key: cord-0983912-6zk0ioep authors: Fintelman-Rodrigues, Natalia; Sacramento, Carolina Q.; Lima, Carlyle Ribeiro; da Silva, Franklin Souza; Ferreira, André C.; Mattos, Mayara; de Freitas, Caroline S.; Soares, Vinicius Cardoso; Gomes Dias, Suelen da Silva; Temerozo, Jairo R.; Miranda, Milene; Matos, Aline R.; Bozza, Fernando A.; Carels, Nicolas; Alves, Carlos Roberto; Siqueira, Marilda M.; Bozza, Patrícia T.; Souza, Thiago Moreno L. title: Atazanavir inhibits SARS-CoV-2 replication and pro-inflammatory cytokine production date: 2020-04-06 journal: bioRxiv DOI: 10.1101/2020.04.04.020925 sha: 309a80ed9980d65445b9bddbbd472f00da2e9875 doc_id: 983912 cord_uid: 6zk0ioep Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors, such as atazanavir (ATV). ATV is of high interest because of its bioavailability within the respiratory tract. Our results show that ATV could dock in the active site of SARS-CoV-2 Mpro, with greater strength than LPV. ATV blocked Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells, human pulmonary epithelial cell line and primary monocytes, impairing virus-induced enhancement of IL-6 and TNF-α levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19. Coronaviruses (CoVs) are single-stranded positive sense RNA viruses with large enveloped nucleocapsids that are able to infect a range of hosts including both animals and humans 1 . Although a number of human CoV are known to circulate seasonally, two highly pathogenic variants emerged in the 21 st century that cause life-threatening infection, the severe acute respiratory syndrome (SARS-CoV) and middle-east respiratory syndrome (MERS-CoV) 2 . At the end of 2019, a novel variant of SARS-CoV (SARS-CoV-2) appeared in the citizens of the City of Wuhan, China that is believed to have spilled over to humans from animal reservoirs, most likely bats and/or pangolins 3 . The novel 2019 CoV is phylogenetically closer to SARS-CoV (from the 2002 outbreak) than MERS-CoV (from 2012 outbreak) 2, 3 . Both SARS-and MERS-CoV raised international public health concerns with rates of mortality of 10 and 35%, respectively 4, 5 . Soon after its discovery, the contemporary SARS-CoV-2 became a pandemic threat, with the number of confirmed infections ramping up globally 6 . To date, SARS-CoV-2 is responsible for 10 times more deaths than the total sum from SARS-and MERS-CoV, with more causalities daily that are continue to scale up 6 . Currently, the most effective response to the SARS-CoV-2 pandemic has been self-quarantining and social distancing to avoid contact between infected and uninfected individuals that can flatten the virus dissemination curve, which aim to reduce the burden on medical resources to prevent loss of service for those with the highest need. While these social actions can disrupt virus transmission rates, they are not expected to reduce the absolute number of infected individuals. Furthermore, these strategies are also provoking a severe reduction in global economic activity 7 . To effectively combat the impact of SARS-CoV-2 on infected individuals, and society as a whole, it is essential to identify antiviral drugs for immediate use, as well as develop new drugs and a vaccine for long-term solutions to the disease associated with SARS-CoV-2 (COVID- 19) . Repurposing of clinically approved drugs is the fastest pathway towards an effective response to a pandemic outbreak 8 9 . This mega trial has been 4 putting forward lopinavir (LPV)/ritonavir (RTV), in combination or not with interferonβ (IFN-β), chloroquine (CQ) and remdesivir to treat COVID-19 9 . LPV, RTV and remdesivir target viral enzymes, while the actions of CQ and IFN-β target host cells. The most successful antiviral drugs often directly target viral enzymes 10 Mpro better than LPV or RTV, that included ATV 17 . Importantly, ATV has been described to reach the lungs after intravenous administration 18, 19 . Moreover, a proposed secondary use of ATV to treat pulmonary fibrosis suggested that this drug could functionally reach the lungs 19 . The seriousness of COVID19 and the need for an immediate intervention, along with this series of observations with LPV, RTV and ATV, motivated us to evaluate the susceptibility of SARS-CoV-2 to ATV. Since ATV is available as a clinical treatment alone or in combination with RTV, both therapies were studied here, which for the first time describes that SARS-CoV-2 Mpro is a target for ATV. Further, ATV alone or withRTV could inhibit viral replication in cell culture models of infection that also prevented the release of a cytokine storm-associated mediators. Our timely data 5 highlights an additional therapeutic approach against COVID-19 that should be considered for clinical trials. The targeting of the enzyme Mpro from SARS-CoV-2 by both ATV and LPV was evaluated by molecular modeling using a representative structure (PDB:6LU7). As shown