一种新型植物类黄酮对 Sars-Cov-2 病毒复制和体内血栓形成的抑制作用,Blood

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一种新型植物类黄酮对 Sars-Cov-2 病毒复制和体内血栓形成的抑制作用
Blood ( IF 21.0 ) Pub Date : 2021-11-05 , DOI: 10.1182/blood-2021-153513
Moua Yang ₁ , Lin Lin ₂ , Christina Scartelli ₂ , Da-Yuan Chen _{3,

4} , Anika Patel ₂ , Roelof Bekendam ₂ , Lijun Sun ₅ , Mohsan Saeed _{3,

4} , Robert Flaumenhaft ₂

Affiliation

摘要基于植物类黄酮的安全性和广泛可用性，已将其作为 β-冠状病毒复制的抑制剂和 COVID19 的潜在治疗剂进行了研究。SARS-CoV-2 病毒复制依赖于一种称为 3CL 蛋白酶或主蛋白酶 (Mpro) 的半胱氨酸蛋白酶，它将从 SARS-CoV-2 ssRNA 翻译的多蛋白切割成 11 种功能蛋白。该蛋白酶在 β-冠状病毒中高度保守，不耐受突变。SARS-CoV、SARS-CoV-2 和 MERS 的主要蛋白质 (Mpro) 已通过计算机和体外方法被鉴定为黄酮类化合物的靶标。我们之前已经表明，选择的类黄酮可抑制蛋白质二硫键异构酶 (PDI)，这对于正常血栓形成至关重要。这些类黄酮 PDI 抑制剂可在体内阻断血栓形成，并在临床研究中显示出作为抗血栓形成剂的功效。鉴于 COVID19 相关凝血病引起的大量发病率和死亡率，我们试图确定一种黄酮类化合物，它可以抑制 SARS-CoV-2 Mpro 和 PDI，从而可能阻断病毒复制和血栓形成。虽然在计算机研究中确定了许多类黄酮作为 SARS-CoV-2 主要蛋白 (Mpro) 抑制剂，但之前尚未对类黄酮针对 SARS-CoV-2 进行全面的体外测试。因此，我们使用高通量筛选评估了 1,020 种不同的黄酮类化合物在基于荧光的 Mpro 底物切割试验中抑制 SARS-CoV-2 Mpro 的能力。该分析确定了四种新的 Mpro 类黄酮抑制剂，其 IC 50 范围为 5-15 µM：amentoflavone, 3, 8'-biapigenin、jaceidin triacetate 和 pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG)。这些化合物与之前鉴定的 SARS-CoV-2 Mpro、黄芩素和杨梅素的黄酮类抑制剂相同或更有效。结构活性关系将芹菜素鉴定为额外的 Mpro 抑制剂。在基于 Vero-E6 的 SARS-CoV-2 复制分析中，PGHG 的抑制 IC 50 = 4.9 µM。在 50 µM 时，芹菜素抑制 94±2.1%，黄芩素抑制 65±8.0%，而杨梅素、阿门黄酮和 3,8'-biapigenin 不抑制病毒复制。Jaceidin triacetate 毒性太大，无法进一步分析。我们接下来评估了新型 Mpro 抑制剂抑制 PDI 的能力。最有效的 PDI 抑制剂是 PGHG，在胰岛素比浊法中阻断 PDI 还原酶活性，IC 50 = 3.99±1.14 µM，在二-曙红-GSSG 试验中，IC 50 = 1.50±0.60 µM。当针对 PDI 的分离片段进行测试时，PGHG 抑制分离的 a 和 a' 片段以及 ab、b'xa' 和 abb'x 片段，表明它作用于 PDI 的 a 和 a' 结构域。由于 PDI 对血栓形成至关重要，我们评估了 PGHG 是否会阻止血管损伤后的血小板积累和纤维蛋白形成。我们给小鼠输注 25 mg/kg PGHG 或载体，随后通过激光诱导的提睾循环内小动脉损伤诱导血栓形成。PGHG 的输注导致 82±6.2% 的血小板积累抑制和 79±3.7% 的纤维蛋白形成抑制。相比之下，与载体对照相比，25 mg/kg 对小鼠尾部出血没有显着影响。靶向治疗仍然是抗击 COVID19 武器的重要组成部分。我们的研究结果表明，在 Penthorum chinense Pursh 中发现的一种天然类黄酮 PGHG 可抑制 SARS-CoV-2 复制和血栓形成，而不会增加出血。这一观察结果为开发用于抑制 β-冠状病毒的植物类黄酮疗法提供了原理证明，并支持进一步评估 PGHG 在 COVID19 中的治疗用途。图 1 图 1. 披露无需声明相关的利益冲突。我们的研究结果表明，在 Penthorum chinense Pursh 中发现的一种天然类黄酮 PGHG 可抑制 SARS-CoV-2 复制和血栓形成，而不会增加出血。这一观察结果为开发用于抑制 β-冠状病毒的植物类黄酮疗法提供了原理证明，并支持进一步评估 PGHG 在 COVID19 中的治疗用途。图 1 图 1. 披露无需声明相关的利益冲突。我们的研究结果表明，在 Penthorum chinense Pursh 中发现的一种天然类黄酮 PGHG 可抑制 SARS-CoV-2 复制和血栓形成，而不会增加出血。这一观察结果为开发用于抑制 β-冠状病毒的植物类黄酮疗法提供了原理证明，并支持进一步评估 PGHG 在 COVID19 中的治疗用途。图 1 图 1. 披露无需声明相关的利益冲突。这一观察结果为开发用于抑制 β-冠状病毒的植物类黄酮疗法提供了原理证明，并支持进一步评估 PGHG 在 COVID19 中的治疗用途。图 1 图 1. 披露无需声明相关的利益冲突。这一观察结果为开发用于抑制 β-冠状病毒的植物类黄酮疗法提供了原理证明，并支持进一步评估 PGHG 在 COVID19 中的治疗用途。图 1 图 1. 披露无需声明相关的利益冲突。

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Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Abstract Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh , inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

更新日期：2021-11-05

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