murine hepatitis virus, delayed brain tumor cell ( EC50 = 30 nM ); SARS-CoV, HAE cell ( EC50 = 74 nM ); MERS-CoV, HAE cell ( EC50 = 74 nM ); SARS-CoV-2 ( IC50 = 3.3 μM ); SARS-CoV-2 alpha ( IC50 = 4.7 μM ); SARS-CoV-2 beta ( IC50 = 32 μM ); SARS-CoV-2 gamma ( IC50 = 3.7 μM ); SARS-CoV-2 delta ( IC50 = 9.2 μM )

GS-5734 demonstrates broad-spectrum antiviral activity against other pathogenic RNA viruses in vitro and antiviral activity against multiple EBOV variants in cell-based assays (EC50=0.06-0.14 μM).[1] With an EC50 of 0.03 μM for the murine hepatitis virus in delayed brain tumor cells and 0.074 μM for SARS-CoV and MERS-CoV in HAE cells, GS-5734 functions as a broad-spectrum therapeutic to protect against CoVs.[2]

The administration of 3 mg/kg GS-5734 results in improved survival regardless of the time at which the treatment is started. Following three days of viral exposure, all animals receiving 10 mg/kg GS-5734 treatments reach the end of their in-life phase. Animals given repeated doses of 10 mg/kg GS-5734, however, consistently exhibit stronger antiviral effects. Clinical disease signs and markers of coagulopathy and end organ pathophysiology related to EVD are associated with improvement when treated with the 10 mg/kg D3 regimen (starting 3 days after virus exposure).[1]

In vitro RSV RNA synthesis assay[1]
RNA synthesis by the RSV polymerase was reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1–14 of the RSV leader promoter31,32,33 (3′-UGCGCUUUUUUACG-5′). RNA synthesis reactions were performed as described previously, except that the reaction mixture contained 250 μM guanosine triphosphate (GTP), 10 μM uridine triphosphate (UTP), 10 μM cytidine triphosphate (CTP), supplemented with 10 μCi [α-32P]CTP, and either included 10 μM adenosine triphosphate (ATP) or no ATP. Under these conditions, the polymerase is able to initiate synthesis from the position 3 site of the promoter, but not the position 1 site. The NTP metabolite of GS-5734 was serially diluted in DMSO and included in each reaction mixture at concentrations of 10, 30, or 100 μM as specified in Fig. 1f. RNA products were analysed by electrophoresis on a 25% polyacrylamide gel, containing 7 M urea, in Tris–taurine–EDTA buffer, and radiolabelled RNA products were detected by autoradiography.

---

RSV A2 polymerase inhibition assay[1]
Transcription reactions contained 25 μg of crude RSV RNP complexes in 30 μL of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl2, 3 mM DTT, 2 mM EGTA, 50 μg ml−1 BSA, 2.5 U RNasin, 20 μM ATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, and 1.5 μCi [α-32P]ATP (3,000 Ci mmol−1)). The radiolabelled nucleotide used in the transcription assay was selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription.
To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds were added using a six-step serial dilution in fivefold increments. After a 90-min incubation at 30 °C, the RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.

---

Inhibition of human RNA polymerase II[1]
For a 25 μl reaction mixture, 7.5 μl 1 × transcription buffer (20 mM HEPES (pH 7.2–7.5), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol), 3 mM MgCl2, 100 ng CMV positive or negative control DNA, and a mixture of ATP, GTP, CTP and UTP was pre-incubated with various concentrations (0–500 μM) of the inhibitor at 30 °C for 5 min. The mixture contained 5–25 μM (equal to Km) of the competing 33P-labelled ATP and 400 μM of GTP, UTP, and CTP. The reaction was started by addition of 3.5 μl of HeLa and extract. After 1 h of incubation at 30 °C, the polymerase reaction was stopped by addition of 10.6 μl proteinase K mixture that contained final concentrations of 2.5 μg μl−1 proteinase K, 5% SDS, and 25 mM EDTA. After incubation at 37 °C for 3–12 h, 10 μl of the reaction mixture was mixed with 10 μl of the loading dye (98% formamide, 0.1% xylene cyanol and 0.1% bromophenol blue), heated at 75 °C for 5 min, and loaded onto a 6% polyacrylamide gel (8 M urea). The gel was dried for 45 min at 70 °C and exposed to a phosphorimager screen. The full length product, 363 nucleotide runoff RNA, was quantified using a Typhoon Trio Imager and Image Quant TL Software.

