Here we present data on the mechanism of action of VP-14637 and JNJ-2408068 (formerly R-170591), two small-molecule inhibitors of respiratory syncytial virus (RSV). Both inhibitors exhibited potent antiviral activity with 50% effective concentrations (EC50s) of 1.4 and 2.1 nM, respectively. A similar inhibitory effect was observed in a RSV-mediated cell fusion assay (EC50=5.4 and 0.9 nM, respectively). Several drug-resistant RSV variants were selected in vitro in the presence of each compound. All selected viruses exhibited significant cross-resistance to both inhibitors and contained various single amino acid substitutions in two distinct regions of the viral F protein, the heptad repeat 2 (HR2; mutations D486N, E487D, and F488Y), and the intervening domain between HR1 and HR2 (mutation K399I and T400A). Studies using [3H]VP-14637 revealed a specific binding of the compound to RSV-infected cells that was efficiently inhibited by JNJ-2408068 (50% inhibitory concentration=2.9 nM) but not by the HR2-derived peptide T-118. Further analysis using a transient T7 vaccinia expression system indicated that RSV F protein is sufficient for this interaction. F proteins containing either the VP-14637 or JNJ-2408068 resistance mutations exhibited greatly reduced binding of [3H]VP-14637. Molecular modeling analysis suggests that both molecules may bind into a small hydrophobic cavity in the inner core of F protein, interacting simultaneously with both the HR1 and HR2 domains. Altogether, these data indicate that VP-14637 and JNJ-2408068 interfere with RSV fusion through a mechanism involving a similar interaction with the F protein.

中文翻译：

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小分子 VP-14637 和 JNJ-2408068 通过类似机制抑制呼吸道合胞病毒融合。

在这里，我们提供了两种呼吸道合胞病毒 (RSV) 小分子抑制剂 VP-14637 和 JNJ-2408068（以前称为 R-170591）的作用机制数据。两种抑制剂均表现出有效的抗病毒活性，50% 的有效浓度 (EC50) 分别为 1.4 和 2.1 nM。在 RSV 介导的细胞融合试验中观察到类似的抑制作用（EC50 分别为 5.4 和 0.9 nM）。在每种化合物存在的情况下，在体外选择了几种抗药性 RSV 变体。所有选定的病毒都对这两种抑制剂表现出显着的交叉耐药性，并在病毒 F 蛋白的两个不同区域、七肽重复序列 2（HR2；突变 D486N、E487D 和 F488Y）以及 HR1 之间的插入域中包含各种单氨基酸取代和 HR2（突变 K399I 和 T400A）。使用 [3H]VP-14637 的研究揭示了该化合物与 RSV 感染细胞的特异性结合，该结合被 JNJ-2408068（50% 抑制浓度 = 2.9 nM）有效抑制，但不受 HR2 衍生肽 T-118 的抑制。使用瞬时 T7 痘苗表达系统的进一步分析表明 RSV F 蛋白足以进行这种相互作用。含有 VP-14637 或 JNJ-2408068 抗性突变的 F 蛋白表现出大大降低的 [3H]VP-14637 结合。分子模型分析表明，这两种分子都可能结合到 F 蛋白内核中的一个小的疏水腔中，同时与 HR1 和 HR2 结构域相互作用。总之，这些数据表明 VP-14637 和 JNJ-2408068 通过涉及与 F 蛋白类似相互作用的机制干扰 RSV 融合。