in Figure 1 , ATV occupied the S1* and S1 regions, whereas LPV occupied S1* and S2 regions with calculated free energy scores for LPV and ATV of -59.87 and -65.49 Kcal/mol, respectively. The more spontaneous binding of ATV, suggested by its lower energy score, may be related to its projected ability to form hydrogens bonds with the amino acid residues Asn142, His164, and Glu166 in Mpro, whereas the binding of LPV depends on hydrophobic interactions ( Figure 2 ). A molecular dynamic analysis revealed that the root-mean-square deviation (RMSD) for the SARS-CoV-2 Mpro backbone presented different conformations in complex with ATV or LPV ( Figure S1 ). LPV was initially at a 3.8 Å distance from the catalytic residue Cys145 ( Figure S2A and S3A), which after conformational changes extended to a distance equivalent to 7.17 Å ( Figure 3A and 4A) that is projected to most likely limit the extent of its antiviral inhibition. Another critical residue, His41, was satisfactorily at a distance of 2.89 Å from bound LPV ( Figure 3A and 4A). While ATV did not interact with His41 or Cys145 ( Figure S2B and S3B), its position remained stable within the active site independent of conformational changes displayed by the enzyme ( Figure 3B and 4B). The steric occupation of the cleft in the enzymatic active site by ATV, which block the residues of the catalytic amino acids, can be explained by its stronger interactions with Mpro, compared to LPV, through multiple hydrogen bonds during stationary docking and molecular dynamics (Tables S1-S3). Next, we evaluated whether ATV could inhibit SARS-CoV We extended our investigation to the inhibition of SARS-CoV-2 replication by ATV using a range of different cellular systems. Vero cells are a well-known model system that produce high virus titers and display visual cytopathic effects to viral infections. ATV alone, or in combination with RTV, inhibited infectious virus production and SARS-CoV RNA levels in Vero cells ( Figure 6A and B, respectively). CQ was used as a positive control because of its inclusion in the SOLIDARITY trial due to its encouraging pre-clinical and clinical results against SARS-CoV-2 replication and COVID-19, respectively 20, 21 . ATV/RTV was the most potent therapy tested; with an EC 50 of 0.5 ± 0.08 µM. ATV alone and CQ's potencies were 2.0 ± 0.12 µM and 1.0 ± 0.07 µM, respectively. SARS-CoV-2 susceptibility to CQ is consistent with recent reports in the literature 20 , validating our analysis. The ATV/RTV, ATV, and CQ cytotoxicity values, CC 50 , were 280 ± 3 µM, 312 ± 8 µM and 259 ± 5 µM, respectively. Our results indicate that the selectivity index (SI, which represents the ratio between the CC 50 and EC 50 values) for ATV/RTV, ATV and CQ were 560, 156 and 259, respectively, which shows that ATV/RTV has a high therapeutic potential that was greater than CQ. Since the results regarding the pharmacologic activity of ATV and ATV/RTV against SARS-CoV-2 replication in Vero cells were promising, we next investigated 7 whether the proposed drug therapies could inhibit virus replication in a human epithelial pulmonary cell line (A549). ATV alone showed a nearly 10-fold increase in potency for inhibiting SARS-CoV-2 replication in A549 ( Figure 6C ) compared to Vero cells ( Figure 6B ). ATV/RTV and CQ were similarly potent in inhibiting virus replication in both cell types ( Figure 6B and C). ATV/RTV, ATV and CQ EC 50 values to inhibit SARS-CoV-2 replication in A549 cells were 0.60 ± 0.05 µM, 0.22 ± 0.02 µM and 0.89 ± 0.02 µM, respectively. In vitro results confirmed the rational that SARS-CoV-2 would be susceptible to ATV that included cells derived from the respiratory tract. Recent reports on the COVID-19 outbreak have implicated that an increase in the levels of lactate dehydrogenase (LDH) and interleukin 6 (IL-6) is associated with mortality 22 . Viral infection in the respiratory tract often trigger the migration of blood monocytes to orchestrate the transition from innate to adaptive immune responses 23 . For these reasons, ATV and ATV/RTV were tested at suboptimal (1 μM) or optimal (10 μM) doses in a SARS-CoV-2-infection model utilizing human primary monocytes. ATV/RTV and CQ were similarly efficient to inhibit viral replication in the human monocytes ( Figure 7A ). Virus infection increased cellular mortality by 75%, which was prevented by ATV, at both doses tested, and by ATV/RTV, at 10 μM ( Figure 7B ). As a control, detergent treatment completely destroyed all cells ( Figure 7B ). Moreover, we observed that infections by SARS-CoV-2 triggered the expected increase in the IL-6 levels in the culture supernatant, which ranged from 20-to 60-fold depending on the cell donor ( Figure 7C , open circles in nil-treated cells). The virus-induced enhancement of IL-6 levels were significantly prevented by treatment with ATV at 10 µM, ATV/RTV at both 1 and 10 µM and CQ at 10 µM ( Figure 7C ). Another biomarker of uncontrolled pro-inflammatory cytokine response, TNF-α, was up-regulated 40-fold during virus infection ( Figure 7D ). Only the combination of ATV/RTV could significantly prevent the induction of TNF-α release ( Figure 7D ). Altogether, our results confirm that ATV and ATV/RTV should not be ignored as an additional therapeutic option against COVID-19. 