去泛素化酶UL36的抑制剂LDN57444抑制马立克氏病病毒的增殖  

Suppression of replication of Marek’s disease virus by deubiquitinase UL36 inhibitor LDN57444

作　　者：王文婧[1] 范青松 刘宇航[1] 王婷[1] 邓国辉[1] 谢知航 王梦涵 吕岩[1] 许家翠 艾永兴[1] WANG Wenjing;FAN Qingsong;LIU Yuhang;WANG Ting;DENG Guohui;XIE Zhihang;WANG Menghan;LYU Yan;XU Jiacui;AI Yongxing(Key Laboratory of Zoonosis Research,Ministry of Education,College of Animal Science,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物科学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2022年第10期1949-1957,共9页Chinese Journal of Veterinary Science

基　　金：吉林省自然科学基金资助项目(20180101244JC);国家自然科学基金资助项目(31272528);吉林大学大学生创新创业训练计划资助项目(202110183X629)。

摘　　要：马立克氏病病毒(MDV)引起的鸡马立克氏病(MD)可用疫苗进行防御,但疫苗无法阻止MDV对鸡的感染以及MDV的传播和毒力的增强,MD仍为养禽业的重要威胁。而抑制细胞内MDV增殖是增强疫苗防御效果的重要策略,化学药物无疑是重要的选择之一。本研究在前期研究基础上,以MDV编码的去泛素化酶UL36为靶点,经高通量筛选,从蛋白酶抑制剂库中筛选出对UL36去泛素化酶的活性具有抑制作用的化合物LDN57444,应用酶抑制动力学方法揭示,LDN57444以非竞争性方式抑制UL36去泛素化酶活性,IC_(50)为3.10μmol/L。同源建模结合分子对接分析发现,LDN57444结合于UL36-480的C末端口袋中,通过氢键、Pi-Sigma作用、Pi-Pi堆积作用、Pi-Alkyl作用等方式与UL36-480互作,进而抑制UL36-480的活性。进一步在细胞水平,应用MDV致CEF的CPE模型分析LDN57444对MDV在CEF细胞中的增殖的抑制作用,结果发现,5,20μmol/L的LDN57444可显著抑制CPE的形成,并抑制MDV在CEF细胞内的增殖(P<0.01)。同时,应用MD鸡模型分析LDN57444是否可以抑制MDV在鸡体内的增殖,结果发现,每日高于0.5 mg/kg给药可显著抑制MDV在鸡体内的增殖(P<0.01)。本研究结果表明,化合物LDN57444可以抑制MDV在细胞及动物体内增殖,为进一步研制抗MD药物提供了可靠的依据。Marek’s disease(MD) caused by Marek’s disease virus(MDV) is preventable by vaccines, while the vaccine does not prevent the infection of MDV on chicken, the transmission and virulence enhancement of MDV.Since MD is still a serious threat to the poultry industry, it is an important strategy to enhance the efficacy of vaccines by inhibiting the intracellular MDV replication, and chemical drugs are the important options.In this study, the MDV-encoded deubiquitinase UL36 was used as the target for high-throughput screening based on the previous research.The results showed that compound LDN57444 screened from the protease inhibitor library had inhibitory effect on the activity of UL36.Enzyme inhibition kinetics results revealed that LDN57444 inhibited the deubiquitinase activity of UL36 in a non-competitive manner with ICof 3.10 μmol/L.Analysis using homology modeling combined with molecular docking showed that LDN57444 bound to the C-terminal pocket of UL36-480,and interacted with UL36-480 through hydrogen bond, Pi-Sigma interaction, Pi-Pi stacked, Pi-Alkyl interaction, etc.,to inhibit the activity of UL36-480.Analysis of the inhibitory effect of LDN57444on the replication of MDV in CEF cells suggested that CPE was suppressed by LDN57444,and the replication of MDV in CEF cells was inhibited significantly as well(P<0.01).Furthermore,significant inhibition of the MDV replication in chickens(P<0.01) was found after daily administration of more than 0.5 mg/kg of LDN57444 using MDV-infected chicken model.In summary,in this study,we demonstrated that the compound LDN57444 inhibit the replication of MDV at the cell and animal levels,which provides a reliable basis for future development of anti-MD drugs.

关 键 词：去泛素化酶UL36 马立克氏病病毒 抑制剂LDN57444 CEF细胞 

分 类 号：S831.7[农业科学—畜牧学]