---

Inhibition of human mitochondrial RNA polymerase[1]
Twenty nanomolar POLRMT was incubated with 20 nM template plasmid (pUC18-LSP) containing POLRMT light-strand promoter region and mitochondrial (mt) transcription factors TFA (100 nM) and mtTFB2 (20 nM) in buffer containing 10 mM HEPES (pH 7.5), 20 mM NaCl, 10 mM DTT, 0.1 mg ml−1 BSA, and 10 mM MgCl234. The reaction mixture was pre-incubated to 32 °C, and the reactions were initiated by addition of 2.5 μM of each of the natural NTPs and 1.5 μCi of [32P]GTP. After incubation for 30 min at 32 °C, reactions were spotted on DE81 paper and quantified.

EBOV assay in HeLa and HFF-1 cells[1]
Antiviral assays were conducted in BSL-4 at USAMRIID. HeLa or HFF-1 cells were seeded at 2,000 cells per well in 384-well plates. Ten serial dilutions of compound in triplicate were added directly to the cell cultures using the HP D300 digital dispenser in twofold dilution increments starting at 10 μM at 2 h before infection. The DMSO concentration in each well was normalized to 1% using an HP D300 digital dispenser. The assay plates were transferred to the BSL-4 suite and infected with EBOV Kikwit at a multiplicity of infection of 0.5 PFU per cell for HeLa cells and with EBOV Makona at a multiplicity of infection of 5 PFU per cell for HFF-1 cells. The assay plates were incubated in a tissue culture incubator for 48 h. Infection was terminated by fixing the samples in 10% formalin solution for an additional 48 h before immune-staining, as described in Supplementary Table 1.[1]

---

EBOV human macrophage infection assay[1]
Antiviral assays were conducted in BSL-4 at USAMRIID. Primary human macrophage cells were seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate were added directly to the cell cultures using an HP D300 digital dispenser in threefold dilution increments 2 h before infection. The concentration of DMSO was normalized to 1% in all wells. The plates were transferred into the BSL-4 suite, and the cells were infected with 1 PFU per cell of EBOV in 100 μl of media and incubated for 1 h. The inoculum was removed, and the media was replaced with fresh media containing diluted compounds. At 48 h post-infection, virus replication was quantified by immuno-staining as described in Supplementary Table 1.[1]

---

RSV A2 antiviral assay[1]
For antiviral tests, compounds were threefold serially diluted in source plates from which 100 nl of diluted compound was transferred to a 384-well cell culture plate using an Echo acoustic transfer apparatus. HEp-2 cells were added at a density of 5 × 105 cells per ml, then infected by adding RSV A2 at a titer of 1 × 104.5 tissue culture infectious doses (TCID50) per ml. Immediately following virus addition, 20 μl of the virus and cells mixture was added to the 384-well cell culture plates using a μFlow liquid dispenser and cultured for 4 days at 37 °C. After incubation, the cells were allowed to equilibrate to 25 °C for 30 min. The RSV-induced cytopathic effect was determined by adding 20 μl of CellTiter-Glo Viability Reagent. After a 10-min incubation at 25 °C, cell viability was determined by measuring luminescence using an Envision plate reader.

Rhesus monkeys (Macaca mulatta)
3 mg/kg, 10 mg/kg
IV

---

In vivo efficacy[1]
Rhesus monkeys (Macaca mulatta) were challenged on day 0 by intramuscular injection with a target dose of 1,000 PFU of EBOV Kikwit (Ebola virus H. sapiens-tc/COD/1995/Kikwit), which was derived from a clinical specimen obtained during an outbreak occurring in the Democratic Republic of the Congo (formerly Zaire) in 1995. Challenge virus was propagated from the clinical specimen using cultured cells (Vero or Vero E6) for a total of four passages. Animals (3–6 years old) were randomly assigned to experimental treatment groups, stratified by sex (with equal number of males and females per group) and balanced by body weight, using SAS statistical software. Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals. The primary endpoint for efficacy studies was survival to day 28 following virus challenge. GS-5734 was formulated at Gilead Sciences in water with 12% sulfobutylether-β-cyclodextrin (SBE- β-CD), pH adjusted to 3.0 using HCl. Formulations were administered to anaesthetized animals by bolus intravenous injection at a rate of approximately 1 min per dose in the right or left saphenous vein. The volume of all vehicle or GS-5734 injections was 2.0 ml kg−1 body weight. Animals were anaesthetized using intramuscular injection of a solution containing ketamine (100 mg ml−1) and acepromazine (10 mg ml−1) at 0.1 ml kg−1 body weight.[1]
Animals were observed at least twice daily to monitor for disease signs, and animals that survived to day 28 were deemed to be protected. Study personnel alleviated unnecessary suffering of infected animals by euthanizing clinically moribund animals. The criteria used as the basis for euthanasia of moribund animals were defined before study initiation and included magnitude of responsiveness, reduced body temperature, and/or specified alterations to serum chemistry parameters35. Serum chemistry was analysed using a Vitros 350 Chemistry System, and coagulation parameters were evaluated using a Sysmex CA-1500 coagulation analyser. Haematology analysis was conducted using a Siemens Advia 120 Hematology System with multispecies software. On days in which GS-5734 or vehicle dosing were scheduled with blood sample collection for clinical pathology or viraemia analysis, blood samples were collected immediately before dose administration.[1]

