Puhl Ana C, Fritch Ethan James, Lane Thomas R, Tse Longping V, Yount Boyd L, Sacramento Carol Queiroz, Tavella Tatyana Almeida, Costa Fabio Trindade Maranhão, Weston Stuart, Logue James, Frieman Matthew, Premkumar Lakshmanane, Pearce Kenneth H, Hurst Brett L, Andrade Carolina Horta, Levi James A, Johnson Nicole J, Kisthardt Samantha C, Scholle Frank, Souza Thiago Moreno L, Moorman Nathaniel John, Baric Ralph S, Madrid Peter, Ekins Sean
Collaborations Pharmaceuticals, Inc., 840 Main Campus Drive, Lab 3510, Raleigh, NC 27606, USA.
Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill NC 27599, USA.
bioRxiv. 2020 Dec 2:2020.12.01.407361. doi: 10.1101/2020.12.01.407361.
SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown activity against Ebola virus and demonstrated activity against SARS-CoV-2 . Most notably the RNA polymerase targeting remdesivir demonstrated activity and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in HeLa cells and of mouse adapted Ebola virus in mouse . We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC values of 180 nM and IC 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with K values of 339 nM and 647 nM, respectively. Human C for pyronaridine and quinacrine is greater than the IC hence justifying evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是一种新发现的病毒，迄今为止已在全球导致超过130万人死亡，全球确诊病例超过5900万例。事实证明，能够逆转疾病严重程度的小分子抑制剂很难被发现。为了加快药物转化，一种被广泛应用的关键方法是药物重新利用。一些药物已显示出对埃博拉病毒有活性，并证明对SARS-CoV-2也有活性。最值得注意的是，靶向RNA聚合酶的瑞德西韦在人类疾病的早期阶段显示出活性和疗效。测试其他对埃博拉病毒有活性的小分子药物似乎是评估其对SARS-CoV-2潜在作用的合理策略。我们之前已将咯萘啶、泰洛龙和奎纳克林（分别来自疟疾、流感和抗原生动物用途）重新用作HeLa细胞中埃博拉病毒和马尔堡病毒的抑制剂，以及小鼠适应性埃博拉病毒在小鼠体内的抑制剂。我们现在已经在感染了SARS-CoV-2以及其他病毒（包括鼠肝炎病毒和人冠状病毒229E）的各种细胞系（VeroE6、Vero76、Caco-2、Calu-3、A549-ACE2、HUH-7和单核细胞）中测试了这三种药物。这些结果的汇总表明，在不同细胞系中观察到的抗病毒活性存在很大差异。我们发现泰洛龙和咯萘啶分别以180 nM和198 nM的IC值抑制A549-ACE2细胞中的病毒复制。我们还在假病毒试验中对它们进行了测试，并使用微量热泳动技术测试这些分子与刺突蛋白的结合。它们分别以339 nM和647 nM的K值与刺突RBD蛋白结合。咯萘啶和奎纳克林的人体C值大于IC值，因此有理由进行评估。我们还对其可能为溶酶体亲和性的作用机制提供了新的见解。
