Kyasanur forest disease is a neglected zoonotic disease caused by a single-stranded RNA-based flavivirus, the incidence of which was first recorded in 1957 in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological complications leading to mortality. The current treatment regimens are symptomatic and supportive, and no targeted therapies are available for this disease. In this study, we evaluated the ability of FDA-approved drugs sofosbuvir (and its active metabolite) and Dasabuvir to inhibit the RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA-dependent RNA polymerase domain was expressed in Escherichia coli, and RNA-dependent RNA polymerase activity was demonstrated with the purified protein. The RNA-dependent RNA polymerase assay conditions were optimized, followed by the determination of apparent Km,ATP to validate the enzyme preparation. Half maximal-inhibitory concentrations against RNA-dependent RNA polymerase activity were determined for Sofosbuvir and its active metabolite. Dasabuvir did not show detectable inhibition with the tested conditions. This is the first demonstration of the inhibition of RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus with small molecule inhibitors. These initial findings can potentially facilitate the discovery and development of targeted therapies for treating Kyasanur forest disease.

中文翻译：

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Sofosbuvir 及其三磷酸代谢物抑制 Kyasanur 森林病病毒非结构蛋白 5 的 RNA 依赖性 RNA 聚合酶活性

Kyasanur 森林病是一种被忽视的人畜共患疾病，由基于单链 RNA 的黄病毒引起，其发病率于 1957 年首次记录在印度南部。Kyasanur 森林病病毒通过被感染的刺血蜱叮咬传播给猴子和人类。Kyasanur 森林病是一种发热性疾病，在严重的情况下，会导致神经系统并发症，从而导致死亡。目前的治疗方案是对症和支持疗法，没有针对该病的靶向治疗。在这项研究中，我们评估了 FDA 批准的药物 sofosbuvir（及其活性代谢物）和 Dasabuvir 抑制 Kyasanur 森林病病毒 NS5 蛋白的 RNA 依赖性 RNA 聚合酶活性的能力。含有 N 末端甲基转移酶结构域和 C 末端 RNA 依赖性 RNA 聚合酶结构域的 NS5 蛋白在大肠杆菌中表达，并用纯化的蛋白质证明了 RNA 依赖性 RNA 聚合酶活性。优化 RNA 依赖性 RNA 聚合酶测定条件，然后测定表观 Km、ATP 以验证酶制剂。确定了 Sofosbuvir 及其活性代谢物对 RNA 依赖性 RNA 聚合酶活性的半数最大抑制浓度。Dasabuvir 在测试条件下未显示可检测的抑制作用。这是小分子抑制剂抑制 Kyasanur 森林病病毒 NS5 蛋白的 RNA 依赖性 RNA 聚合酶活性的首次证明。