Development of therapeutics for SARS-CoV-2 infection

SARS-CoV-2 感染疗法的开发

基本信息

批准号：
10262620
负责人：
Hiroaki Mitsuya
金额：
$ 48.22万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10262620
关键词：
2019-nCoV Active Sites Affect Amino Acid Sequence Antibodies Antiviral Agents Appearance Biological Assay COVID-19 Cell Culture Techniques Cells Cessation of life China Chloroquine Clinical Complex Coughing Country Cultured Cells Cytostatics Data Disease Disease Outbreaks Dyspnea Esters Exposure to Fatigue Fever Genome Hour Hydroxychloroquine Hypoxia Individual Indoles Infection Intubation Light Lopinavir Mechanical ventilation Microfilaments Middle East Respiratory Syndrome Middle East Respiratory Syndrome Coronavirus Monoclonal Antibodies Mus Nelfinavir Nucleotides Papain Pathogenicity Pattern Peptide Hydrolases Pharmaceutical Chemistry Pharmaceutical Preparations Polyproteins Process Province RNA RNA Viruses Reporting Roentgen Rays Role SARS coronavirus Series Severe Acute Respiratory Syndrome Specificity Structure System TMPRSS2 gene Testing Therapeutic Time Toxic effect Viral Viral Genome Viral Physiology Viral Pneumonia Virion Virus Virus Replication base cellular imaging common symptom covalent bond culture plates cytotoxic cytotoxicity cytotoxicity test design drug discovery immunocytochemistry indoline inhibitor/antagonist lead optimization microscopic imaging nitazoxanide novel novel coronavirus nucleotide analog overexpression pandemic disease pathogen remdesivir replicase small molecule synergism therapeutic development virtual

项目摘要

The genome of SARS-CoV-2 has overall 80% nucleotide identity with that of SARS-CoV and the main proteases (Mpro) of these two viruses have 96% amino acid sequence identity. Superimposition of the structures of the main protease of both SARS-CoV-2 and SARS-CoV showed near identity in their tertiary structures. Structurally, the 5' two-thirds of the viral genome encodes two overlapping polyproteins, pp1a and pp1ab, which are processed to generate the viral replication complex, replicase polyprotein, which undergoes processing by two viral proteases, the Mpro and papain-like protease (PLpro). The role of the two proteases is essential for replication of SARS-CoV and both proteases have been recognized as attractive targets for developing antiviral agent. Based on the high structure homology, we examined potential activity of experimental Mpro inhibitors, which we newly synthesized, and those previously shown to be active against SARS-CoV-2 in a VeroE6 cell- or TMPRSS2-overexpressing VeroE6 (VeroE6TMPRSS2) cell-based assay using two SARS-CoV-2 strains, JPN/TY/WK-521 (SARS-CoV-2WK-521) and NCGM-nCoV-05-2N (SARS-CoV-205-2N), employing quantitative RNA-qPCR assay with cell culture supernatants, cytotoxicity assays, and immunocytochemistry. We first tested the antiviral activity against SARS-CoV-2WK-521 employing a series of compounds that had previously been reported to be active against SARS-CoV and identified GRL-0920 as well as a newly synthesized GRL-0820 as potent inhibitors of SARS-CoV-2. When VeroE6 cells were exposed to SARS-CoV-2WK-521 at MOI of 0.05 and cultured in the presence of various concentrations of the two indole chloropyridinyl esters, GRL-0820 and GRL-0920, were found to be most potent against SARS-CoV-2WK-521 with IC50 values of 15 and 2.8 microM, respectively, using RNA-qPCR. To corroborate the observed activity of GRL-0820 and GRL-0920, we further examined the antiviral activity of these two compounds together with other selected compounds that had previously been reported to be active against SARS-CoV and/or SARS-CoV-2, including remdesivir, lopinavir, nelfinavir, favipiravir, hydroxychloroquine, nitazoxanide, and nafamostat. To compare the antiviral activity of the 9 compounds as accurately as possible, the compounds were examined in one assay done at the same time. We confirmed that GRL-0820 and GRL-0920 were active against SARS-CoV-2WK-521 with IC50 values of 23 and 8.6 microM, respectively. Remdesivir was also found to be active against SARS-CoV-2WK-521 (IC50=1.2 microM). The IC50 values of lopinavir, nelfinavir, and nitazoxanide turned out to be 19, 3.1, and 31 microM, however, their specificity indeces were only 2.7, 17, and 2.8, respectively. Thus, such IC50 