Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics

Nils C. Gassen; Jan Papies; Thomas Bajaj; Frederik Dethloff; Jackson Emanuel; Katja Weckmann; Daniel E. Heinz; Nicolas Heinemann; Martina Lennarz; Anja Richter; Daniela Niemeyer; Victor M. Corman; Patrick Giavalisco; Christian Drosten; Marcel A. Müller

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.15.997254 doi: bioRxiv preprint Interestingly, putrescine (Figure 3c, red) was increased whereas its downstream products of the 143 polyamine biosynthesis pathway, spermidine and spermine, were strongly reduced. The putrescine 144 increase might be explained by a SARS-CoV-2-induced inhibition of spermidine synthase, the enzyme 145 that synthesizes spermidine from putrescine (Figure 3c, red) The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.15.997254 doi: bioRxiv preprint (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.