key: cord-0914567-2ejg2ngh
authors: Vitner, Einat. B.; Avraham, Roy; Achdout, Hagit; Tamir, Hadas; Agami, Avi; Cherry, Lilach; Yahalom-Ronen, Yfat; Politi, Boaz; Erez, Noam; Melamed, Sharon; Paran, Nir; Israely, Tomer
title: Antiviral activity of Glucosylceramide synthase inhibitors against SARS-CoV-2 and other RNA virus infections
date: 2020-05-19
journal: bioRxiv
DOI: 10.1101/2020.05.18.103283
sha: 663fa2faaeb4feae622e7f120d0b9ecbbbb5a35a
doc_id: 914567
cord_uid: 2ejg2ngh

The need for antiviral drugs is real and relevant. Broad spectrum antiviral drugs have a particular advantage when dealing with rapid disease outbreaks, such as the current COVID-19 pandemic. Since viruses are completely dependent on internal cell mechanisms, they must cross cell membranes during their lifecycle, creating a dependence on processes involving membrane dynamics. Thus, in this study we examined whether the synthesis of glycosphingolipids, biologically active components of cell membranes, can serve as an antiviral therapeutic target. We examined the antiviral effect of two specific inhibitors of GlucosylCeramide synthase (GCS); (i) Genz-123346, an analogue of the FDA-approved drug Cerdelga®, (ii) GENZ-667161, an analogue of venglustat which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit the replication of four different enveloped RNA viruses of different genus, organ-target and transmission route: (i) Neuroinvasive Sindbis virus (SVNI), (ii) West Nile virus (WNV), (iii) Influenza A virus, and (iv) SARS-CoV-2. Moreover, GCS inhibitors significantly increase the survival rate of SVNI-infected mice. Our data suggest that GCS inhibitors can potentially serve as a broad-spectrum antiviral therapy and should be further examined in preclinical and clinical trial. Analogues of the specific compounds tested have already been studied clinically, implying they can be fast-tracked for public use. With the current COVID-19 pandemic, this may be particularly relevant to SARS-CoV-2 infection. One Sentence Summary An analogue of Cerdelga®, an FDA-approved drug, is effective against a broad range of RNA-viruses including the newly emerging SARS-CoV-2.

Viral infections create a significant burden to human health worldwide, making the development of antiviral drugs a pressing need. Despite the rapid advancement in pharmaceutical and biotechnological approaches (e.g., RNA interference [RNAi] (1)), the development of successful antiviral treatments remains a challenge (2) . Historically, drug research has mainly focused on targeting viral components, because of the perceived specificity of such an approach (3).

However, since viral life cycle is dependent on the host, specific host mechanisms can also be explored as antiviral targets. There are distinct advantages for this approach such as creating a high barrier to resistance, providing broad coverage of different genotypes/serotypes, possibly even multiple viruses, and expanding the list of potential targets for a drug, when druggable viral targets are limited (4) .

While side effects may be of particular concern for such treatments, another advantage of targeting host protein is the availability of many approved drugs against host proteins, allowing for drug repurposing. The main advantage of repurposing approved drugs is that they have already proven to be sufficiently safe, they have successfully passed clinical trials and regulatory scrutiny, and they have already undergone post-marketing surveillance (5) .

This leads to significantly reduced timelines and required investment in making treatment available. In cases of major pandemic outbreaks caused by new viruses, shortening the time to treatment can have a major impact on public health and economics, making drug repurposing particularly desirable.

Sphingolipids (SLs) are biologically active components of cell membranes and as such are tightly linked to all processes involving membrane dynamics, making them potential key regulators in the life cycle of obligatory intracellular pathogens such as viruses.

