key: cord-0991592-apdwdolw
authors: Lo, Michael K.; Shrivastava-Ranjan, Punya; Chatterjee, Payel; Flint, Mike; Beadle, James R.; Valiaeva, Nadejda; Murphy, Joyce; Schooley, Robert T.; Hostetler, Karl Y.; Montgomery, Joel M.; Spiropoulou, Christina F.
title: Broad-Spectrum In Vitro Antiviral Activity of ODBG-P-RVn: An Orally-Available, Lipid-Modified Monophosphate Prodrug of Remdesivir Parent Nucleoside (GS-441524)
date: 2021-11-24
journal: Microbiol Spectr
DOI: 10.1128/spectrum.01537-21
sha: 690581392914e86d7cdfab6ae24e154176d58058
doc_id: 991592
cord_uid: apdwdolw

The necessity for intravenous administration of remdesivir confines its utility for treatment of coronavirus disease 2019 (COVID-19) to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524), against viruses that cause diseases of human public health concern, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had activity nearly equivalent to that of remdesivir in primary-like human small airway epithelial cells. Our results warrant in vivo efficacy evaluation of ODBG-P-RVn. IMPORTANCE While remdesivir remains one of the few drugs approved by the FDA to treat coronavirus disease 2019 (COVID-19), its intravenous route of administration limits its use to hospital settings. Optimizing the stability and absorption of remdesivir may lead to a more accessible and clinically potent therapeutic. Here, we describe an orally available lipid-modified version of remdesivir with activity nearly equivalent to that of remdesivir against emerging viruses that cause significant disease, including Ebola and Nipah viruses. Our work highlights the importance of such modifications to optimize drug delivery to relevant and appropriate human tissues that are most affected by such diseases.

R emdesivir (RDV; Veklury, GS-5734) is an adenosine nucleotide analog phosphoramidate prodrug with broad-spectrum antiviral activity in vitro and in vivo (1) and is currently the only FDA-approved therapeutic for treating coronavirus 2019 disease in hospitalized patients over the age of 12 (2) . While RDV did not significantly reduce COVID-19 mortality, it shortened the time to recovery compared to the time for placebo controls (3) . The short half-life of RDV in human and animal plasma (4) (5) (6) (7) , alongside the in vivo efficacy of the RDV parent nucleoside (RVn; GS-441524) against coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (8) (9) (10) (11) , have driven proposals to utilize the RVn instead of RDV to treat COVID-19 (12) . A recent comparative pharmacokinetic study in nonhuman primates, however, demonstrated higher levels of the active metabolite RVn-triphosphate (RVn-TP) in lower respiratory tract tissues of RDV-dosed animals than in RVn-dosed animals (6) . A significant drawback of RDV is the requirement for intravenous administration, which limits its use to hospital contexts. To develop an orally bioavailable form of remdesivir, we recently synthesized a 1-O-octadecyl-2-O-benzyl-sn-glycerylester (ODBG) lipid-modified monophosphate prodrug of RVn (ODBG-P-RVn) (C 40 H 62 N 5 O 9 P) (Fig. 1A) , which demonstrated more favorable in vitro antiviral activity than RVn and RDV against SARS-CoV-2 in Vero-E6 cells (13) .

In this study, we extended our in vitro comparisons to include 14 viruses from 7 virus families responsible for causing diseases of significant human public health concern. These were Ebola virus (EBOV) and Marburg virus (MARV) from the family Filoviridae, Nipah virus (NiV), Hendra virus (HeV), human parainfluenza virus 3 (hPIV3), measles virus (MV), mumps virus (MuV), and Sosuga virus (SoSuV) from the family Paramyxoviridae, respiratory syncytial virus (RSV) from the family Pneumoviridae, yellow fever virus (YFV) from the family Flaviviridae, Lassa virus (LASV) from the family Arenaviridae, Crimean-Congo hemorrhagic fever virus (CCHFV) from the family Nairoviridae, and SARS-CoV-2 from the family Coronaviridae (14) (15) (16) (17) (18) . We utilized the following three previously described assays to compare the antiviral activities of RVn, RDV, and ODBG-P-RVn against this panel of viruses (15, 17) : (i) directly measuring the fluorescence of a reporter protein expressed by recombinant viruses (REP) (Fig. 1B) , (ii) quantitating focus-forming units (FFU) via fluorescent-reporter imaging (Fig. 1C) , and (iii) indirectly measuring cytopathic effect (CPE) based on cellular ATP levels (CellTiterGlo 2.0; Promega) ( Fig. 1D) , which was also used to evaluate compound cytotoxicity (Fig. 1E ). Assay conditions varied based on virus replication kinetics and on the specific assay used; the multiplicities of infection (MOIs) ranged from 0.01 to 0.25, and endpoint measurements were conducted between 72 and 144 h postinfection (hpi) (see Methods in the supplemental material). We conducted dose-response experiments using 8-point, 3-fold serial dilutions of RVn, RDV, and ODBG-P-RVn against our panel of viruses in Vero-E6 cells and showed that ODBG-P-RVn consistently had greater antiviral activity than RVn and RDV against all viruses susceptible to RVn/RDV inhibition, with 50% effective concentration (EC 50 ) values ranging from 0.026 to 1.13 mM (Fig. 1B to D, left; Table 1 ; Fig. S1 in the supplemental material). RVn and ODBG-P-RVn induced partial cytotoxicity but only at the highest concentration tested (100 mM) and without reaching 50% cytotoxic concentration (CC 50 ). To understand the comparatively greater potency of ODBG-P-RVn in Vero E6 cells, we measured the levels of RVn-TP in cells treated with RVn, RDV, or ODBG-P-RVn. We observed that RVn-TP levels indeed correlated with antiviral activity, with ODBG-P-RVn consistently accumulating to higher levels than both RVn and RDV across 3 time points (Fig. 1F ). We then compared these antivirals in human hepatoma (Huh7) and bronchioalveolar carcinoma (NCI-H358) cell lines, which represent more-relevant cell types that are targeted by subsets of viruses used in our study. In both human cell lines, although ODBG-P-RVn showed EC 50 values comparable to those observed in Vero-E6 cells and was 3-to 5-fold more active than RVn, it consistently showed 6-to 20-fold less activity than RDV (Fig. 1B to D, middle left and middle right; Table 1 ; Fig. S2 and S3). Whereas the CC 50 values for RDV in Huh7 and NCI-H358 cells were 54.2 and 77.2 mM, respectively, ODBG-P-RVn was less cytotoxic in Huh7 cells (CC 50 = 93.4 mM) and did not show measurable cytotoxicity in NCI-H358 cells even at the highest concentration tested (100 mM) (Fig. 1E , middle right; Table 1) .

