key: cord-0881551-kej61hbu authors: Zapata-Cardona, María I.; Flórez-Álvarez, Lizdany; Zapata-Builes, Wildeman; Guerra-Sandoval, Ariadna L.; Guerra-Almonacid, Carlos M.; Hincapié-García, Jaime; Rugeles, María T.; Hernandez, Juan C. title: Atorvastatin effectively inhibits late replicative cycle steps of SARS-CoV-2 in vitro date: 2021-03-02 journal: bioRxiv DOI: 10.1101/2021.03.01.433498 sha: e852f86f05d08a44769f3eb7e37487ab59b4420b doc_id: 881551 cord_uid: kej61hbu Introduction SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a pandemic of historic proportions and continues to spread worldwide. Currently, there is no effective therapy against this virus. This article evaluated the in vitro antiviral effect of Atorvastatin against SARS-CoV-2 and also identified the interaction affinity between Atorvastatin and three SARS-CoV-2 proteins, using in silico structure-based molecular docking approach. Materials and methods The antiviral activity of Atorvastatin against SARS-CoV-2 was evaluated by three different treatment strategies using a clinical isolate of SARS-CoV-2. The interaction of Atorvastatin with Spike, RNA-dependent RNA polymerase (RdRp) and 3C-like protease (3CLpro) was evaluated by molecular docking. Results Atorvastatin showed anti-SARS-CoV-2 activity of 79%, 54.8%, 22.6% and 25% at 31.2, 15.6, 7.9, and 3.9 µM, respectively, by pre-post-treatment strategy. In addition, atorvastatin demonstrated an antiviral effect of 26.9% at 31.2 µM by pre-infection treatment. This compound also inhibited SARS-CoV-2 in 66.9%, 75%, 27.9% and 29.2% at concentrations of 31.2, 15.6, 7.9, and 3.9 µM, respectively, by post-infection treatment. The interaction of atorvastatin with SARS-CoV-2 Spike, RdRp and 3CL protease yielded a binding affinity of −8.5 Kcal/mol, −6.2 Kcal/mol, and −7.5 Kcal/mol, respectively. Conclusion Our study demonstrated the in vitro anti-SARS-CoV-2 activity of Atorvastatin, mainly against the late steps of the viral replicative cycle. A favorable binding affinity with viral proteins by bioinformatics methods was also shown. Due to its low cost, availability, well-established safety and tolerability, and the extensive clinical experience of atorvastatin, it could prove valuable in reducing morbidity and mortality from COVID-19. Importance The COVID-19 pandemic constitutes the largest global public health crisis in a century, with enormous health and socioeconomic challenges. Therefore, it is necessary to search for specific antivirals against its causative agent (SARS-CoV-2). In this sense, the use of existing drugs may represent a useful treatment option in terms of safety, cost-effectiveness, and timeliness. Atorvastatin is widely used to prevent cardiovascular events. This compound modulates the synthesis of cholesterol, a molecule necessary in different stages of the viral replicative cycle. Our study demonstrated the antiviral potential of atorvastatin against SARS-CoV-2, using an in vitro model. Furthermore, the ability of Atorvastatin to directly interfere with three viral targets (Spike, RdRp and 3CL protease) was demonstrated by bioinformatic methods. This compound is a well-studied, low-cost, and generally well-tolerated drug, so it could be a promising antiviral for the treatment of COVID-19. declared COVID-19 a pandemic [2] . Since then, it has affected 218 countries and 75 territories worldwide, causing enormous human health consequences [1, 3] . 76 Currently, disease control has been based on symptom management, including the 77 use of convalescent plasma, synthetic antibodies, interferon, low-dose 78 corticosteroids, IL-1 and IL-16 inhibitors, Azithromycin, Remdesivir, Baricitinib, 79 Lopinavir/Ritonavir, Hydroxychloroquine, and, in severe cases, supportive care 80 (oxygen and mechanical ventilation) [4, 5] . Although there may be approved drugs affect the viral replicative cycle [11, 12] . 