key: cord-295765-c7o2ukm6
authors: Silvas, Jesus A.; Jureka, Alexander S.; Nicolini, Anthony M.; Chvatal, Stacie A.; Basler, Christopher F.
title: Inhibitors of VPS34 and lipid metabolism suppress SARS-CoV-2 replication
date: 2020-07-20
journal: bioRxiv
DOI: 10.1101/2020.07.18.210211
sha: 
doc_id: 295765
cord_uid: c7o2ukm6

Therapeutics targeting replication of SARS coronavirus 2 (SARS-CoV-2) are urgently needed. Coronaviruses rely on host membranes for entry, establishment of replication centers and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we tested small molecule inhibitors that target membrane dynamics or lipid metabolism. Included were inhibitors of the PI3 kinase VPS34, which functions in autophagy, endocytosis and other processes; Orlistat, an inhibitor of lipases and fatty acid synthetase, is approved by the FDA as a treatment for obesity; and Triacsin C which inhibits long chain fatty acyl-CoA synthetases. VPS34 inhibitors, Orlistat and Triacsin C inhibited virus growth in Vero E6 cells and in the human airway epithelial cell line Calu-3, acting at a post-entry step in the virus replication cycle. Of these the VPS34 inhibitors exhibit the most potent activity.

SARS-CoV-2, a member of the Betacoronavirus genus, is an enveloped positive-sense, 47 RNA virus responsible for a current pandemic 1 . Because of its profound impact on society and 48 human health there is an urgent need to understand SARS-CoV-2 replication requirements and to 49 identify therapeutic strategies 2 . Repurposing drugs developed for other purposes may provide a 50 shortcut to therapeutic development 3-6 . The use of compounds known to target specific host 51 factors may also elucidate key pathways needed for virus replication. In murine embryonic stem cell lines, autophagy was found to be critical for DMV formation and 60 replication of the beta-coronavirus mouse hepatitis virus 7 . However, studies in bone marrow 61 derived macrophages or primary mouse embryonic fibroblasts lacking ATG5 indicated that 62 autophagy is not essential for DMV formation or MHV replication 11 . An alternate model 63

indicates that beta coronaviruses usurp vesicles known as EDEMosomes, which associate with 64 compound. Infections with SARS-CoV-2 at an MOI of 0.01 were carried out by directly adding 

Development of 96-well format assay to measure SARS-CoV-2 cytopathic effects 167 SARS-CoV-2 induces significant cytopathic effects in infected Vero E6 cells. Based on 168 this property, we standardized a 96-well format assay that provides continuous real-time, label-169 free monitoring of the integrity of cell monolayers, thereby providing assessment of virus growth 170 through decreased cell viability. This assay was standardized using the Maestro Z platform 171 (Axion BioSystems, Atlanta, GA), an instrument that uses 96-well plates containing electrodes in 172 each well (CytoView-Z plates). The electrodes measure electrical impedance across the cell 173 monolayer every minute throughout the course of the experiment. As SARS-CoV-2 replication 174 damages the cell monolayer, impedance measurements decrease over time, providing a detailed 175 assessment of infection kinetics. 176

The capacity of the system to differentiate different levels of virus replication was first 177 assessed. Confluent Vero E6 monolayers in CytoView-Z plates were infected with SARS-CoV-2 178 . CC-BY-NC-ND 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted July 20, 2020. . https://doi.org/10.1101/2020.07.18.210211 doi: bioRxiv preprint at multiple MOIs (10 to 0.0001) and resistance measurements were acquired for 72 hours post-179 infection. As shown in Figure 1A , the progression of infection at each MOI was clearly distinct. 180

A decrease in resistance could be observed as early as 18-20 h.p.i. at an MOI of 10 and 1, and as 181 late as 56 h.p.i. at an MOI of 0.0001. Depending on MOI, signals reached their nadirs between 182 32 to 72 h.p.i. To correlate with a decrease in resistance, the raw kinetic data was used to 183 determine the median time to cell death for each MOI ( Figure 1B ). Based on its desirable 184 kinetics, the MOI of 0.01 was chosen for the screening of compounds for antiviral activities. 185