To further evaluate cell type-specific effects on the antiviral activities of RVn, RDV, and ODBG-P-RVn, we tested them against a smaller subset of reporter viruses in primary-like human telomerase reverse transcriptase (hTERT)-immortalized human microvascular endothelial (TIME) cells (19, 20) . In TIME cells, we observed a trend in antiviral activity similar to those in Huh7 and NCI-H358 cells, with ODBG-P-RVn showing 15-to 22-fold greater activity than RVn but 5-to 8-fold less activity than RDV in reporterbased assays ( Table 2 ; Fig. S4A ). We further compared the activities of RDV and ODBG-P-RVn by infectious yield assay and observed that both compounds equivalently reduced the infectious yield of EBOV expressing ZsGreen protein (EBOV-ZsG), by up to 4 log 10 , and that of of NiV-ZsG, by approximately 2 log 10 , in a dose-dependent manner, with EC 50 values closely mirroring the values determined in reporter assays (Table 2 ; Fig. S4B ). However, RDV was more cytotoxic (CC 50 = 17.2 mM) than ODBG-P-RVn (CC 50 . 50 mM), which is reflected in its biphasic inhibition of NiV-ZsG, with cytotoxic inhibition by RDV shown at 16.6 mM (Fig. S4C) .

Since ODBG lipid nucleoside modification enhances in vivo lung tissue distribution via the chylomicron pathway (21, 22) , we compared the activities of the three compounds against filoviruses, paramyxoviruses, and RSV in another primary-like, hTERTimmortalized small airway epithelial cell line (HSAEC1-KT) (23) . Notably, the doseresponse curves of RDV and ODBG-P-RVn were strikingly similar, with EC 50 values in the submicromolar range within a 3-fold range of each other; the EC 50 values for some viruses were almost identical ( Fig. 1B to D, right; Table 2 ; Fig. S5 ). Furthermore, RDV and ODBG-P-RVn reduced the infectious yields of EBOV-ZsG and NiV-ZsG in HSAEC1-KT cells equivalently, by 5 log 10 and 3 log 10 , respectively, and their EC 50 values reflected the limited differential in antiviral activities between them ( Fig. 1G ; Table 2 ). Although ODBG-P-RVn was more cytotoxic (CC 50 = 20.5) than RDV (CC 50 . 100) in HSAEC1-KT cells (Fig. 1D , right; Table 2 ), it also effectively reduced the virus yields at noncytotoxic concentrations. We also evaluated the antiviral activity of the ODBG lipid alone and observed no detectable antiviral activity against any of the viruses tested in HSAEC1-KT cells (data not shown).

Our results demonstrate that ODBG-P-RVn has greater antiviral activity than RVn and has cell type-dependent activity levels that range from moderately lower than to nearly equal to those of RDV. In vivo, RDV is converted rapidly to RVn (4-7), which has 0.5 to 2 log 10 less activity than RDV against most of the viruses tested. In contrast, ODBG-P-RVn was stable in plasma for .24 h and reached therapeutic plasma levels (above the EC 90 for SARS-CoV-2) after oral administration of 16.9 mg/kg of body weight to Syrian hamsters; it also did not produce virologically significant levels of RVn (13) . Thus, one would predict sustained in vivo antiviral activity with ODBG-P-RVn, without substantial generation of RVn, the less active metabolite, in plasma. Taken together, our results support further optimization of ODBG-P-RVn and future in vivo evaluation of such monophosphate lipid-modified analogs of RVn for their efficacy against viruses significant to human health.

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 6.8 MB.

We thank Tatyana Klimova for helpful comments in reviewing the manuscript. We thank Pei-Yong Shi (University of Texas Medical Branch) for the kind gift of the SARS-CoV-2 reporter strain expressing mNeonGreen.

FIG 1 Comparison of antiviral activities of RVn, RDV, and ODBG-P-RVn in African green monkey (Vero-E6), human hepatoma (Huh7), human bronchioalveolar carcinoma (NCI-H358), and primary-like human telomerase reverse transcriptase-immortalized small airway epithelial (HSAEC1-KT) cell REFERENCES

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The findings and conclusions in this report are those of the authors and do not necessarily represent those of the Centers for Disease Control and Prevention. This work was supported by CDC core funding and by the National Institute of Allergy and Infectious Diseases (grant number RO1-AI131424).