---

View More

Pharmacokinetic evaluations[1]
Three uninfected male rhesus monkeys were used for the pharmacokinetic study. GS-5734 was formulated in solution at 5 mg ml−1 with 12% sulfobutylether-β-cyclodextrin in water, pH 3.5–4.0, and 2 ml kg−1 was administered by slow bolus (approximately 1 min) for a final dose of 10 mg kg−1. Blood samples for plasma and PBMCs were collected from a femoral vein/artery and were taken from each monkey over a 24-h period. Plasma samples were obtained at predose and at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose. PBMC samples were obtained at 2, 4, 8, and 24 h. Blood samples for plasma were collected into chilled collection tubes containing sodium fluoride/potassium oxalate as the anticoagulant and were immediately placed on wet ice, followed by centrifugation to obtain plasma. Blood samples for PBMC isolation were collected at room temperature into CPT vacutainer tubes containing sodium heparin for isolation. Plasma and isolated PBMC samples were frozen immediately and stored at ≤60 °C until analysed.[1]
For plasma analysis, an aliquot of 25 μl of each plasma sample was treated with 100 μl of 90% methanol and acetonitrile mixture (1:1, v:v) and 10% water with 20 nM 5-(2-aminopropyl)indole as an internal standard. Then, 100 μl of samples were filtered through an Agilent Captiva 96 well 0.2 μm filter plate. Filtered samples were dried down completely for approximately 20 min and reconstituted with 1% acetonitrile and 99% water with 0.01% formic acid. An aliquot of 10 μl was injected for LC-MS/MS using a HTC Pal autosampler. Analytes were separated on a Phenomenex Synergi Hydro-RP 30A column (75 × 2.0 mM, 4.0 μm) using a Waters Acquity ultra performance LC, a flow rate of 0.26 ml min−1, and a gradient from Mobile phase A containing 0.2% formic acid in 99% water and 1% acetonitrile to mobile phase B containing 0.2% formic acid in 95% acetonitrile and 5% water over 4.5 min. For MS/MS analysis, we used a Waters Xevo TQ-S in positive multiple reaction monitoring mode using an electrospray probe. Plasma concentrations of GS-734, alanine metabolite and Nuc were determined using an 8-point calibration curve spanning a concentration range of over three orders of magnitude. Quality control samples were run at the beginning and end of the run to ensure accuracy and precision within 20%. Intracellular metabolites in PBMCs were quantified by LC-MS/MS as described above for in vitro activation studies.[1]

---

Radiolabelled tissue distribution[1]
Six cynomolgus monkeys were administered a single dose of [14C]GS-5734 at 10 mg kg−1 (25 μCi kg−1) by intravenous administration (slow bolus). Tissues were collected from three animals at 4 and 168 h postdose. The tissues were excised, rinsed with saline, blotted dry, weighed, and placed on wet ice. Tissues (testes, epididymis, eyes and brain; following homogenization) and plasma were analysed by liquid scintillation counting. Concentrations were converted to ng equivalents of GS-5734 per gram of sample.

---

Assessment of resistant virus virulence in vivo. [2]
Groups of 10 to 12 10-week old female BALB/c (Charles River, Inc.) mice were anesthetized with ketamine-xylazine and intranasally infected with either 104 or 103 PFU/50 µl wild-type mouse-adapted SARS-CoV expressing nanoluciferase (WT SARS-CoV) or SARS-MA15 NanoLuc engineered to harbor resistance mutations in nsp12 (F480L + V557L SARS-CoV). Animals were weighed daily to monitor virus-associated weight loss. On days 2 and 4 postinfection, 5 to 6 animals per group were sacrificed by isoflurane overdose and the inferior right lobe was harvested and frozen at −80°C until the titer was determined by plaque assay as described previously (38). A 5- to 6-animal cohort was monitored out to 7 days postinfection in order to compare the kinetics of recovery, after which lung samples were harvested and the titer determined as described for previous samples.