Glucosylceramide (GlcCer) is the backbone of more than 300 structurally different Glycosphingolipids (GSLs) including gangliosides and sulfatides. Its accumulation leads to Gaucher diseases accompanied by chronic brain inflammation and activation of the antiviral immune response (6) . GSLs are involved in lateral and vertical segregation of receptors required for attachment, membrane fusion and endocytosis, as well as in intracellular replication, assembly and release of viruses. In addition, GSLs and their metabolites are inseparably interwoven in signal transduction processes, and the regulation of innate and intrinsic responses of infected target cells (7) . Viral-induced elevation of SL levels was shown to be associated with a number of viruses; elevation of GM2-ganglioside and Lactosylceramide was shown upon infection with Zika virus and Hepatitis C virus (HCV), respectively (8, 9) . Human Cytomegalovirus (HCMV) induces elevation of ceramide and GM2-ganglioside (10) , and Dengue virus induces elevation of ceramide and sphingomyelin (11) . Additionally, Influenza virus was shown to induce Sphingomyelin and GlcCer elevation (12, 13) and suppression of the biosynthesis of cellular sphingolipids results in the inhibition of the maturation of influenza virus particles in vitro (14, 15) . Moreover, iminosugars are known for their broad-spectrum antiviral activity, presumably because of their mechanism of action as endoplasmic reticulum (ER)-resident α-glucosidases I and II inhibitors (16) . 1-Deoxynojirimycin (DNJ) iminosugar derivatives inhibit in vitro production of infectious viruses including dengue virus (DENV) (17, 18) , hepatitis B virus (HBV) (19, 20) , hepatitis C virus (HCV) (21), human immunodeficiency virus (HIV) (22, 23) , and influenza A virus (24) . Antiviral efficacy of the iminosugar N-butyl-DNJ (NB-DNJ, Miglustat, Zavesca) has been further demonstrated in vivo against DENV infection (25) .

Although these reports present strong circumstantial evidence that inhibition of ER α-glucosidase activity is the cause of iminosugar antiviral activity (26) , the ubiquity of D-glucose in metabolism suggests that other pathways may be equally affected by iminosugar treatment.

Indeed, NB-DNJ has been approved for clinical use since 2002 as a second line treatment for Gaucher's disease (27) -a lysosomal storage disease (LSD). In this context, NB-DNJ is used as an inhibitor of UDP-glucose:ceramide glucosyltransferase (glucosylceramide synthase (GCS)), (EC 2.4.1.80) to reduce production of GSLs that accumulate due to a deficiency in GlcCer degradation (28) . Thus, GSL synthetic pathways may be therapeutic targets for a broad-range of viral infection. Drugs targeting SL metabolizing enzymes are currently in use and constantly being developed for treating LSDs and other disorders in which alteration in SL levels are involved in disease pathology ( 29 -31 ) . This allows a potential repurposing of these already approved drugs as antivirals. While the inhibitor NB-DNJ affects multiple host targets, specific inhibition of GCS is now possible using GCS inhibitors which are currently available. In this study, we examined the antiviral activity of two specific inhibitors of GCS, which catalyze the biosynthesis of GlcCer. These inhibitors block the conversion of ceramide to GlcCer, the first step in the biosynthesis of gangliosides and other glycosphingolipids. 

To determine whether GCS inhibitors block the replication of SVNI, Vero cells were incubated in Vero and N2a cells, respectively ( Fig 1B) . 

To determine which stage of SVNI infection cycle was affected by GCS inhibitors, a time-ofaddition assay was performed. As shown in Fig 

To evaluate whether GZ-161 could protect SVNI-infected mice, at a concentration comparable with the pre-clinical/clinical studies for drug approval, mice were treated with GZ-161 (20mg/kg/day, i.p) and infected with a lethal dose of SVNI. GZ-161 significantly protected pretreated infected mice from SVNI-induced mortality (Fig 3) . Virtually all SVNI-infected mice 

Next, we examined whether the antiviral activity of GCS inhibitors is specific to SVNI or 

Since GCS inhibitors showed antiviral activity towards two neuronopathic viruses, and given the current COVID-19 pandemic, we checked whether GZ-161 and GZ-346 will block also non- GZ-161 and GZ-346 significantly reduced PR8-induced cytotoxicity by 65% and 90% respectively (Fig. 5B) . 

To test for antiviral activity of GCS inhibitors against SARS-CoV-2, Vero E6 cells were incubated with 10 μM GZ-161 or GZ-346 one hour prior to infection with SARS-CoV-2.

Supernatants were harvested 24 hpi and analyzed by plaque forming units assay to measure the effect of the drugs on SARS-CoV-2 replication (Fig 6A and B) . Data are means of six replicates ± SEM. *** p <0.001, **** p <0.0001.

The need for antiviral drugs is real and relevant. This is especially true for diseases without an effective vaccine. Antiviral drugs with a broad spectrum have a particular advantage dealing with emerging disease outbreaks, such as the current COVID-19 pandemic.

