active d omain of c aspase-1 w ith high a ffinity to inhibit i ts cleavage activity.Dialysis experiments showed that CIB-1710 reversibly inhibited the activity of caspase-1.The in vitro screening model for caspase-1 inhibitors established in this study provides an efficient and low-cost way to discover novel and efficient caspase-1 small-molecule inhibitors,and the active compound CIB-1710 warrants further investigation as a novel caspase-1 inhibitor.