To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 186 therapeutics, we first tested Remdesivir, a well-described inhibitor of SARS-CoV-2 that has been 187 granted emergency use authorization (EUA) for the treatment of COVID-19 24, 25 . Vero E6 cells 188

were seeded on a CytoView-Z plate, incubated overnight to allow cells to stabilize, pretreated 189 with 6-fold dilutions of Remdesivir for 1 hour and infected with SARS-CoV-2. Resistance 190 measurements were recorded for 48 h.p.i. ( Figure 1C ). In agreement with previous studies, we 191 determined an 50% inhibitory concentration (IC50) for Remdesivir of 1.54 µM ( Figure 1D ) 24 . 192 Taken together, these data validate the impedance-based assay described as a tool for screening 193 of potential SARS-CoV-2 therapeutics. 194

Inhibitors of VPS34 activity impair SARS-CoV-2 growth 195 VPS34 is a multifunctional protein involved in autophagy and membrane trafficking. 196

Since coronaviruses induce formation of double membrane vesicles for replication, we wanted to 197 determine if VPS34 activity was essential for SARS-CoV-2 replication. Therefore, we tested two CoA synthetases. To test these against SARS-CoV-2, VeroE6 cells were pre-seeded onto a 219

CytoView-Z plate, allowed to stabilize and then pre-treated with Triacsin C or Orlistat for 1 hour 220 before infection with SARS-CoV-2 at an MOI of 0.01. Based on the toxicity window of 1-20 221 h.p.t. determined with the VPS34 inhibitors, neither Triacsin C nor Orlistat induced early 222 cytotoxic effects, even at the highest concentrations of 50uM and 500uM, respectively ( Figure  223 3A and 3C). Both compounds exhibited inhibition at the higher concentrations tested, although 224 . CC-BY-NC-ND 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted July 20, 2020. . https://doi.org/10.1101/2020.07.18.210211 doi: bioRxiv preprint complete inhibition was not achieved even with 500 µM of Orlistat. Based on the data we 225 extrapolated an IC50 of 422.3uM for Orlistat and calculated an IC50 of 19.5uM for Triacsin C 226 ( Figure 3B and 3D ). Viruses such as HCV and rotavirus that are sensitive to inhibition by 227

Triacsin C are also impaired by inhibitors of DGATs 14, 31 . Therefore, we tested the effects of 228 DGAT1 and DGAT2 inhibitors T863 and PF06424439 32, 33 . Neither compound displayed any 229 inhibitory activity (Supplemental Figure 1) . This data suggests that metabolism of fatty acids Supernatants were collected at 48 h.p.i. and titered on VeroE6 cells by plaque assay. In parallel, 259

to determine cytotoxicity of these compounds, Calu-3 cells were seeded onto 96-well black 260 walled 96-well plates, allowed to reach 95% confluency and treated with VPS34-IN1, PIK-III, 261

Triacsin C, Orlistat, DMSO, or mock treated with media alone. CellTox Green was added at the 262 time of dosing and fluorescence measured at 48 h.p.i. in order to assess cytotoxicity. Each of the 263 compounds inhibited production of infectious virus, as measured by plaque assay on Vero E6 264 cells Figure 5A , C, E, and G). In contrast to VeroE6 cells, no cytotoxicity was observed even at 265 the highest dose for each compound in Calu-3 cells. We observed IC50s of 0.55µM (VPS34-266 IN1), 0.12µM (PIK-III), 21.25µM (Orlistat), and 0.04µM (Triacsin C), as shown in Figure 5B , Because each compound exhibited inhibitory effects when added after viral entry, we 294 next asked whether the compounds altered the establishment of viral replication centers. Calu-3 295 cells were seeded onto fibronectin coated glass cover slips and allowed to reach 95% confluency. 296

Cells were pre-treated with approximately the IC90 of VPS34-IN1 (5 µM), PIK-III (5 µM), 297

Orlistat (500 µM), or Triacsin C (50 µM) and infected with SARS-CoV-2 at a MOI of 3. At 24 298 h.p.i. cells were fixed, permeabilized, and indirect immunofluorescence performed using primary 299 antibodies against SARS-CoV-2 nucleoprotein (N) and dsRNA. We observed that when 300 compared to the media only or DMSO controls, N became completely cytoplasmic and did not 301 form any large inclusion like formations in the presence of the compounds (Figure 6 ). 302

Additionally, even though dsRNA could be detected both distributed throughout the cytoplasm 303 and associated with N in large inclusion like formations in the media only and DMSO controls, 304

in the cells treated with inhibitors, dsRNA was only found distributed throughout the cytoplasm. 305

