Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells

Abstract

Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Keywords: CES1; COVID-19; CatA; HINT1; SARS-CoV-2; nucleotide analogs; prodrug activation; remdesivir.

FIG 1

Structures of RDV and major metabolites.

FIG 2

Expression of enzymes involved in the metabolism of phosphoramidate prodrug in human lung tissue and in single cells of human lung and airways. (A) Bulk mRNA expression of genes in normal human lung tissue in the GTEx data set. (B) Bulk mRNA expression of genes in normal human lung tissue in the HPA data set. (C) Expression profiles of genes in human healthy lung cells based on scRNA-seq data. (D) Expression profiles of genes in human healthy airway cells based on scRNA-seq data.

FIG 3

Expression of enzymes involved in metabolism of nucleoside, NMP, NDP, and NTP in human lung tissue and in single cells of human lung and airways. (A) Bulk mRNA expression of genes in normal human lung tissue in the GTEx data set. (B) Bulk mRNA expression of genes in normal human lung tissue in the HPA data set. (C) Expression profiles of genes in human healthy lung cells based on scRNA-seq data. (D) Expression profiles of genes in human healthy airway cells based on scRNA-seq data.

FIG 4

mRNA expression of CES1, CatA, and HINT1 in human NHBE cells. The levels are shown as average ± standard deviation from three independent measurements. One-way ANOVA was used for statistical analysis.

FIG 5

Protein expression of CES1, CatA, and HINT1 in lung NHBE cells and human lung and liver S9 fractions. (A) Western blot analysis of human cell and tissue samples. The amounts of lung S9 samples loaded for detection of CES1, CatA, and HINT1 proteins were 5 μg, 10 μg, and 10 μg, respectively. The amounts of human liver S9 samples loaded for detection of CES1, CatA, and HINT1 proteins were 0.2 μg, 10 μg, and 10 μg, respectively. The recombinant proteins were loaded at a range of 0.3 to 100 ng per lane. (B) Graphical representation of the protein levels. The levels are shown as average ± standard deviation from three independent measurements. One-way ANOVA was used for statistical analysis.

FIG 6

Effect of CES1 inhibitor BNPP and CatA inhibitor telaprevir on formation of active metabolite GS-443902 in RDV-treated NHBE (A) and antiviral activity against SARS-CoV-2 in A549-hACE2 cells (B). (A) Cells from three different donors were incubated with 1 μM RDV and DMSO (gray dotted bar), BNPP (filled bar), and telaprevir (open bar) and harvested at 24 h post-compound addition. Triphosphate (TP) levels are shown as average ± standard deviation of the percentage of the DMSO control across the three donors. One-way ANOVA was used for statistical analysis. (B) Effect of increasing telaprevir concentrations on potency of RDV against SARS-CoV-2 replication in A549-hACE2 cells. The RDV titration curve in the presence of either DMSO alone (black circles) or 0.5 (green boxes), 2.5 (blue diamonds), or 10 μM telaprevir (red triangles) from one of three experiments is displayed. The levels are shown as average ± standard deviation from three replicates. Two-way ANOVA was used for statistical analysis.