key: cord-0801008-467lldzm authors: Akaberi, Dario; Krambrich, Janina; Ling, Jiaxin; Luni, Chen; Hedenstierna, Göran; Järhult, Josef D.; Lennerstrand, Johan; Lundkvist, Åke title: Mitigation of the replication of SARS-CoV-2 by Nitric Oxide in Vitro date: 2020-09-21 journal: Redox Biol DOI: 10.1016/j.redox.2020.101734 sha: a39df404aaa7afa036a8f6ef39accbc3a4faa11d doc_id: 801008 cord_uid: 467lldzm The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations’ health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 μM and 400 μM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine. Organization on March 11, 2020 [1] . SARS-CoV-2 belongs to the betacoronavirus genus and 46 is, together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and the 47 middle-east respiratory syndrome (MERS), the third coronavirus (CoV) emerging from 48 animals to humans within less than two decades [1] . The nucleotide sequence similarity 49 between SARS-CoV-2 and SARS-CoV is about 79% and between SARS-CoV-2 and MERS-50 Given the severity and the rapid spread of SARS-CoV-2, including a high proportion of 52 asymptomatic carriers [3], it is of clear importance to find effective therapeutics against 53 COVID-19. Despite extensive efforts to treat the disease and given the limitation in usage and 54 effectiveness of remdesivir [4], the development of therapeutic interventions is hindered by 55 the lack of effective antiviral drugs against SARS-CoV-2. 56 However, nitric oxide (NO), known to have a broad antimicrobial effect against bacteria, 57 fungi, helminths, protozoa and viruses, is a promising compound [5] . Antiviral against SARS-CoV in vitro [9, 10] and in vivo by inhalation in very low concentrations in a 61 small clinical trial [11] . Furthermore, inhaled NO improved arterial oxygenation in 62 hypoxemic patients by redistributing blood flow in the lung to better ventilated regions, and 63 counteracted blood clotting, both effects being of importance for COVID-19 patients [12] . 64 The role of NO-inhalation in the prevention and treatment of COVID-19 has been proposed 65 [13], but no clinical data has yet been reported. 66 In the present study the antiviral effect of NO on SARS-CoV-2 infected cells was tested in 67 vitro. The potential mechanism of action of NO as cysteine protease inhibitor, previously 68 reported for Coxsackievirus 3C cysteine protease [7], was furthermore examined on 69 SARS-CoV-2 3CL cysteine protease. The corresponding viral genome copy number to each Ct value was calculated based on a 112 curve equation generated with known standards: DNA gene fragments corresponding to the 113 amplified region of the viral genome were designed as templates and diluted to known 114 concentrations of 10 6 to 10 0 copies per µl. A standard curve plotted to the known concentra-115 tions was then created by performing qPCR on serial dilutions of the templates. 116 The recombinant SARS-CoV-2 3CL protease was produced adopting a published construct 118 used for the expression of SARS-CoV 3CL protease [17], containing nucleotide sequences 119 corresponding to residues S1-Q306 (Chinese isolate, NCBI accession number 120 YP_009725301). A detailed protocol is described in the supporting information. Statistical analysis was performed using GraphPad Prism (v.6.0). 141 The inhibitory effect of NO on the replication of SARS-CoV-2 was evaluated by performing a 143 yield reduction assay by RT-qPCR for the quantification of the viral RNA (Supplementary 144 Table 1 ). Viral replication kinetics and development of CPE were followed up to 72 hpi. NO+ ion) to the protease active site cysteine. In fact, the more enzyme is S-nitrosated and 190 therefore removed from the pool of active protease, the less substrate will be cleaved. 191 However, the proposed S-nitrosation mechanism of action remains to be proven. Moreover, 192 whether inhaled NO exerts antiviral effects by a similar mechanism, and at what 193 concentration, remains also to be shown, an issue recently touched by Ignarro [13] . 194 In this study, we demonstrated that NO can inhibit the replication of SARS-CoV-2 in Vero E6 196 and we identified the SARS-CoV-2 main protease as a target for NO. There is a great need for 197 J o u r n a l P r e -p r o o f effective antivirals against SARS-CoV-2 to be used in the on-going COVID-19 pandemic. 198 Based on this study and previous studies on SARS-CoV in vitro [8, 9] , and in a small clinical 199 trial [10] , we conclude that NO may be applied Three Emerging Coronaviruses in Two DecadesThe Story of SARS SARS and MERS: recent 253 insights into emerging coronaviruses Presymptomatic viral shedding and 257 infective ability of Severe Acute Respiratory Syndrome coronavirus 2 Remdesivir for the Treatment of Covid-19 -Preliminary Report Nitric oxide synthases: roles, tolls, and controls Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex 270 virus type 1 replication An Antiviral Mechanism of Nitric Oxide: Inhibition of a Viral Protease, 274 Immunity Nitric oxide and peroxynitrite have different 277 antiviral effects against hantavirus replication and free mature virions Inhibition 280 of SARS-coronavirus infection in vitro by S-nitroso-N-acetylpenicillamine, a nitric oxide 281 donor compound Nitric Oxide Inhibits the Replication Cycle of Severe Acute Respiratory Syndrome 285 Inhalation of Nitric Oxide in the Treatment of Severe Acute Respiratory Syndrome: 289 A Rescue Trial in Beijing Inhaled nitric oxide Inhaled NO and Covid-19 Mechanism of vascular smooth muscle relaxation by organic nitrates, 297 nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as 298 active intermediates Long-distance airborne dispersal of SARS-CoV-2 in COVID-19 wards Detection of 2019 novel 306 coronavirus (2019-nCoV) by real-time RT-PCR Production of Authentic SARS-CoV Mpro with Enhanced Activity: Application as a 310 Novel Tag-cleavage Endopeptidase for Protein Overproduction Viral mutation accelerated by nitric oxide 314 production during infection in vivo Inhibition of vesicular stomatitis virus infection by nitric oxide S-Nitrosylation of Viral Proteins: 319 Molecular Bases for Antiviral Effect of Nitric Oxide