values were thought possibly to be affected by their cytotoxicity. While favipiravir and nafamostat were apparently non-toxic at all concentrations tested but their IC50 values were both greater than 100 microM, suggesting that neither compounds were inert against the virus. Hydroxychloroquine appeared to be nontoxic at up to 100 microM and its IC50 turned out to be 5.1 microM, however, its cytotoxicity was suspected to have contributed to its apparent anti-viral activity (vide infra). As described above, cytostatic and cytotoxic effects of test compounds in cell-based assays is often mistakenly judged to be the apparent antiviral activity being associated with the multitudes of toxicity-caused reduction of virion copies produced in the assays. Indeed, none of such toxic agents did not become of clinical utility as antiviral agents. Thus, we carefully asked if the three compounds, GRL-0920 and remdesivir exerted antiviral activity at 1, 10, and 100 microM without significant cytostatic or cytotoxic effects. We first examined the light microscopic images of VeroE6 cells exposed to SARS-CoV-2WK-521 and cultured in the presence of GRL-0920 or remdesivir. VeroE6 cells were exposed to SARS-CoV-2WK-521 for 1 hour, the virus was washed off, and the cells were cultured in the presence of various concentrations of test compound for 3 days. VeroE6 cells cultured alone appear to be robust; however, those exposed to SARS-CoV-2WK-521 and cultured in the absence of test compounds, many cells got granular patterns. VeroE6 cells exposed to the virus and cultured with GRL-0920 or remdesivir (at 100 microM), all the cells appeared to be protected. GRL-0920 reproducibly and completely blocked the infectivity and cytopathic effect of SARS-CoV-2WK-521 when examined using the 'low stringent" assay using VeroE6 cells. When VeroE6 cells were exposed to SARS-CoV-2WK-521 and cultured in the presence of remdesivir or GRL-0920 at 0.01, 0.1, 1, 10, and 100 microM, there was increasing suppression of viral replication at 10 microM and either near complete (remdesivir) or complete (GRL-0920) suppression at 100 microM as assessed using either of the convIgG m or murine anti-spike monoclonal antibody as the primary antibody. When the activity of 100 microM remdesivir and GRL-0920 against SARS-CoV-2WK-521 was examined using VeroE6 cells, the appearance of actin filaments proved to be virtually the same as seen in the "high stringent" assay using VeroE6TMPRSS2 cells. These data strongly suggest that the VeroE6 cells protected by remdesivir and GRL-0920 are viable and likely to have maintained the integrity of the cells. We have also identified two small molecule compounds, GRL-1720 and GRL-2420, both of which target Mpro of SARS-CoV-2 and potently block the infectivity, replication, and cytopathicity of SARS-CoV-2WK-521. As assessed using the quantitative VeroE6 cell-based assay with RNA-qPCR, the EC50 values of GRL-1720 and GRL-2420 were 15 and 4.2 microM, respectively, and apparent CC50 values were both greater than 100 microM. Since the cytotoxicity of test compounds in cell-based assays is often mistakenly judged to be the apparent antiviral activity being estimated by the multitudes of toxicity-associated reduction of the number of virion copies produced in the assays. Indeed, none of such toxic agents did not become of clinical utility as antiviral agents. Thus, we carefully asked if the three compounds, GRL-1720, GRL-2420, and remdesivir exerted cytotoxicity at 1, 10, and 100 microM. VeroE6 cells cultured alone appeared to be robust and had spread at the bottom of the microtiter culture plates; however, VeroE6 cells exposed to SARS-CoV-2WK-521 and cultured in the absence of test compounds, many cells got granular patterns and got detached from the bottom of the culture plate, indicating the cells were infected and killed by the cytopathicity of the virus. In contrast, when the SARS-CoV-2WK-521-exposed cells were cultured in the presence of each of the three compounds at 10 microM, the cells appeared to be moderately protected and at 100 microM, all the cells appeared to be completely protected by each compound and all three images of the cells cultured with 100 microM of each compound appeared to be similar to the image of the cells cultured alone.