8 In these two decades of the 21st century, the human vulnerability to emerging viral diseases has been notable 24 . The emergence of infectious disease highlights the undeniable fact that existing countermeasures are inefficient to prevent virus spill over and diseases outbreak. Preclinical data on the susceptibility of an emerging virus to clinically approved drugs can allow for the rapid mobilization of resources towards clinical trials 8 . This approach proved feasible for combating the Zika, yellow fever and chikungunya outbreaks experienced in Brazil over the past 5 years, when our group demonstrated that sofosbuvir, a blockbuster drug against hepatitis C, could represent a compassionate countermeasure against these diseases [25] [26] [27] [28] [29] . Currently, the rate of SARS-CoV-2 dissemination has become one of the most rapidly evolving pandemics known in modern times with the number of cases and deaths doubling every week and the peak of the pandemic has yet to arrive 6 ATV was approved in 2003, and become a wider prescribed drug among HIVinfected individuals, than LPV, including for critically ill patients 16 . ATV shows a safer profile than LPV in both short-and long-tem therapeutic regimens 15,33 . ATV has a 9 documented bioavailability to reach the respiratory tract 18, 34 , which lead to its proposed use against pulmonary fibrosis 19 Highly pathogenic respiratory viruses, such as influenza A virus, have been associated with a cytokine storm that describes an uncontrolled pro-inflammatory cytokine response 38, 39 . Cytokine storms also seem to be highly relevant for pathogenic human CoVs 40 . Contemporary investigations on SARS-CoV-2 strongly suggest the involvement of cytokine storm with disease severity 22 . COVID-19 mortality is associated with enhanced IL-6 levels and consistent cell death, as measured by LDH 1 0 release 22 . We showed that ATV and ATV/RTV decreased IL-6 release in SARS-CoV-2infected human primary monocytes. Moreover, we also included in our analysis TNF-α, another hallmark of inflammation during respiratory virus infections 22, 41 . Our results reveled that cellular mortality and cytokine storm-associated mediators were reduced after treatment with the repurposed antiretroviral drugs used in this study. Among the most promising anti-SARS-CoV-2 drugs, CQ, IFN-β and LPV displayed a higher toxic profile than ATV. Moreover, ATV and ATV/RTV have in vitro antiviral potencies comparable to CQ and remdesivir, which were superior to LPV/RTV. In summary, our study highlights a new option among clinically approved drugs that should be considered in ongoing clinical trials for an effective treatment for COVID-19. The antiviral ATV, ATV/RTV and CQ were received as donations from Instituto de 1 1 African green monkey kidney (Vero, subtype E6) and A549 (human lung epithelial cells) cells were cultured in high glucose DMEM with 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin and 100 μ g/mL streptomycin (Pen/Strep; ThermoFisher) at 37 °C in a humidified atmosphere with 5% CO 2 . Human primary monocytes were obtained after 3 h of plastic adherence of Virus titers were determined as the tissue culture infectious dose at 50% (TCID 50 /mL). Virus stocks were kept in -80 °C ultralow freezers. The virus strain was sequenced to confirm the virus identity and its complete genome is publicly deposited (https://nextstrain.org/ncov: Brazil/RJ-314/2020 or GISAID EPI ISL #414045). The levels of TNF-α, IL-6 and LDH were quantified in the monocyte supernatants from infected and uninfected cells. ELISA for TNF-α and IL-6 required 100 µL of supernatants to be exposed to capture antibody in 96-well plates. After a 2h incubation period at room temperature (RT), the detection antibody was added. Plates were incubated for another 2h at RT. Streptavidin-HRP and its substrate were added, incubated for 20 minutes and the optical density was determined using a microplate reader set to 450 nm. Extracellular lactate dehydrogenase (LDH) was quantified using Doles ® kit according to manufacturer's` instructions. Supernatant was centrifuged at 5,000 rpm for The docking of ligands was performed in a box of 10 Å edges with its mass center matching that of the complexed peptide. Each scan produced 20 conformations for each ligand with the best score being used for molecular dynamics simulations. 