There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine—which also targets 2C—but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

非常需要抗病毒药物来治疗肠道病毒（EV）和鼻病毒（RV）感染，这种感染可能很严重，有时甚至危及生命。保守的非结构蛋白2C是AAA + ATPase，是药物开发的有希望的目标。在这里，我们目前针对化合物的EV-A71和几个EV-B物种成员的2C蛋白，但不是脊髓灰质炎病毒（PV）（EV-C物种）的化合物的结构-活性关系研究。该化合物在结构上与美国食品药品监督管理局（FDA）批准的药物氟西汀（也针对2C）有关，但具有良好的化学性质。我们鉴定了几种具有增强的抗病毒效力和广泛活性的化合物。四种化合物具有广谱的EV和RV活性，并能抑制当代引起公众关注的EV菌株，包括EV-A71，柯萨奇病毒（CV）-A24v和EV-D68。重要的是，与（S）-氟西汀，这些化合物不再具有神经活性。通过提高针对这些抑制剂之一的抗性EV-A71，CV-B3和EV-D68变体，我们确定了新的2C抗性突变。这些突变的逆向工程揭示了抗性发展的保守机制。抗药性病毒首先在2C的α2螺旋中或附近具有突变。这种突变破坏了化合物的结合并提供了抗药性，但这是以病毒适应为代价的。然后在远距离定位的2C残基处获得额外的突变，以增加抗性和/或补偿适应性的丧失。利用计算方法确定EV-A71和PV 2C晶体结构中α2螺旋附近的溶剂可及通道，提出了抑制剂的保守结合口袋。