key: cord-255552-k1retwa4 authors: Gassen, Nils C.; Papies, Jan; Bajaj, Thomas; Dethloff, Frederik; Emanuel, Jackson; Weckmann, Katja; Heinz, Daniel E.; Heinemann, Nicolas; Lennarz, Martina; Richter, Anja; Niemeyer, Daniela; Corman, Victor M.; Giavalisco, Patrick; Drosten, Christian; Müller, Marcel A. title: Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics date: 2020-04-15 journal: bioRxiv DOI: 10.1101/2020.04.15.997254 sha: doc_id: 255552 cord_uid: k1retwa4 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an acute threat to public health and the world economy, especially because no approved specific drugs or vaccines are available. Pharmacological modulation of metabolism-dependent cellular pathways such as autophagy reduced propagation of highly pathogenic Middle East respiratory syndrome (MERS)-CoV. Here we show that SARS-CoV-2 infection limits autophagy by interfering with multiple metabolic pathways and that compound-driven interventions aimed at autophagy induction reduce SARS-CoV-2 propagation in vitro. In-depth analyses of autophagy signaling and metabolomics indicate that SARS-CoV-2 reduces glycolysis and protein translation by limiting activation of AMP-protein activated kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1). Infection also downregulates autophagy-inducing spermidine, and facilitates AKT1/SKP2-dependent degradation of autophagy-initiating Beclin-1 (BECN1). Targeting of these pathways by exogenous administration of spermidine, AKT inhibitor MK-2206, and the Beclin-1 stabilizing, antihelminthic drug niclosamide inhibited SARS-CoV-2 propagation by 85, 88, and >99%, respectively. In sum, SARS-CoV-2 infection causally diminishes autophagy. A clinically approved and well-tolerated autophagy-inducing compound shows potential for evaluation as a treatment against SARS-CoV-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an acute threat to public health 22 and the world economy, especially because no approved specific drugs or vaccines are available. Compound-based targeting of cellular proteins that are essential for the virus life cycle has led to the 50 discovery of broadly reactive drugs against a range of CoVs (3-6). As virus propagation strongly 51 depends on energy and catabolic substrates of host cells, drug target identification should consider 52 the metabolism of infected cells (3). Autophagy, a highly conserved cytosolic degradation process of 53 long-lived proteins, lipids, and organelles in eukaryotic cells, is tightly controlled by metabolism (7, 8). During autophagy, intracellular macromolecules are recycled by incorporation into LC3B-lipidated 55 autophagosomes (AP) and degradation into their monomers, such as fatty and amino acids, after 56 fusion with low pH lysosomes (9). In the case of highly pathogenic Middle East respiratory syndrome 57 (MERS)-CoV, we recently showed that autophagy is limited by a virus-induced AKT1-dependent 58 activation of the E3-ligase S-phase kinase-associated protein 2 (SKP2), which targets the key autophagy 59 initiating protein Beclin-1 (BECN1) for proteasomal degradation (10). Congruently, inhibition of SKP2 propagation up to 50% (Figure 4a, lower right, Figure S3d,e) . AKT1 blocks mTORC1 inhibitor TSC2 (30) 172 and further supports the suggestion that up-regulation of mTORC1 components has antiviral effects. As AKT1 inhibition results in BECN1 up-regulation and autophagy induction (10, 31), SARS-CoV-2 174 growth inhibition was expected. Direct blocking of the negative BECN1 regulator SPK2 by previously 175 described inhibitors SMIP004, SMIP004-7, valinomycin, and niclosamide (10) showed SARS-CoV-2 176 growth inhibition from 50 (SMIP004, SMIP004-7) to over 99% in case of valinomycin and niclosamide 177 (Figure 4a, lower panel, Figure S3d,e) . We further confirmed that the dominant intervention of 178 niclosamide during SARS-CoV-2 infection acts on autophagy induction, as adding BafA1 after 179 niclosamide treatment showed an enhancing effect on the lipidation of LC3B as reflected by 180 comparable LC3B-II/I ratios between mock-and SARS-CoV-2-infected cells (Figure 4b) . However, we 181 cannot exclude that the activity of niclosamide as a hydrogen ionophore has additional inhibitory 182 functions, e.g. by blocking endosomal acidification (32), which is important for SARS-CoV-2 entry (6). Twenty-four hours later, cells were fixed and analyzed by fluorescence microscopy. Vesicles with both green and red fluorescence (APs) and with red fluorescence only (autolysosomes, AL) were counted. In all panels error bars denote standard error of mean derived from n = 3 biologically independent experiments. Tp < 0.1, *p < 0.05, ***p < 0.001 (two-way ANOVA in a,b, one-way ANOVA in c,d, t-test in e. Abbreviations: LC3B, microtubule-associated protein 1A/1B light chain 3B; mRFP, monomeric red fluorescent protein; EGFP, enhanced green fluorescent protein. Clinical Characteristics of Coronavirus Disease 2019 in China. 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