In this study we demonstrate that the GCS inhibitors GZ To advance the pre-clinical development of GCS inhibitors as antiviral drugs, we further examined whether GZ-161 is effective in vivo. While both GZ-161 and GZ-346 target GCS, only GZ-161 can penetrate the brain, making it the preferred choice for diseases involving the CNS (35) . While GZ-161 significantly improved the survival, GZ-346 had no effect when given starting from 2dpi (data not shown), highlighting the necessity of the inhibitor to penetrate the brain in viral-infections of the CNS.

Treatments of SVNI-infected mice with GZ-161 were carried out at 20mg/kg/day, the same dosage that was administered during the preclinical development of GZ-161 for Gaucher disease (49) and Fabry (50) . The preclinical studies using this dosage supported the clinical dossier to approve the safe and effective use of venglustat (GZ-161 analogue) and eliglustat (GZ-346 analogue) in humans at 168 mg/day to treat Gaucher disease. Thus, as a proof of concept, our findings consistently show that GCS inhibitors, at a pragmatic concentration, possesses antiviral activity robust enough to provide protection against severe SVNI infection.

While GZ161 was only tested in-vivo on SVNI, in-vitro comparison of GZ-161 on the different viruses suggests it is even more potent against Influenza (~90% inhibition) and SARS-COV-2 (~100% inhibition) compared to SVNI (~60% inhibition). Furthermore, the mouse model of SVNI disease is acute with a mortality rate of about 90% and significant CNS damage, and yet GZ-161 treatment was found to be effective.

We therefore believe it is important to test GZ-161 in-vivo also against Influenza virus and COVID-19. Naturally, the best means of administration of these drugs for respiratory diseases also needs to be established (Intraperitoneal, intravenous, intranasal, etc.). While treatment with GZ-161 was effective also when administration started post viral exposure, effect was more significant when administration began pre exposure. It is therefore worth considering both therapeutic and prophylactic treatment for populations at high risk. CO2/95% air atmosphere.

The original strain of Sindbis virus (SV) was isolated in 1990 from mosquitoes in Israel.

This strain was used as a source for variants which differ in their neuro-invasiveness and virulence, generated by a serial passages of SV in suckling and weanling mouse brain. At the 15th passage a neurovirulent variant was observed and designated SVN (neurovirulent). After 7 more passages in weanling mouse brains, another variant was observed and designated SVNI (neuroinvasive). The SVNI strain used is both virulent and CNS-invasive [13] . 

The Cell viability of >95% of non-infected cells was determined by XTT assay (Merck, a colorimetric cell proliferation assay, for quantification of cellular proliferation, viability, and cytotoxicity). Evaluation of the half maximal inhibitory concentration (IC50) was performed by GraphPad Prism 6. Percentage of inhibition was calculated by subtracting the ratio of PFU between treated and untreated cells from 1. 

Vero cells were seeded at a density of 5 × 10 5 cells per well in 6-well plates. After incubating overnight, cells were treated in 4-replicates GZ-161 or GZ-346. One hour later, cells were infected with WNV at MOI 0.1 for 24 h. Supernatant was collected for qPCR. Percentage of inhibition was calculated by subtracting the ratio of PFU between treated and untreated cells from 1.

MDCK cells were seeded at a density of 5 × 10 5 cells per well in 6-well plates. After incubating overnight, cells were treated in 4-replicates with GZ-161 or GZ-346. One hour later, cells were infected in serum-free medium containing 0.5 μg/mL TPCK-trypsin with PR8 at MOI 0.1. Supernatants were collected 8 hpi for qPCR. Cell cytotoxicity was determined 24 hpi by LDH Assay (Cytotoxicity) (ab65393) according to manufacturer's protocol. Percentage of inhibition was calculated by subtracting the ratio of PFU between treated and untreated cells from 1.

Vero E6 cells were seeded at a density of 1. Mice were weighed daily throughout the experiment, and dosages were adjusted accordingly.

Control mice received a similar volume of injection buffer without the active agent.

Statistical analyses were performed with a two-tailed unpaired t-test, or as indicated in the legends. P values are indicated by asterisks in the figures as follows: *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Differences with a P value of 0.05 or less were considered significant. The exact value of n, representing the number of mice in the each experiment, is indicted in the figure legends. Data for all measurements are expressed as means ± SEM.

For mouse survival, Kaplan-Meier survival curves were generated and analyzed for statistical significance with GraphPad Prism 6.0 [Log-rank (Mantel-Cox) test (conservative)].

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