[1]. Nature . 2016 Mar 17;531(7594):381-5.

[2]. mBio . 2018 Mar 6;9(2):e00221-18.

Remdesivir is a carboxylic ester resulting from the formal condensation of the carboxy group of N-[(S)-{[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl]methoxy}(phenoxy)phosphoryl]-L-alanine with the hydroxy group of 2-ethylbutan-1-ol. A broad-spectrum antiviral prodrug with potent in vitro antiviral activity against a diverse panel of RNA viruses such as Ebola virus, MERS-CoV and SARS-CoV. It is currently in Phase III clinical trials for the treatment of Covid-19 in adults. It has a role as an antiviral drug, a prodrug and an anticoronaviral agent. It is a carboxylic ester, a pyrrolotriazine, a nitrile, a phosphoramidate ester, a C-nucleoside and an aromatic amine. It is functionally related to a GS-441524.

---

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which is a respiratory disease that is capable of progressing to viral pneumonia and acute respiratory distress syndrome (ARDS); COVID-19 can be fatal. Like other RNA viruses, SARS-CoV-2 depends on an RNA-dependent RNA polymerase (RdRp) enzyme complex for genomic replication, which can be inhibited by a class of drugs known as nucleoside analogues. Remdesivir (GS-5734) is an adenosine triphosphate analogue first described in the literature in 2016 as a potential treatment for Ebola. Broad antiviral activity of remdesivir is suggested by its mechanism of action, and to date, it has demonstrated in vitro activity against the Arenaviridae, Flaviviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, and Coronaviridae viral families. Remdesivir activity against the Coronaviridae family was first demonstrated in 2017, leading to considerable interest in remdesivir as a possible treatment for COVID-19. Remdesivir was confirmed as a non-obligate chain terminator of RdRp from SARS-CoV-2 and the related SARS-CoV and MERS-CoV, and has been investigated in multiple COVID-19 clinical trials. After initially being granted an FDA Emergency Use Authorization (EUA) on May 1st, 2020, remdesivir was fully approved by the FDA for the treatment of COVID-19 on October 22, 2020. Remdesivir is currently marketed under the trademark name VEKLURY by Gilead Sciences Inc. Remdesivir was also approved by the European Commission on July 3, 2020. Remdesivir in combination with [baricitinib] for the treatment of COVID-19, was granted an FDA Emergency Use Authorization on November 19, 2020.

---

View More

Remdesivir is a SARS-CoV-2 Nucleotide Analog RNA Polymerase Inhibitor.

---

Remdesivir is an antiviral nucleotide analogue used for therapy of severe novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) infection. Remdesivir therapy is given intravenously for 3 to 10 days and is frequently accompanied by transient, reversible mild-to-moderate elevations in serum aminotransferase levels but has been only rarely linked to instances of clinically apparent liver injury, its hepatic effects being overshadowed by the systemic effects of COVID-19.

---

Remdesivir is a prodrug of an adenosine triphosphate (ATP) analog, with potential antiviral activity against a variety of RNA viruses. Upon administration, remdesivir, being a prodrug, is metabolized into its active form GS-441524. As an ATP analog, GS-441524 competes with ATP for incorporation into RNA and inhibits the action of viral RNA-dependent RNA polymerase. This results in the termination of RNA transcription and decreases viral RNA production.

---

Remdesivir is indicated for the treatment of adult and pediatric patients 28 days of age and older and weighing at least 3 kg for coronavirus disease 2019 (COVID-19) infection requiring hospitalization. It is also indicated for the treatment of non-hospitalized patients with mild-to-moderate COVID-19, who are at high risk for progression to severe COVID-19, including hospitalization or death. Remdesivir was originally granted FDA Emergency Use Authorization (EUA) on May 1, 2020, for use in adults and children with suspected or confirmed COVID-19 in a hospital setting with an SpO2 ≤94%. Following the FDA approval, this EUA was revised to cover hospitalized pediatric patients between 3.5 and 40 kg, as well as those under 12 years of age that weigh at least 3.5 kg, with suspected or laboratory-confirmed COVID-19. Under both the on-label and EUA indications, patients not needing invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO) should be treated for 5 days (including the loading dose on day 1) and may be extended up to 10 days if they do not show improvement. Patients requiring invasive mechanical ventilation or ECMO should be treated for 10 days. In Europe, remdesivir is approved for the treatment of adults and adolescents weighing at least 40 kg with pneumonia requiring supplemental oxygen (low- or high-flow oxygen or other non-invasive ventilation at start of treatment). It is also indicated for the treatment of COVID-19 in adults who do not require supplemental oxygen and who are at increased risk of progressing to severe COVID-19.