1 4 Since the tertiary structure (3D) of the SARS-CoV-2 Mpro is a homodimer, we focused the molecular dynamics only one chain, henceforward chain A. Molecular dynamics calculations were performed using NAMD 2.9 49 and Charmm27* force field 50 at pH 7, i.e., with deprotonated Glu and Asp, protonated Arg and Lys, and neutral His with a protonated Nε atom. This all-atom force field has been able to fold properly many soluble proteins [51] [52] [53] Proteinases were assayed after electrophoresis on 10% SDS-PAGE with 0.1% copolymerized gelatin 56 The assays were performed blinded by one professional, codified and then read by another professional. All experiments were carried out at least three independent times, including a minimum of two technical replicates in each assay. The doseresponse curves used to calculate EC 50 A representative structure of Mpro (PDB:6LU7) was color coded to show the electrostatic potential of residues in the active site for negative (blue) and positive (red) charges. Panel A, the cavities of ligand interaction designated S1*, S1 and S2 in the absence of inhibitors. Panel B, placement of LPV (cyan) docked in the S1* and S2 regions of the active site. Panel C, placement of ATV (orange) docked in the S1* and S1 regions of the active site. The molecular biology of coronaviruses Origin and evolution of pathogenic coronaviruses Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins WHO | Middle East respiratory syndrome coronavirus An interactive web-based dashboard to track COVID-19 in real time Opinion | Will Our Economy Die From Coronavirus? Coronavirus puts drug repurposing on the fast track WHO R&D Blueprint: informal consultation on prioritization of candidate therapeutic agents for use in novel coronavirus 2019 infection Approved Antiviral Drugs over the Past 50 Years Small molecules targeting severe acute respiratory syndrome human coronavirus Coronaviruses: an overview of their replication and pathogenesis Pharmacokinetics and pharmacodynamics of cytochrome P450 inhibitors for HIV treatment A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19 Effects of Switching from Lopinavir/ritonavir to Atazanavir/ritonavir on Muscle Glucose Uptake and Visceral Fat in HIV Infected Patients Treatment Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents: An Update Old Drugs for Newly Emerging Viral Disease Preclinical pharmacokinetics and tissue distribution of longacting nanoformulated antiretroviral therapy Protective Effect of Atazanavir Sulphate Against Pulmonary Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study The host immune response in respiratory virus infection: balancing virus clearance and immunopathology Zika virus and neurological diseaseapproaches to the unknown Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo Beyond members of the Flaviviridae family, sofosbuvir also inhibits chikungunya virus replication Sofosbuvir protects Zika virus-infected mice from mortality, preventing short-and long-term sequelae The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication Efficacy of sofosbuvir as treatment for yellow fever: protocol for a randomised controlled trial in Brazil (SOFFA study) UN health chief announces global 'solidarity trial' to jumpstart search for COVID-19 treatment A rapid advice guideline for the diagnosis and treatment of 2019 2019-nCoV) infected pneumonia (standard version) Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV HIV protease inhibitors: a review of molecular selectivity and toxicity UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues through a drug-target interaction deep learning model The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor Inhibition of the Main Protease 3CL-pro of the Coronavirus Disease 19 via Structure-Based Ligand Design and Molecular Modeling Cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza A (H1N1): role of the host immune response in pathogenesis Role of macrophage cytokines in influenza A virus infections Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology Induction of TNF-alpha in human macrophages by avian and human influenza viruses A simple method of estimating fifty percent endpoints COVID-19) Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation Development and testing of a general amber force field DOCK 6: Impact of new features and current docking performance Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting UCSF Chimera--a visualization system for exploratory research and analysis Scalable molecular dynamics with NAMD Development and current status of the CHARMM force field for nucleic acids Folding Atomistic Proteins in Explicit Solvent Using Simulated Tempering Communication: Multiple atomistic force fields in a single enhanced sampling simulation How fast-folding proteins fold Comparison of simple potential functions for simulating liquid water Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major 1 6 The authors declare no competing financial interests.