K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

中文翻译：

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半胱氨酸蛋白酶抑制剂可阻断人和猴细胞的SARS-CoV-2感染

K777是一种二肽类似物，包含一个亲电的乙烯基砜部分，并且是组织蛋白酶的有效共价灭活剂。将Vero E6，HeLa / ACE2，Caco-2，A549 / ACE2和Calu-3细胞暴露于SARS-CoV-2，然后用K777处理。K777降低了病毒感染性，其EC50值抑制了病毒感染：Vero E6为74 nM，A549 / ACE2为<80 nM，HeLa / ACE2细胞为4 nM。相反，Calu-3和Caco-2细胞的EC50值在低微摩尔范围内。对于任何宿主细胞，在10-100μM抑制剂下均未观察到K777的毒性。K777不抑制由SARS-CoV-2编码的木瓜蛋白酶样半胱氨酸蛋白酶和3CL半胱氨酸蛋白酶的活性，浓度≤100μM。这些结果表明，K777通过灭活对于病毒感染性必不可少的哺乳动物半胱氨酸蛋白酶来发挥其强大的抗病毒活性。使用K777的炔丙基衍生物作为基于活性的探针，K777在Vero E6细胞中选择性靶向组织蛋白酶B和组织蛋白酶L。然而，仅组织蛋白酶L切割SARS-CoV-2刺突蛋白，并且K777阻断该蛋白水解。组织蛋白酶L切割刺突蛋白的位点在SARS-CoV-2的S1结构域中，不同于在SARS CoV-1刺突蛋白中观察到的裂解位点。这些数据支持以下假设：K777的抗病毒活性是通过抑制宿主组织蛋白酶L的活性并随后丧失病毒突波蛋白加工过程介导的。K777在Vero E6细胞中选择性靶向组织蛋白酶B和组织蛋白酶L。然而，仅组织蛋白酶L切割SARS-CoV-2刺突蛋白，并且K777阻断该蛋白水解。组织蛋白酶L切割刺突蛋白的位点在SARS-CoV-2的S1结构域中，不同于在SARS CoV-1刺突蛋白中观察到的裂解位点。这些数据支持以下假设：K777的抗病毒活性是通过抑制宿主组织蛋白酶L的活性并随后丧失病毒突波蛋白加工过程介导的。K777在Vero E6细胞中选择性靶向组织蛋白酶B和组织蛋白酶L。然而，仅组织蛋白酶L切割SARS-CoV-2刺突蛋白，并且K777阻断该蛋白水解。组织蛋白酶L切割刺突蛋白的位点在SARS-CoV-2的S1结构域中，不同于在SARS CoV-1刺突蛋白中观察到的裂解位点。这些数据支持以下假设：K777的抗病毒活性是通过抑制宿主组织蛋白酶L的活性并随后丧失病毒突波蛋白加工过程介导的。