Mechanism of Action

Remdesivir is an inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), which is essential for viral replication. Remdesivir is an adenosine nucleotide prodrug that distributes into cells where it is metabolized to a nucleoside monophosphate intermediate by carboxyesterase 1 and/or cathepsin A, depending upon the cell type. The nucleoside monophosphate is subsequently phosphorylated by cellular kinases to form the pharmacologically active nucleoside triphosphate metabolite (GS-443902). Remdesivir triphosphate (RDV-TP) acts as an analog of adenosine triphosphate (ATP) and competes with high selectivity (3.65-fold) over the natural ATP substrate for incorporation into nascent RNA chains by the SARS-CoV-2 RdRp, which results in delayed chain termination (position i+3) during replication of the viral RNA. In a biochemical assay assessing RDV-TP incorporation by the MERS-CoV RdRp complex, RDV-TP inhibited RNA synthesis with an IC50 value of 0.032 µM. RDV-TP can also inhibit viral RNA synthesis following its incorporation into the template viral RNA as a result of read-through by the viral polymerase that may occur at higher nucleotide concentrations. When remdesivir nucleotide is present in the viral RNA template, the efficiency of incorporation of the complementary natural nucleotide is compromised, thereby inhibiting viral RNA synthesis. Remdesivir triphosphate is a weak inhibitor of mammalian DNA and RNA polymerases, including human mitochondrial RNA polymerase.

Antiviral Activity

In Cell Culture

Remdesivir exhibited cell culture antiviral activity against a clinical isolate of SARS‑CoV‑2 in primary human airway epithelial (HAE) cells with a 50% effective concentration (EC50) of 9.9 nM after 48 hours of treatment. Remdesivir inhibited the replication of SARS-CoV-2 in the continuous human lung epithelial cell lines Calu-3 and A549-hACE2 with EC50 values of 280 nM after 72 hours of treatment and 115 nM after 48 hours of treatment, respectively.

Remdesivir EC50 values for SARS-CoV-2 in A549-hACE2 cells were not different when combined with chloroquine phosphate or hydroxychloroquine sulfate at concentrations up to 2.5 µM. In a separate study, the antiviral activity of remdesivir was antagonized by chloroquine phosphate in a dose-dependent manner when the two drugs were co-incubated at clinically relevant concentrations in HEp-2 cells infected with respiratory syncytial virus (RSV). Higher remdesivir EC50 values were observed with increasing concentrations of chloroquine phosphate. Increasing concentrations of chloroquine phosphate or hydroxychloroquine sulfate reduced formation of remdesivir triphosphate in A549-hACE2, HEp-2, and normal human bronchial epithelial cells.

Based on cell culture susceptibility testing by virus yield reduction assay and/or N protein ELISA assay, remdesivir retained similar antiviral activity against clinical isolates of SARS-CoV-2 variants compared to an earlier lineage SARS-CoV-2 (lineage A) isolate, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Epsilon (B.1.429), Zeta (P.2), Iota (B.1.526), Kappa (B.1.617.1), Lambda (C.37), and Omicron variants (including B.1.1.529/BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, BA.4.6, BA.5, BF.5, BF.7, BQ.1, BQ.1.1, CH.1.1, EG.1.2, EG.5.1, FL.22, XBB, XBB.1.5, XBB.1.16, XBB.2.3.2, and XBF). For these variants, the EC50 fold change values ranged between 0.2 and 2.3 compared to an earlier lineage SARS-CoV-2 (lineage A) isolate. Using the SARS-CoV-2 replicon system, remdesivir retained similar antiviral activity against Omicron subvariants BA.2.86 and XBB.1.9.2 compared to the wildtype reference replicon (lineage B).

In Clinical Trials

SARS-CoV-2 RNA shedding results from GS-US-540-5776 (ACTT-1) indicate that remdesivir does not significantly reduce the amount of detectable SARS-CoV-2 RNA in oropharyngeal or nasopharyngeal swabs or plasma samples in hospitalized patients compared to placebo, and SARS-CoV-2 RNA shedding results from GS-US-540-9012 indicate that remdesivir does not significantly reduce the amount of detectable SARS-CoV-2 RNA in nasopharyngeal swabs in non-hospitalized patients compared to placebo.

