The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of infections worldwide. While the search for an effective antiviral is still ongoing, experimental therapies based on repurposing of available antivirals is being attempted, of which HIV protease inhibitors (PIs) have gained considerable interest. Inhibition profiling of the PIs directly against the viral protease has never been attempted in vitro, and while few studies reported an efficacy of lopinavir and ritonavir in SARS-CoV-2 context, the mechanism of action of the drugs remains to be validated. We carried out an in-depth analysis of the efficacy of HIV PIs against the main protease of SARS-CoV-2 (Mpro) in cell culture and in vitro enzymatic assays, using a methodology that enabled us to focus solely on any potential inhibitory effects of the inhibitors against the viral protease. For cell culture experiments a dark-to-bright GFP reporter substrate system was designed. Lopinavir, ritonavir, darunavir, saquinavir, and atazanavir were able to inhibit the viral protease in cell culture, albeit in concentrations much higher than their achievable plasma levels, given their current drug formulations. While inhibition by lopinavir was attributed to its cytotoxicity, ritonavir was the most effective of the panel, with IC50 of 13.7 µM. None of the inhibitors showed significant inhibition of SARS-CoV-2 Mpro in our in vitro enzymatic assays up to 100 µM concentration. Targeting of SARS-CoV-2 Mpro by some of the HIV PIs might be of limited clinical potential, given the high concentration of the drugs required to achieve significant inhibition. Therefore, given their weak inhibition of the viral protease, any potential beneficial effect of the PIs in COVID-19 context might perhaps be attributed to acting on other molecular target(s), rather than SARS-CoV-2 Mpro.

中文翻译：

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HIV蛋白酶抑制剂对SARS-CoV-2主要蛋白酶的功效分析


由严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 引起的大流行已导致全球数百万人感染。虽然寻找有效的抗病毒药物仍在进行中，但基于现有抗病毒药物重新利用的实验性疗法正在尝试，其中 HIV 蛋白酶抑制剂 (PI) 引起了相当大的兴趣。从未在体外尝试过直接针对病毒蛋白酶进行 PI 的抑制分析，虽然很少有研究报告洛匹那韦和利托那韦在 SARS-CoV-2 中的功效，但这些药物的作用机制仍有待验证。我们在细胞培养和体外酶测定中深入分析了 HIV PI 对 SARS-CoV-2 (Mpro) 主要蛋白酶的功效，使用的方法使我们能够只关注任何潜在的抑制作用病毒蛋白酶抑制剂。对于细胞培养实验，设计了从暗到亮的 GFP 报告基因底物系统。洛匹那韦、利托那韦、达芦那韦、沙奎那韦和阿扎那韦能够抑制细胞培养物中的病毒蛋白酶，尽管考虑到其当前的药物配方，其浓度远高于其可达到的血浆水平。虽然洛匹那韦的抑制作用归因于其细胞毒性，但利托那韦是该组中最有效的，IC50 为 13.7 µM。在我们的体外酶测定中，浓度高达 100 µM 时，没有一种抑制剂显示出对 SARS-CoV-2 Mpro 的显着抑制作用。鉴于实现显着抑制所需的药物浓度较高，一些 HIV PI 靶向 SARS-CoV-2 Mpro 的临床潜力可能有限。 因此，鉴于它们对病毒蛋白酶的抑制较弱，PI 在 COVID-19 背景下的任何潜在有益作用可能归因于作用于其他分子靶标，而不是 SARS-CoV-2 Mpro。