SARS-CoV-2 的基因组与 SARS-CoV 的基因组总体上具有 80% 的核苷酸同一性，并且这两种病毒的主要蛋白酶 (Mpro) 具有 96% 的氨基酸序列同一性。 SARS-CoV-2 和 SARS-CoV 的主要蛋白酶结构的叠加显示出它们的三级结构接近同一性。从结构上看，病毒基因组的 5' 三分之二编码两个重叠的多蛋白 pp1a 和 pp1ab，它们经过加工后生成病毒复制复合物、复制酶多蛋白，该多蛋白经过两种病毒蛋白酶（Mpro 和木瓜蛋白酶）的处理。 PLpro）。这两种蛋白酶的作用对于 SARS-CoV 的复制至关重要，并且这两种蛋白酶已被认为是开发抗病毒药物的有吸引力的靶标。基于高度结构同源性，我们检查了我们新合成的实验性 Mpro 抑制剂的潜在活性，以及之前在 VeroE6 细胞或 TMPRSS2 过表达 VeroE6 (VeroE6TMPRSS2) 细胞中显示出对 SARS-CoV-2 有活性的抑制剂。使用两种 SARS-CoV-2 毒株 JPN/TY/WK-521 (SARS-CoV-2WK-521) 和NCGM-nCoV-05-2N (SARS-CoV-205-2N)，采用细胞培养上清液定量 RNA-qPCR 测定、细胞毒性测定和免疫细胞化学。我们首先使用一系列先前报道对 SARS-CoV 有活性的化合物测试了对 SARS-CoV-2WK-521 的抗病毒活性，并确定 GRL-0920 以及新合成的 GRL-0820 是 SARS 的有效抑制剂-CoV-2。当 VeroE6 细胞以 0.05 MOI 暴露于 SARS-CoV-2WK-521 并在不同浓度的两种吲哚氯吡啶酯 GRL-0820 和 GRL-0920 存在下培养时，发现对 SARS-CoV 最为有效-2WK-521，使用 RNA-qPCR 时的 IC50 值分别为 15 和 2.8 microM。为了证实观察到的 GRL-0820 和 GRL-0920 的活性，我们进一步检查了这两种化合物以及先前报道的对 SARS-CoV 和/或 SARS-CoV-2 有活性的其他选定化合物的抗病毒活性，包括瑞德西韦、洛匹那韦、奈非那韦、法匹拉韦、羟氯喹、硝唑尼特和萘莫司他。为了尽可能准确地比较 9 种化合物的抗病毒活性，在同时进行的一项测定中对这些化合物进行了检查。我们证实 GRL-0820 和 GRL-0920 对 SARS-CoV-2WK-521 有活性，IC50 值分别为 23 和 8.6 microM。瑞德西韦还被发现对 SARS-CoV-2WK-521 有活性（IC50=1.2 microM）。洛匹那韦、奈非那韦和硝唑尼特的 IC50 值分别为 19、3.1 和 31 µM，但其特异性指数分别仅为 2.7、17 和 2.8。因此，这样的IC50值被认为可能受到它们的细胞毒性的影响。虽然法匹拉韦和那法莫司他在所有测试浓度下均明显无毒，但它们的 IC50 值均大于 100 µM，表明这两种化合物对病毒均不具有惰性。羟氯喹在浓度高达 100 µM 时似乎无毒，其 IC50 为 5.1 µM，但怀疑其细胞毒性导致了其明显的抗病毒活性（见下文）。如上所述，基于细胞的测定中测试化合物的细胞抑制和细胞毒性作用经常被错误地判断为与测定中产生的大量由毒性引起的病毒体拷贝减少相关的明显抗病毒活性。