Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

中文翻译：

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一种小分子通过靶向人类巨细胞病毒核出口复合物发挥选择性抗病毒活性。

人类巨细胞病毒（HCMV）是一种重要的病原体，需要新的抗病毒药物。与其他疱疹病毒一样，HCMV 编码由两个亚基 UL50 和 UL53 组成的核出口复合物 (NEC)，这两个亚基的相互作用对于病毒复制至关重要。为了探索小分子是否可以通过抑制 NEC 亚基相互作用来发挥选择性抗病毒活性，我们建立了这些相互作用的均相时间分辨荧光 (HTRF) 测定法，并用它来筛选超过 200,000 个含有化合物的孔。命名为 GK1 和 GK2 的两种化合物在 HTRF 测定中选择性抑制这种相互作用，GK1 在免疫共沉淀测定中也具有活性，表现出比细胞毒性或针对另一种疱疹病毒的活性更有效的抗 HCMV 活性。在显着减少 HCMV 噬斑形成的剂量下，GK1 和 GK2 对病毒蛋白的表达影响很小或没有影响，并减少了细胞亚群中 UL53 与 UL50 在核边缘的共定位。GK1 和 GK2 含有丙烯酰胺部分，预计会与半胱氨酸共价相互作用，而没有这种潜力的类似物缺乏活性。质谱分析显示 GK2 与 UL50 和 UL53 上的多个半胱氨酸结合。然而，用丝氨酸 (C214S) 取代 UL53 的半胱氨酸 214 消除了体外可检测到的 GK1 和 GK2 抑制活性，并且工程化到 HCMV 中的 C214S 取代赋予了对 GK1 的抗性，GK1 是两种抑制剂中更有效的。因此，GK1 通过靶向 NEC 发挥选择性抗病毒活性。对接研究表明，丙烯酰胺将 GK1 或 GK2 的一端与 UL53 口袋内的 C214 相连，从而使分子的另一端在空间上阻碍 UL50，从而防止 NEC 形成。我们的结果证明了用小分子靶向 NEC 可以选择性阻断 HCMV 复制的概念。此类化合物可作为开发抗 HCMV 药物的基础和研究 HCMV 的化学工具。