key: cord-0910656-19o75w1r
authors: Ohashi, Hirofumi; Wang, Feng; Stappenbeck, Frank; Tsuchimoto, Kana; Kobayashi, Chisa; Saso, Wakana; Kataoka, Michiyo; Kuramochi, Kouji; Muramatsu, Masamichi; Suzuki, Tadaki; Sureau, Camille; Takeda, Makoto; Wakita, Takaji; Parhami, Farhad; Watashi, Koichi
title: Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro
date: 2021-02-01
journal: bioRxiv
DOI: 10.1101/2021.01.31.429001
sha: f326f17baac34136b158323234572497bccd6699
doc_id: 910656
cord_uid: 19o75w1r

Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

Coronavirus disease 2019 , caused by infection with the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), has drastically impacted public health and, on a global scale, caused enormous harm to human societies and their economic vitality. In the search for effective treatments for COVID-19, understandably, the repurposing of existing FDAapproved drugs has been given high priority due to their known safety profiles [1] . For example, remdesivir (RDV), which was originally designed as an anti-ebola virus agent, has been repurposed to become the first and, to date, the only FDA-approved drug treatment for SARS-CoV-2 infection. Similarly, chloroquine and hydroxychloroquine, which are used to control malaria, have been investigated as COVID-19 treatments [2] . Beyond drug repurposing [3] , other approaches are urgently needed to invigorate discovery research for new, specific, and potent anti-COVID-19 drugs.

Naturally occurring oxysterols include metabolites of cholesterol involved in the biosynthesis of steroid hormones, vitamin D, bile acids and other crucial signaling molecules [4, 5] . Beyond their role as passive and transient metabolites, endogenous oxysterols are increasingly recognized as lipid signaling molecules that can regulate a range of physiological processes, including lipid homeostasis, transport and metabolism as well as immune response [5] . In recent years, numerous reports have ascribed broad spectrum antiviral properties to naturally occurring oxysterols. against herpes simplex virus [7] , human papillomavirus-16, human rhinovirus [8] , murine norovirus [9] , rotavirus [10] , and zika virus [11] .

In this study, we focused on oxysterols, including naturally occurring and semi-synthetic oxysterols, to identify potent anti-SARS-CoV-2 agents, since we have already developed various semi-synthetic oxysterols as drug candidates in the context of cancer, fibrotic diseases and bone regeneration: Oxy133, an allosteric activator of Hedgehog (Hh) signaling, was designed for orthopedic applications, such as bone repair and spine fusion [12] [13] [14] ; Oxy186, an inhibitor of Hh signaling that acts downstream of the Smoothened (Smo) receptor, was designed as a potential anti-tumorigenic agent [15] , and Oxy210 was designed for application in cancer and fibrosis through dual inhibition of Hh and transforming growth factor-beta (TGF-b) signaling [16] . In the present report, using cell-based analysis, we demonstrate that Oxy210 and its analogue, viruses [17] [18] [19] . Oral administration of a single dose of Oxy210 at 200 mg/kg in mice resulted in a peak plasma concentration (Cmax) of about 19 µM, which exceeds both the IC50 (5.6 µM) and IC90 (8.6 µM), respectively, determined in our cell-based assay. These data provide foundational evidence for Oxy210 and Oxy232 as potential anti-COVID-19 candidates for further therapeutic development in the future.

In this study, we used a cell-based SARS-CoV-2 infection system previously reported [20] . 

Although the natural oxysterols, 7-KC, 22(R)-OHC, 24(S)-OHC, and 27-OHC showed modest anti-SARS-CoV-2 activities, their physiological concentrations are at far below µM ranges [21, 22] in the circulation of healthy humans, suggesting their limited role, if any, in preventing viral infection in physiological condition. In search for oxysterols with improved antiviral activity, we evaluated the potential of semi-synthetic oxysterol derivatives for SARS-CoV-2 inhibition.

SARS-CoV-2-induced CPE and virus propagation were blocked when treated with Oxy210 but not Oxy133 ( Figure was almost equivalent to that of the natural oxysterols shown earlier; the maximum reduction in viral RNA was 83% when used at 12 µM as compared to control ( Figure 2C , note that the viral RNA shown in logarithm scale). On the other hand, Oxy210 and Oxy232 showed much higher antiviral potencies; viral RNA production was reduced by 99.4% (Oxy210) and 99.9% (Oxy232)

at the maximum at 15 µM ( Figure 2C ). No significant cytotoxicity by Oxy186 and Oxy210 was found up to 15 µM, the maximum concentration in the infection assay, however, Oxy232 slightly reduced cell viability when used at concentrations above 10 µM ( Figure 2D ). Due to the greater availability of Oxy210 we performed further studies with this oxysterol analogue. The 50% and 90% maximal inhibitory concentration (IC50, IC90) and 50% maximal cytotoxic concentration (CC50) of Oxy210 were 5.6 µM, 8.6 µM, and >15 µM, respectively.

We previously reported that Oxy210 inhbited Hedgehog (Hh) and transforming growth factor b (TGFb) signalings in fibroblastic cells and tumor cells [16] . In contrast, Oxy232, a close structural analogue of Oxy210, is devoid of significant TGFb inhibitory properties ( Figure 2E ), but retains inhibitory activity toward Hh signaling ( Figure 2F ), suggesting that inhibition of TGFb signaling is not resposible for the anti-SARS-CoV-2 activity. Consistent with this observation, treatment with the TGFb signaling inhibitor, SB431542, did not significantly inhibit the production of viral RNA ( Figure 2G ). In addition, inactivation of Hh pathway by either HPI-1 or GDC0449 did not decrease the viral RNA levels ( Figure 2G ). These data suggest that Oxy210, Oxy232 and other antiviral oxysterol analogues inhibit SARS-CoV-2 production independent of the inhibition of Hh or TGFb signaling pathways.