This data suggests that the compound disrupt replication center formation. 306

Here, we demonstrate that two VPS34 inhibitors, Orlistat, and Triacsin C each have clear effects 308 on SARS-CoV-2 replication and the morphology of viral replication centers. Generation of 309 replication centers is a key feature of the replication of many viruses 36-38 . These can serve as sites 310

where required components concentrate within a relatively closed environment and hide viral 311 replication products from the host innate immune response 39 . In order to generate these centers, 312 many viruses usurp host cellular pathways that are used to generate membranes or organelles 38 . 313

Betacoronaviruses have been shown to target the ERAD-EDEMosome-ER pathways to generate formation. That antiviral activity against HCV and rotavirus is connected to lipid droplet 382 formation is supported by the fact that these viruses are sensitive to inhibition by the DGAT 383 inhibitors, T863 and PF06424439. In contrast, the compounds did not exhibit any activity against 384 SARS-CoV-2 in Vero E6 cells whereas Triacsin C did. This suggests an alternate role for long 385 . CC-BY-NC-ND 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted July 20, 2020. . https://doi.org/10.1101/2020.07.18.210211 doi: bioRxiv preprint chain fatty acyl CoA or its downstream metabolites other than triacylglycerol and lipid droplets. 386

It is notable that the IC50 for Triacsin C was substantially lower in the Calu-3 cell assay as 387 compared to the Vero cell assay. A lesser decrease in IC50 was also noted for Orlistat in the 

Coronavirus Pandemic-Therapy and Vaccines

Review of the 2019 novel coronavirus

CoV-2) based on current evidence

Drug repurposing for new, efficient, 418 broad spectrum antivirals

Drug Repurposing Approaches 420 for the Treatment of Influenza Viral Infection: Reviving Old Drugs to Fight Against a 421 Long-Lived Enemy

Repurposing anticancer drugs for COVID-19-induced inflammation, 423 immune dysfunction, and coagulopathy

Coronavirus 427 replication complex formation utilizes components of cellular autophagy

Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived 430 vesicles exporting short-lived ERAD regulators, for replication

Unconventional use of LC3 by coronaviruses 433 through the alleged subversion of the ERAD tuning pathway

A unifying structural and functional model of the coronavirus 436 replication organelle: Tracking down RNA synthesis

Coronavirus replication does not require the autophagy gene ATG5

Rab5 and class III phosphoinositide 3-kinase Vps34 are involved in 440 hepatitis C virus NS4B-induced autophagy

Recruitment of Vps34 PI3K and enrichment 442 of PI3P phosphoinositide in the viral replication compartment is crucial for replication of 443 a positive-strand RNA virus

Modulation 445 of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C 446 virus

Novel triacsin C analogs as potential antivirals against rotavirus infections

Rotaviruses associate with cellular lipid droplet components to 450 replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit 451 viroplasm formation and viral replication

Evaluation of the antiviral activity of orlistat (tetrahydrolipstatin) 453 against dengue virus, Japanese encephalitis virus, Zika virus and chikungunya virus

Involvement of fatty acid synthase in dengue virus infection

The anti-obesity drug orlistat reveals anti-viral activity

Lipase inhibitor orlistat prevents hepatitis B virus infection by targeting 460 an early step in the virus life cycle

Orlistat, a new lipase inhibitor for the 462 management of obesity

Impedance-based cell monitoring: barrier properties 464 and beyond

Identification and 466 characterization of severe acute respiratory syndrome coronavirus replicase proteins

Remdesivir is a direct-acting antiviral that inhibits RNA-dependent 469 RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high 470 potency

Compassionate Use of Remdesivir in Covid-19

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals 474 that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream 475 target of class III phosphoinositide 3-kinase

Fatty acid metabolism: target for metabolic syndrome

Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved 482 druggable cofactors of Old World Alphavirus genome replication

Modulation of fatty acid synthase enzyme activity and expression 485 during hepatitis C virus replication

Efficient hepatitis C virus particle formation requires diacylglycerol 487 acyltransferase-1

Targeting Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small 489 molecule inhibitors for the treatment of metabolic diseases

Discovery and Optimization of Imidazopyridine-Based Inhibitors of 492

Diacylglycerol Acyltransferase 2 (DGAT2)

Severe acute respiratory syndrome coronavirus infection of human 494 ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of 495 the lungs

SARS-CoV replication and 497 pathogenesis in an in vitro model of the human conducting airway epithelium

Building Viral Replication Organelles: 500 Close Encounters of the Membrane Types

Making of viral replication organelles by remodeling interior 502 membranes

Acknowledgments. This work was supported by NIH grants R01AI125453 and P01AI120943