DataSheet_1_Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit cytopathic effect, papain-like protease, and MPRO enzymes of SARS-CoV-2.pdf

BackgroundAlthough tremendous success has been achieved in the development and deployment of effective COVID-19 vaccines, developing effective therapeutics for the treatment of those who do come down with the disease has been with limited success. To repurpose existing drugs for COVID-19, we previously showed, qualitatively, that erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit SARS-COV-2-induced cytopathic effect (CPE) in Vero cells.
AimThis study aimed to quantitatively explore the inhibition of SARS-CoV-2-induced CPE by erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin and to determine the effect of these drugs on SARS-CoV-2 papain-like protease and 3CL protease (MPRO) enzymes.
MethodsNeutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride) cell viability assay was used to quantify CPE after infecting pre-treated Vero cells with clinical SARS-Cov-2 isolates. Furthermore, SensoLyte® 520 SARS-CoV-2 papain-like protease and SensoLyte® 520 SARS-CoV-2 MPRO activity assay kits were used to evaluate the inhibitory activity of the drugs on the respective enzymes.
ResultsErythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibit SARS-CoV-2-induced CPE in Vero cells, with inhibitory concentration-50 (IC50) values of 3.27 µM, 4.23 µM, 9.29 µM, 3.19 µM, and 84.31 µM, respectively. Furthermore, erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibited SARS-CoV-2 papain-like protease with IC50 values of 0.94 µM, 0.88 µM, 1.14 µM, 1.07 µM, and 1.51 µM, respectively, and inhibited the main protease (MPRO) with IC50 values of 1.35 µM, 1.25 µM, 7.36 µM, 1.15 µM, and 2.44 µM, respectively.
ConclusionThe IC50 for all the drugs, except ivermectin, was at the clinically achievable plasma concentration in humans, which supports a possible role for the drugs in the management of COVID-19. The lack of inhibition of CPE by ivermectin at clinical concentrations could be part of the explanation for its lack of effectiveness in clinical trials.

背景 尽管高效COVID-19疫苗的研发与应用已取得巨大成功，但针对确诊患者的有效治疗药物开发却进展有限。为筛选可用于COVID-19治疗的现有老药，我们此前通过定性实验证实，红霉素、瑞他莫林、吡哆醇、叶酸与伊维菌素可在Vero细胞中抑制SARS-CoV-2诱导的细胞病变效应（CPE）。
研究目的 本研究旨在定量探究红霉素、瑞他莫林、吡哆醇、叶酸及伊维菌素对SARS-CoV-2诱导细胞病变效应的抑制作用，并明确上述药物对SARS-CoV-2木瓜样蛋白酶（papain-like protease）与3CL蛋白酶（MPRO）的活性影响。
实验方法 本研究采用中性红（3-氨基-7-二甲氨基-2-甲基吩嗪盐酸盐）细胞活力实验，对预先经药物处理的Vero细胞接种临床分离SARS-CoV-2毒株后，定量检测其细胞病变效应。此外，本研究使用SensoLyte® 520 SARS-CoV-2木瓜样蛋白酶检测试剂盒与SensoLyte® 520 SARS-CoV-2 MPRO活性检测试剂盒，评估上述药物对对应酶的抑制活性。
实验结果 实验结果显示，红霉素、瑞他莫林、吡哆醇、叶酸与伊维菌素均可剂量依赖性地抑制Vero细胞中SARS-CoV-2诱导的细胞病变效应，其半数抑制浓度（IC50）分别为3.27 μM、4.23 μM、9.29 μM、3.19 μM与84.31 μM。同时，上述药物可剂量依赖性抑制SARS-CoV-2木瓜样蛋白酶活性，对应IC50值分别为0.94 μM、0.88 μM、1.14 μM、1.07 μM与1.51 μM；且可抑制SARS-CoV-2主蛋白酶（MPRO）活性，对应IC50值分别为1.35 μM、1.25 μM、7.36 μM、1.15 μM与2.44 μM。
结论 除伊维菌素外，其余四种药物的半数抑制浓度均处于人体临床可达到的血浆药物浓度范围内，提示上述药物或可用于COVID-19的临床管理。伊维菌素在临床浓度下未表现出细胞病变效应抑制活性，这或可部分解释其在临床试验中疗效不佳的原因。