To determine which steps in the SARS-CoV-2 life cycle ( Figure. Coronaviruses generally induce the formation of unique membrane compartments, called double membrane vesicles (DMVs), which enables an efficient viral RNA replication [18, 26] . We found that DMV formation occurs with infection by SARS-CoV-2 in VeroE6/TMPRSS2 cells ( Figure. drive replication in a DMV-dependent and -independent manner, respectively [26, 27] . Similar to the effect of an HCV polymerase inhibitor, sofosbuvir, used as a positive control, Oxy210 reduced the DMV-dependent RNA replication of HCV ( Figure 3C ), while the antiviral activity was not observed in HDV infection that was inhibited by the positive control, MyrB ( Figure 3D ).

These data are consistent with the idea that Oxy210 specifically inhibits the DMV-dependent virus replication, although it remains to be determined whether Oxy210 directly inhibits the DMV formation machinery, which we will examine in future studies (see the discussion below).

Given its higher anti-SARS-CoV-2 potency compared to the natural oxysterols, we questioned 

In Therefore, Oxy210 and its analogues, such as Oxy232, could potentially serve as drug candidates targeting COVID-19.

We have previously characterized Oxy210 as a Hh and TGFb signaling inhibitor [16] and have Oxy210. This notion was further supported by the lack of antiviral activity displayed by a TGFb pathway inhibitor, SB431542 (10 µM). Also, the antiviral activity is not likely to be due to the inhibition of Hh pathway, as suggested by the lack of antiviral activity of Hh pathway inhibitors, HPI-1 (10 µM) and GDC0449 (10 µM). Given the absence of unrelated biological activities, such as TGFb inhibition, Oxy232 may be a preferable drug candidate compared to Oxy210.

A recent publication reported significant anti-SARS-CoV-2 activity of 27-OHC; low concentrations of 27-OHC inhibited post-entry and higher concentration inhibited viral entry process [21] . Our time-of-addition analysis suggests that Oxy210 predominantly inhibits the post-entry process, which includes viral RNA replication in the replication factory and the following assembly of progeny virus and its secretion. We observed the formation of DMVs in SARS-CoV-2-infected cells, as previously reported [18, 28] . DMVs, membrane compartments separated from the nuclease/protease-rich cytosol, are generally considered to be sites for efficient replication of genomic RNA of coronaviruses and of certain other RNA viruses, such as HCV [26] . DMVs are also very likely to be important in SARS-CoV-2 replication [17] . We showed that the production of DMVs, induced by SARS-CoV-2, was substantially reduced with Oxy210 treatment. Antiviral effects of Oxy210 were also observed during the replication of HCV, a virus that depends on DMVs for replication, but not with HDV, a virus that replicates independently of DMVs. These findings suggest that Oxy210 specifically reduces DMV-dependent virus replication. It is not clear, however, whether Oxy210 directly inhibits the formation of DMVs.

Further studies will be performed in the future to analyze the mode of action for the antiviral activity of Oxy210 and its analogues, and to elucidate the molecular mechanisms underlying their inhibitory effects on SARS-CoV-2 replication and DMV formation.

It is noteworthy to outline the potential advantages of semi-synthetic oxysterols as anti-SARS-CoV-2 agents, compared to established antiviral compounds, such as RDV:

1) RDV has to be administered intravenously, most often in a hospital setting, whereas the 3) The scale up and manufacturing of oxysterol-based drug candidates is expected to be straightforward and process friendly, especially when compared to the manufacturing process of RDV, which is rather difficult to prepare on scale.

We conclude that semi-synthetic oxysterol derivatives, such as Oxy210 and Oxy232, could be promising leads in the search for COVID-19 drug candidates, used alone, or in combination with other therapies currently FDA approved or under investigation, such as RDV, convalescent plasma or antibody treatments.

Compounds and the synthesis of oxysterol derivatives. Commercially available oxysterols were obtained from Sigma Aldrich. Oxy133, Oxy186 and Oxy210 were prepared as previously described [12, 15, 16] . Oxy232 was prepared via a similar three-step synthesis described for 

We used the SARS-CoV-2 Wk-521 strain, a clinical isolate from a COVID-19 patient, and obtained viral stocks by infecting VeroE6/TMPRSS2 cells [20] . VeroE6/TMPRSS2 cells were inoculated with SARS-CoV-2 at an MOI of 0.001 ( Figure 1B, 1C 

LucNeo#2 cells, carrying a subgenomic replicon RNA for an HCV NN strain (genotype-1b) and the luciferase gene driven by the HCV replication [30] . LucNeo#2 cells were treated with the compounds indicated in Figure 3C 

We perfomed the pharmacokinetics analysis in mice by oral administration with Oxy210 as described previously [16] . 

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Inhibition of Non-Small Cell Lung Cancer Cells by Oxy210, an

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Coronavirus replication factories

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Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

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Increased plasma levels of oxysterols, in vivo markers of oxidative stress, in patients with familial combined hyperlipidemia: reduction during atorvastatin and fenofibrate therapy

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Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

Architecture and biogenesis of plus-strand RNA virus replication factories

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SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

Consensus and variations in cell line specificity among human metapneumovirus strains

Evaluation of the anti-hepatitis C virus effects of cyclophilin inhibitors, cyclosporin A, and NIM811

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Phosphorylation of p38 MAPK and its downstream targets in SARS coronavirus-infected cells