---

Remdesivir is an antiviral nucleotide analogue used for therapy of severe novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) infection. Remdesivir therapy is given intravenously for 3 to 10 days and is frequently accompanied by transient, reversible mild-to-moderate elevations in serum aminotransferase levels but has been only rarely linked to instances of clinically apparent liver injury, its hepatic effects being overshadowed by the systemic effects of COVID-19.

---

Remdesivir is a nucleoside analog used to inhibit the action of RNA polymerase. The duration of action is moderate, as it is given once daily. Due to much higher selectivity of mammalian DNA and RNA polymerases, including human mitochondrial RNA polymerase, for ATP over remdesivir triphosphate, remdesivir is not a significant inhibitor of these enzymes, which contributes to its overall tolerability and safety profile. Despite this, remdesivir carries risks for hypersensitivity reactions, including anaphylaxis and other infusion-related reactions, elevated transaminase levels, and potential decreased efficacy when combined with [hydroxychloroquine] or [chloroquine].

---

Absorption: Remdesivir is absorbed quickly; maximal plasma concentrations following a single 30-minute intravenous infusion are reached within 0.67-0.68 hours (Tmax). Repeated dosing yields a Cmax (coefficient of variation as a percent) of 2229 (19.2) ng/mL and an AUCtau of 1585 (16.6) ng\*h/mL. Remdesivir metabolite [GS-441524] has measured values: Tmax 1.51-2.00 hours, Cmax 145 (19.3) ng/mL, AUCtau 2229 (18.4) ng\*h/mL, and Ctrough 69.2 (18.2) ng/mL. Another metabolite, GS-704277, has measured values: Tmax 0.75 hours, Cmax 246 (33.9) ng/mL, AUCtau 462 (31.4) ng\*h/mL, and an undetermined Ctrough. A 10mg/kg intravenous dose given to cynomolgus monkeys distributes to the testes, epididymis, eyes, and brain within 4h.

---

Route of Elimination: Remdesivir is 74% eliminated in the urine and 18% eliminated in the feces. 49% of the recovered dose is in the form of the metabolite [GS-441524], and 10% is recovered as the unmetabolized parent compound. A small amount (0.5%) of the [GS-441524] metabolite is found in feces.

---

Volume of Distribution: Data regarding the volume of distribution of remdesivir is not readily available.

---

Clearance: Data regarding the clearance of remdesivir is not readily available.

---

Metabolism / Metabolites: Remdesivir is a phosphoramidate prodrug that must be metabolized within host cells to its triphosphate metabolite to be therapeutically active. Upon cell entry, remdesivir is presumed to undergo first esterase-mediated hydrolysis to a carboxylate form followed by cyclization to eject the phenoxide moiety and finally hydrolysis of the cyclic anhydride to yield the detectable alanine metabolite (GS-704277). The alanine metabolite is subsequently hydrolyzed to yield the monophosphate form of remdesivir, which is either hydrolyzed again to yield the bare nucleoside metabolite [GS-441524] or phosphorylated by cellular kinases to yield the active triphosphate form.

---

Biological Half-Life: Remdesivir has an elimination half-life of 1 hour following a single 30-minute intravenous infusion. Under the same conditions, the elimination half-lives of the remdesivir metabolites [GS-441524] and GS-704277 are 27 hours and 1.3 hours, respectively. A 10mg/kg intravenous dose in non-human primates has a plasma half-life of 0.39h. The nucleoside triphosphate metabolite has a half-life of 14h in non-human primates. The nucleoside triphosphate metabolite has a half-life of approximately 20 hours in humans.