98 It has been proposed that statins could be an effective therapeutic strategy against 99 SARS-CoV-2 infection [16] . Recently, it has been reported that statin treatment was 100 associated with a reduced risk of mortality in patients diagnosed with 101 18]. Furthermore, during in silico analysis, ATV interacts with the 3CL protease of 102 SARS-CoV-2 [19] . However, there is no in vitro evidence of its antiviral effect against 103 SARS-CoV-2 or its binding affinity for other viral proteins such as Spike and RNA-104 dependent RNA polymerase (RdRp). This article evaluated the in vitro antiviral effect 105 of the ATV against SARS-CoV-2, and identified the interaction affinity between ATV 106 and three SARS-CoV-2 proteins, using an in silico structure-based molecular 107 docking approach. Atorvastatin showed a low cytotoxicity on Vero E6. 110 Before the antiviral activity evaluation, the cytotoxic effect of ATV on Vero E6 cells 111 was determined by MTT assay. As shown in Figure 1 , the Vero E6 viability was 112 higher than 90% at a concentration of 31.2 µM or less of ATV. At higher ATV 113 concentrations (62.5 to 250 µM), cell viability was lower or equal to 48.4%. The CC50 114 calculated for ATV was 50.3 µM. Cell viability was not affected by CQ and Heparin (positive controls of viral inhibition) 116 at the concentrations that were evaluated (Supplementary figure 1) . The CC50 117 obtained by CQ was more than 100 µM, and by Heparin was more than 100 µg/mL. ATV exhibited antiviral effects against SARS-CoV-2 in a dose-dependent 119 manner. To evaluate the antiviral activity of ATV against SARS-CoV-2, a pre-post treatment 121 strategy was performed in Vero E6 cells. ATV showed inhibition percentages of 122 SARS-CoV-2 of 79% (p=0.002), 54.8% (p=0.002), 22.6% (p=0.04) and 25% 123 (p=0.03) at concentrations of 31.2, 15.6, 7.9, and 3.9 µM, respectively ( Figure 2) . 124 Based on these data, the EC50 calculated for ATV was 15.4 µM, with a selectivity 125 index of 3.3. Chloroquine (positive control of viral inhibition) showed inhibition percentages of 127 100% (p=0.002), 99.9%(p=0.002), 97.5% (p=0.002) and 55.7% (p=0.002) at 100, 128 50, 25, and 12.5 µM concentrations, respectively (Supplementary figure 2 ). An 129 EC50 value of 13.5 µM was found by CQ, with a selectivity index of >7.4. Once an antiviral effect against SARS-CoV-2 was observed, the pre-infection and Other types of interactions were also seen, such as donor hydrogen bonds that were 182 part of the hydrophobic pocket. In the same way, hydrophobic interactions were 183 found with the type of bonds pi-sigma, pi-alkyl, Pi-pi T shaped and Pi-cation. Amino It should be kept in mind that the best candidate for a drug against SARS-CoV-2 is 262 the molecule that can specifically bind to one of the targets mentioned above to form 263 a thermodynamically stable complex. As shown in Figure 4 , complexes between 264 ATV and viral proteins were stabilized mainly by hydrogen bonds, in which ATV can 265 act as an electron acceptor or a simultaneous donor and acceptor. Additionally, 266 hydrophobic interactions (π-alkyl interaction, π-Sulfure, π-π Stached) also 267 contributed to the stability of the complexes. Considering the above, ATV could be 268 a plausible candidate against SARS-CoV-2. In fact, an in silico study showed that COVID-19: Transmission, prevention, and potential 514 therapeutic opportunities WHO Coronavirus Disease (COVID-19) Dashboard A comprehensive review of COVID-19 characteristics Recent Advances in Pathophysiology, Drug Development and Future 522 Perspectives of SARS-CoV-2. 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