Resistance

In Cell Culture

SARS-CoV-2 isolates with reduced susceptibility to remdesivir have been selected in cell culture. In a selection with GS-441524, the parent nucleoside of remdesivir, virus pools emerged expressing amino acid substitutions at V166A, N198S, S759A, V792I, C799F, and C799R in the viral RdRp (nsp12). When these substitutions were individually introduced into a wild-type recombinant virus by site-directed mutagenesis, 1.7- to 3.5-fold reductions in susceptibility to remdesivir were observed. In a cell culture resistance selection experiment with remdesivir, nsp12 amino acid substitution E802D emerged, resulting in a 1.4- to 2.5-fold reduction in susceptibility to remdesivir. In another selection with remdesivir using a SARS-CoV-2 isolate containing the P323L substitution in the viral polymerase, a single amino acid substitution at V166L emerged. Recombinant SARS-CoV-2 with substitutions at P323L alone or P323L+V166L in combination exhibited 1.3- and 1.5-fold reductions in remdesivir susceptibility, respectively.

Cell culture resistance profiling of remdesivir using the rodent CoV murine hepatitis virus identified two substitutions (F476L and V553L) in the viral RdRp (nsp12) at residues conserved across CoVs. Introduction of the corresponding substitutions (F480L and V557L) into SARS-CoV resulted in 6-fold reduction in susceptibility to remdesivir in cell culture and attenuated SARS-CoV pathogenesis in a mouse model. When individually introduced into a SARS-CoV-2 recombinant virus, the corresponding substitutions at F480L and V557L each conferred 2-fold reduced susceptibility to remdesivir.

In Clinical Studies

In a literature publication, the SARS-CoV-2 nsp12 E802D substitution previously identified in a resistance selection experiment emerged in one individual treated with remdesivir. The E802D substitution resulted in a 1.4- to 2.5-fold increase in the remdesivir EC50 value.

In Study CO-US-540-5776 (ACTT-1), among 61 subjects with baseline and post-baseline sequencing data available, the rate of emerging substitutions in the viral RdRp (nsp12) was similar in subjects treated with VEKLURY compared to placebo. Two subjects treated with VEKLURY had an emergent substitution previously identified in resistance selection experiments (nsp12 V792I in one and C799F in the other). These substitutions are associated with 2.2- and 2.5-fold decreases in remdesivir susceptibility, respectively, based on assessments of clinical isolates. In one subject treated with VEKLURY, nsp12 V792F emerged at low frequency and was associated with a 1.8-fold decrease in remdesivir susceptibility.

In Study GS-US-540-5773, among 19 subjects treated with VEKLURY with baseline and post-baseline sequencing data available, the V792F substitution in viral RdRp (nsp12) emerged at low frequency in one subject.

In Study GS-US-540-9012, among 244 subjects with baseline and post-baseline sequencing data available, the rate of emerging substitutions in the viral RdRp (nsp12) was similar in subjects treated with VEKLURY compared to placebo. In one subject treated with VEKLURY, one substitution in the RdRp (nsp12 A376V) emerged and was associated with a 12.6-fold decrease in remdesivir susceptibility in a subgenomic replicon assay. This subject was not hospitalized and showed alleviation of all baseline symptoms, except loss of taste and smell, prior to or on Day 14.

In Study GS-US-540-5912, among 60 subjects with baseline and post-baseline sequencing data available, substitutions in the viral RdRp (nsp12) emerged in 8 subjects treated with VEKLURY. In 4 subjects treated with VEKLURY, three substitutions in the RdRp (nsp12 E136V, M794I, or C799F) emerged and were associated with 2.9-, 2.9-, and 3.4-fold reduced susceptibility to remdesivir in a subgenomic replicon assay.

In Study GS-US-540-5823, among pediatric subjects with baseline and post-baseline sequencing data available, treatment-emergent substitutions in the viral RdRp (nsp12) were observed in 3 of 27 subjects treated with VEKLURY and were evaluated for susceptibility to remdesivir. In one subject, two substitutions (nsp12 substitutions V166L and V792I) emerged and were associated with 1.85- and 3.6-fold decreases in remdesivir susceptibility relative to reference, respectively. This subject was hospitalized at baseline, recovered from COVID-19, and was released from the hospital on Day 13. None of the substitutions observed in any of the other genes (nsp9–10, nsp13–14) encoding for proteins of the viral replication-transcription complex have been associated with reduced susceptibility to remdesivir.

The relationship between the level of reduced susceptibility to remdesivir observed in subgenomic replicon assays and the inhibition of SARS-CoV-2 replication by remdesivir in humans has not been fully established.