---

Mechanism of Action: COVID-19 is caused by the positive-sense RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of the viral genome is a key step in the infectious cycle of RNA viruses, including those of the _Filoviridae_, _Paramyxoviridae_, _Pneumoviridae_, and _Coronaviridae_ families, and is carried out by viral RNA-dependent RNA polymerase (RdRp) enzymes or enzyme complexes. For both SARS-CoV and SARS-CoV-2, the RdRp comprises nsp7, nsp8, and nsp12 subunits under physiological conditions, although functional RdRp complexes can be reassembled _in vitro_ that incorporate only the nsp8 and nsp12 subunits, similar to the Middle East respiratory syndrome coronavirus (MERS-CoV). Remdesivir is a phosphoramidite prodrug of a 1'-cyano-substituted adenosine nucleotide analogue that competes with ATP for incorporation into newly synthesized viral RNA by the corresponding RdRp complex. Remdesivir enters cells before being cleaved to its monophosphate form through the action of either carboxylesterase 1 or cathepsin A; it is subsequently phosphorylated by undescribed kinases to yield its active triphosphate form remdesivir triphosphate (RDV-TP or GS-443902). RDV-TP is efficiently incorporated by the SARS-CoV-2 RdRp complex, with a 3.65-fold selectivity for RDV-TP over endogenous ATP. Unlike some nucleoside analogues, remdesivir provides a free 3'-hydroxyl group that allows for continued chain elongation. However, modelling and _in vitro_ experiments suggest that at _i_ + 4 (corresponding to the position for the incorporation of the fourth nucleotide following RDV-TP incorporation), the 1'-cyano group of remdesivir sterically clashes with Ser-861 of the RdRp, preventing further enzyme translocation and terminating replication at position _i_ + 3. This mechanism was essentially identical between SARS-CoV, SARS-CoV-2, and MERS-CoV, and genomic comparisons reveal that Ser-861 is conserved across alpha-, beta-, and deltacoronaviruses, suggesting remdesivir may possess broad antiviral activity. Considerations for the use of nucleotide analogues like remdesivir include the possible accumulation of resistance mutations. Excision of analogues through the 3'-5' exonuclease (ExoN) activity of replication complexes, mediated in SARS-CoV by the nsp14 subunit, is of possible concern. Murine hepatitis viruses (MHVs) engineered to lack ExoN activity are approximately 4-fold more susceptible to remdesivir, supporting the proposed mechanism of action. However, the relatively mild benefit of ExoN activity to remdesivir resistance is proposed to involve its delayed chain termination mechanism, whereby additional endogenous nucleotides are incorporated following RDV-TP. In addition, serial passage of MHV in increasing concentrations of the remdesivir parent molecule [GS-441524] led to the development of resistance mutations F476L and V553L, which maintain activity when transferred to SARS-CoV. However, these mutant viruses are less fit than wild-type in both competition assays and _in vivo_ in the absence of selective pressure. To date, no clinical data on SARS-CoV-2 resistance to remdesivir have been described.

---

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.[1]

---

Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.[2]

C27H35N6O8P

602.5760

602.23

C, 53.82; H, 5.85; N, 13.95; O, 21.24; P, 5.14

1809249-37-3

1355149-45-9 [GS443902 (GS-441524 triphosphate)]; 1809249-37-3 (Remdesivir); 1191237-69-0 (GS-441524, an active metabolite of Remdesivir); 1191237-80-5 (Remdesivir O-desphosphate acetonide impurity); 1911578-74-9 (Remdesivir nucleoside monophosphate); 2250110-53-1;1911579-04-8 (GS-704277)

121304016

Off-white to yellow solid powder

1.5±0.1 g/cm3

1.652

2.10

204Ų

CCC(CC)COC(=O)[C@H](C)N[P@](=O)(OC[C@@H]1[C@H]([C@H]([C@](O1)(C#N)C2=CC=C3N2N=CN=C3N)O)O)OC4=CC=CC=C4

RWWYLEGWBNMMLJ-YSOARWBDSA-N

InChI=1S/C27H35N6O8P/c1-4-18(5-2)13-38-26(36)17(3)32-42(37,41-19-9-7-6-8-10-19)39-14-21-23(34)24(35)27(15-28,40-21)22-12-11-20-25(29)30-16-31-33(20)22/h6-12,16-18,21,23-24,34-35H,4-5,13-14H2,1-3H3,(H,32,37)(H2,29,30,31)/t17-,21+,23+,24+,27-,42-/m0/s1

2-ethylbutyl (2S)-2-[[[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxyoxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate

GS-5734; GS 5734; Prodrug of GS441524; Prodrug of GS441524; Remdesivir; GS5734; Prodrug of GS-441524; 3QKI37EEHE; GS 5734; GS 5734 [WHO-DD]; GS-5734; GS5734; Remdesivir; REMDESIVIR [INN];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~166 mM)
Ethanol: ~100 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.17 mg/mL (3.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.17 mg/mL (3.60 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (8.30mM)

---

 (Please use freshly prepared in vivo formulations for optimal results.)