We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC50 values ranging from 35 pM to 35 nM; the most potent of these - 56 and 64 residue hyperstable proteins made using the second approach - are roughly six times more potent on a per mass basis (IC50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.

中文翻译：

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从头设计皮摩尔SARS-CoV-2小蛋白抑制剂。

我们使用两种方法设计与人ACE2受体结合位点附近的SARS-CoV-2 Spike蛋白的受体结合结构域（RBD）形状和化学互补的蛋白质。支架围绕与RBD相互作用的ACE2螺旋构建，或者从头设计的支架与RBD对接以识别新的结合模式。在这两种情况下，首先通过计算机优化设计的序列，然后通过实验优化靶点的结合，折叠和稳定性。九种设计以100pM至10nM的亲和力结合RBD，并阻断了Vero E6细胞的真实SARS-CoV-2感染，IC50值范围为35 pM至35 nM。其中最有效的-使用第二种方法制备的56和64个残基超稳定蛋白-按质量计算的效价大约高六倍（IC50〜0。比目前报道的最佳单克隆抗体高23 ng / ml）。SARS-CoV-2钉胞外域三聚体与两种最有效的微型结合剂的复合物的低温电子显微镜结构表明，设计的结构及其与RBD的结合相互作用几乎与计算模型相同，并且所有三个RBD单个Spike中的蛋白质可以同时参与。这些超稳定的迷你结合剂为新的SARS-CoV-2治疗剂提供了有希望的起点，并说明了计算蛋白设计的功能，可快速产生针对大流行性疾病威胁的潜在治疗剂。SARS-CoV-2钉胞外域三聚体与两种最有效的微型结合剂的复合物的低温电子显微镜结构表明，设计的结构及其与RBD的结合相互作用几乎与计算模型相同，并且所有三个RBD单个Spike中的蛋白质可以同时参与。这些超稳定的迷你结合剂为新的SARS-CoV-2治疗剂提供了有希望的起点，并说明了计算蛋白设计的功能，可快速产生针对大流行性疾病威胁的潜在治疗剂。SARS-CoV-2钉胞外域三聚体与两种最有效的微型结合剂的复合物的低温电子显微镜结构表明，设计的结构及其与RBD的结合相互作用几乎与计算模型相同，并且所有三个RBD单个Spike中的蛋白质可以同时参与。这些超稳定的迷你结合剂为新的SARS-CoV-2治疗剂提供了有希望的起点，并说明了计算蛋白设计的功能，可快速产生针对大流行性疾病威胁的潜在治疗剂。