Zika virus (ZIKV) is a mosquito-borne flavivirus that causes severe neurological disorders, such as microcephaly in fetuses. Most recently, an outbreak of ZIKV started in Brazil in 2015. To date, no therapeutic agents have been approved to treat ZIKV infection in the clinic. Here, we screened a small molecule inhibitor that can inhibit the function of ZIKV non-structural protein 2B (NS2B)-NS3 protease (ZIKV NS2B-NS3 protease), thereby interfering with viral replication and spread. First, we identified the half maximal inhibitory concentration (IC50) of compound 3 (14.01 μM), 8 (6.85 μM), and 9 (14.2 μM) and confirmed that they are all non-competitive inhibitors. In addition, we have used the blind molecular docking method to simulate the inhibition area of three non-competitive inhibitors (compound 3, 8, and 9) with the ZIKV NS2B-NS3 protease. The results indicated that the four allosteric binding residues (Gln139, Trp148, Leu150, and Val220) could form hydrogen bonds or non-bonding interactions most frequently with the three compounds. The interaction might induce the reaction center conformation change of NS2B-NS3 protease to reduce catalyzed efficiency. The concentration of compounds required to reduce cell viability by 50% (CC50), and the concentration of compounds required to inhibit virus-induced cytopathic effect by 50% (EC50) of three potential compounds are >200 μM, 2.15 μM (compound 3), > 200 μM, 0.52 μM (compound 8) and 61.48 μM, 3.52 μM (compound 9), and Temoporfin are 61.05 μM, 2 μM, respectively. To select candidate compounds for further animal experiments, we analyzed the selectivity index (SI) of compound 3 (93.02), 8 (384.61), 9 (17.46), and Temoporfin (30.53, FDA-approved drug against cancer). Compound 8 has the highest SI value. Therefore, compound 8 was selected for verification in animal models. In vivo, compound 8 significantly delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in a ZIKV-infected suckling mouse model. We conclude that compound 8 is worth further investigation for use as a potential future therapeutic agent against ZIKV infection.

中文翻译：

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开发损害寨卡病毒复制的 NS2B-NS3 蛋白酶抑制剂。

寨卡病毒 (ZIKV) 是一种由蚊子传播的黄病毒，可引起严重的神经系统疾病，例如胎儿的小头畸形。最近，ZIKV 于 2015 年在巴西爆发。迄今为止，临床上尚未批准用于治疗 ZIKV 感染的治疗药物。在这里，我们筛选了一种小分子抑制剂，可以抑制 ZIKV 非结构蛋白 2B (NS2B)-NS3 蛋白酶（ZIKV NS2B-NS3 蛋白酶）的功能，从而干扰病毒的复制和传播。首先，我们确定了化合物 3 (14.01 μM)、8 (6.85 μM) 和 9 (14.2 μM) 的半数最大抑制浓度 (IC50)，并确认它们都是非竞争性抑制剂。此外，我们还使用盲分子对接方法模拟了三种非竞争性抑制剂（化合物 3、8 和 9）与 ZIKV NS2B-NS3 蛋白酶的抑制区域。结果表明，四个变构结合残基（Gln139、Trp148、Leu150 和 Val220）最常与这三种化合物形成氢键或非键相互作用。这种相互作用可能会导致 NS2B-NS3 蛋白酶的反应中心构象发生变化，从而降低催化效率。三种潜在化合物的使细胞活力降低 50% (CC50) 所需的化合物浓度和抑制病毒引起的细胞病变效应 50% (EC50) 所需的化合物浓度分别为 >200 μM、2.15 μM（化合物 3） , > 200 μM，0.52 μM（化合物 8）和 61.48 μM，3.52 μM（化合物 9），替莫泊芬分别为 61.05 μM，2 μM。为了选择用于进一步动物实验的候选化合物，我们分析了化合物 3 (93.02)、8 (384.61)、9 (17.46) 和 Temoporfin (30.53, FDA 批准的抗癌药物）。化合物 8 具有最高的 SI 值。因此，选择化合物8进行动物模型验证。在体内，化合物 8 在 ZIKV 感染的乳鼠模型中显着延迟了 ZIKV 诱导的致死率和疾病症状，并减少了 ZIKV 诱导的体重减轻。我们得出结论，化合物 8 值得进一步研究，用作未来潜在的 ZIKV 感染治疗剂。