key: cord-0958593-1zbt8iqs authors: Halfon, Philippe; Bestion, Eloïne; Zandi, Keivan; Andreani, Julien; Baudoin, Jean-Pierre; La Scola, Bernard; Mege, Jean-Louis; Mezouar, Soraya; Schinazi, Raymond F. title: GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition date: 2020-10-06 journal: bioRxiv DOI: 10.1101/2020.10.06.327635 sha: c28c57ad3534dabbee65a1d82f72bc8d0e58c5cc doc_id: 958593 cord_uid: 1zbt8iqs Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection. Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-45 2/2019-nCoV, also called coronavirus disease of 2019, Covid-19) has spread quickly worldwide 46 and has qualified as a pandemic (1) . As of October 2020, more than 29 million cases and 927,000 47 deaths have been reported worldwide (World Health Organization data). SARS-CoV-2 infection 48 leads to a range of symptoms from mild fever to acute respiratory distress syndrome classifying 49 this disease into several clinical categories, including asymptomatic, mild, moderate, severe and 50 critical infection (2). 51 Thus, there is an urgent need for a safe and powerful, accessible and affordable drug to (4) that has recently been given emergency approval as a Covid-19 treatment. 60 The diversion of the autophagy mechanism by viruses, including coronaviruses, allows for 61 viral replication (5). More specifically, coronaviruses were found to use double membrane 62 vesicles to enhance the efficiency of virus replication (6). Additionally, it has been suggested that flaviviruses, retroviruses, and coronaviruses, including SARS-CoV-2 (9). It turned out that the 71 autophagy mechanism acts as an antiviral defense in the early step of viral infection (5). 72 Our team developed an autophagy inhibitor, GNS561, which is a small basic lipophilic 73 molecule that induces lysosomal dysregulation, as proven by the inhibition of late-stage 74 autophagy (10). GNS561 is currently in development for oncology indications (NCT03316222) 75 and it is being tested in a clinical trial of patients infected by Covid-19 (NCT04333914). The 76 objectives of the current study were i) to evaluate the in vitro antiviral activity of GNS561 against 77 SARS-CoV-2, ii) to assess the mechanism of blocking the viral replication by inhibition of 78 autophagy, and iii) to demonstrate a synergistic combination with remdesivir. As illustrated in Fig. 1 and Table 2 respectively. More precisely, the GNS561 inhibitory concentration was 16 and 200 times higher 181 than CQ and remdesivir, respectively. Moreover, we confirmed the strong antiviral effect of 182 GNS561 using another SARS-CoV-2 strain (IHU-MI6). As illustrated in Table 2 , GNS561 183 presented a 0.03 EC50 and 0.07 μM EC90. To summarize, our data showed that GNS561 activity 184 is higher compared to CQ and remdesivir respectively. with nascent particles (red asterisk) ( Fig. 2A) . GNS561 treatment leads to an accumulation and 192 an increase in the volume of autophagic vacuoles in the cytoplasm with the presence of 193 multilamellar bodies characteristic of a complexed autophagy ( Fig. 2A) . 194 Based on these results, we next investigated the location of SARS-CoV-2 in the presence of our 195 antiviral compound in the cellular compartments associated with the autophagy mechanism. As 196 depicted in Fig. 2B , we noted that SARS-CoV-2 was located in LAMP2-positive lysosomes similar 197 to GNS561. Next, we showed that SARS-CoV-2 infection leads to an increase in size of LAMP2 198 particles associated with a reorganization of intracellular LC3-II expression, also called spots (Fig. 199 2C) . In a dose-dependent manner, the size of LC3-II spots increased following GNS561 (Fig. 3B) . Interestingly, normalized LC3-II protein expression was increased in SARS- CoV-2-infected cells treated with GNS561 and decreased after Baf-A1 treatment, suggesting that 211 the compound altered autophagosome synthesis (Fig. 3C) . Overall, our results indicate that 212 GNS561 impacts on autophagy mechanism and subsequent inhibition of viral multiplication. 77 (14, 15) . Moreover, CQ and HCQ have 228 been proposed as treatment for SARS- CoV-2 infection (14, 16, 17) . At every tested MOI (0.01, 229 0.02, 0.2, and 0.8), the EC50 for CQ (2.71, 3.81, 7.14 and 7.36 μM) was lower than that of HCQ show broad-spectrum antiviral activities. Thus, there is some interest in using drug combinations 257 against SARS-CoV-2. Interestingly, it was recently reported that nitazoxanide in combination with 258 amodiaquine, umifenovir or remdesivir presented a significant synergy against SARS-CoV-2 (28). 259 It was also reported that remdesivir and HCQ exhibited a strong antagonism (28). Here, we The epidemiology and pathogenesis of coronavirus disease 283 (COVID-19) outbreak Estimates of the severity of COVID-19 disease (Epidemiology The Global Phosphorylation Landscape of SARS-CoV-2 301 Arguments 303 in favour of remdesivir for treating SARS-CoV-2 infections Autophagy during viral infection -a double-edged sword Virus factories, double membrane vesicles and viroplasm 308 generated in animal cells The SARS-CoV-2 cytopathic effect is blocked with 312 autophagy modulators Chloroquine analogues in drug discovery: new directions of uses, mechanisms 315 of actions and toxic manifestations from malaria to multifarious diseases Role of Chloroquine and Hydroxychloroquine in the Treatment of 318 COVID-19 Infection-A Systematic Literature Review (Infectious Diseases (except HIV/AIDS) GNS561, a new lysosomotropic small 322 molecule, for the treatment of intrahepatic cholangiocarcinoma Quantitative analysis of dose-effect relationships: the combined 325 effects of multiple drugs or enzyme inhibitors The use of lead citrate at high pH as an electron-opaque stain in electron 327 microscopy A three-dimensional model to analyze drug-drug interactions Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-332 nCoV) in vitro Compassionate Use of Remdesivir for Patients with Severe Covid-19, N Engl 341 Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 344 infection in vitro In Vitro Antiviral Activity and Projection of Optimized Dosing 347 Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome 348 Coronavirus 2 (SARS-CoV-2) Targeting the Endocytic Pathway and Autophagy Process as a Novel 350 Therapeutic Strategy in COVID-19 Focusing on the Unfolded Protein 352 Response and Autophagy Related Pathways to Reposition Common Approved Drugs against Replication Complex Formation Utilizes Components of Cellular Autophagy Involvement of Autophagy in Coronavirus Replication Autophagy/virophagy: a "disposal strategy SKP2 attenuates autophagy 365 through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection Chloroquine inhibits autophagic flux by decreasing autophagosome-369 lysosome fusion New insights into the antiviral 371 effects of chloroquine Remdesivir in adults with severe COVID-19: a randomised, double-blind, 377 placebo-controlled, multicentre trial Remdesivir for the Treatment 384 of Covid-19 -Preliminary Report Antagonistic Drug Combinations against SARS-CoV-2 In Vitro Hydroxychloroquine and azithromycin as a treatment of COVID-392 19: results of an open-label non-randomized clinical trial Increased Incidence of Gastrointestinal Side Effects 395 in Patients Taking Hydroxychloroquine: A Brand-related Issue? Retinal toxicity in 398 long term hydroxychloroquine treatment The ocular safety of hydroxychloroquine, Seminars in Arthritis and 400 Restrictive cardiomyopathy 402 caused by chloroquine Heart conduction disorders related to antimalarials toxicity: an analysis of 405 electrocardiograms in 85 patients treated with hydroxychloroquine for connective tissue diseases Acknowledgments. This work was supported in part by the funding from the Emory Center for 411 AIDS Research (5P30-AI-50409 to RFS).