事实上，这些毒性剂都没有成为抗病毒剂的临床用途。因此，我们仔细询问了 GRL-0920 和瑞德西韦这三种化合物在 1、10 和 100 microM 浓度时是否发挥抗病毒活性，而没有显着的细胞抑制或细胞毒性作用。我们首先检查了暴露于 SARS-CoV-2WK-521 并在 GRL-0920 或瑞德西韦存在下培养的 VeroE6 细胞的光学显微镜图像。将 VeroE6 细胞暴露于 SARS-CoV-2WK-521 1 小时，洗掉病毒，并将细胞在不同浓度的测试化合物存在下培养 3 天。单独培养的 VeroE6 细胞似乎很强大；然而，那些暴露于 SARS-CoV-2WK-521 并在没有测试化合物的情况下培养的细胞，许多细胞出现了颗粒状图案。 VeroE6 细胞暴露于病毒并与 GRL-0920 或瑞德西韦（100 µM）一起培养，所有细胞似乎都受到保护。当使用 VeroE6 细胞使用“低严格”检测进行检查时，GRL-0920 可重复且完全阻断 SARS-CoV-2WK-521 的感染性和细胞病变效应。当 VeroE6 细胞暴露于 SARS-CoV-2WK-521 并在0.01、0.1、1、10 和存在瑞德西韦或 GRL-0920 100 µM，在 10 µM 时对病毒复制的抑制增加，并且使用 convIgG m 或鼠抗刺突单克隆抗体作为一抗进行评估，在 100 µM 时几乎完全（瑞德西韦）或完全（GRL-0920）抑制当使用 VeroE6 检查 100 microM 瑞德西韦和 GRL-0920 对抗 SARS-CoV-2WK-521 的活性时。细胞中，肌动蛋白丝的外观被证明与使用 VeroE6TMPRSS2 细胞的“高严格”测定中看到的几乎相同。这些数据强烈表明，受瑞德西韦和 GRL-0920 保护的 VeroE6 细胞是可行的，并且可能保持了完整性。我们还鉴定了两种小分子化合物 GRL-1720 和 GRL-2420，它们都针对 SARS-CoV-2 的 Mpro，并有效阻断SARS-CoV-2WK-521 的感染性、复制性和细胞病变性。使用基于 VeroE6 细胞的 RNA-qPCR 定量测定进行评估，GRL-1720 和 GRL-2420 的 EC50 值分别为 15 和 4.2 µM，表观 CC50 值均大于 100 µM。由于基于细胞的测定中测试化合物的细胞毒性经常被错误地判断为通过测定中产生的与毒性相关的病毒体拷贝数的大量减少来估计的表观抗病毒活性。事实上，这些毒性剂都没有成为抗病毒剂的临床用途。因此，我们仔细询问了 GRL-1720、GRL-2420 和瑞德西韦这三种化合物在 1、10 和 100 microM 浓度时是否发挥细胞毒性。单独培养的 VeroE6 细胞似乎很坚固，并且已扩散到微量滴定培养板的底部；然而，VeroE6 细胞暴露于 SARS-CoV-2WK-521 并在没有测试化合物的情况下培养，许多细胞呈颗粒状并从培养板底部脱落，表明细胞被 SARS-CoV-2WK-521 的细胞病变性感染并杀死。病毒。相比之下，当暴露于 SARS-CoV-2WK-521 的细胞在三种化合物浓度分别为 10 µM 的情况下培养时，细胞似乎受到中等保护，而在 100 µM 浓度下，所有细胞似乎都受到完全保护每种化合物和用 100 µM 每种化合物培养的细胞的所有三个图像似乎与单独培养的细胞的图像相似。