3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), paraformaldehyde, propidium iodide (PI), and 0.05% RNase A were provided by Sigma-Aldrich Inc. (St. Louis, MO, United States). RDV was purchased from Manhey Chemical Limited (Hong Kong, China). For our experiments, the stock solution of RDV was prepared in DMSO, stored at −20°C, and serially diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) when needed. The final DMSO concentration did not exceed 0.1% throughout this study. 5-ethynyl uridine (EU) and 5-ethynyl-2′-deoxyuridine (EdU) were supplied by Tokyo Chemical Industry Co., Ltd. (Shanghai, China). 4′, 6-Diamidino-2-phenylindole (DAPI) and Alexa Fluor™ 594 were purchased from Invitrogen Co. (Carlsbad, CA, United States). Glycine, Tris, CuSO₄, ascorbic acid, EDTA, Triton™ X-100, and TWEEN^®20 were also obtained from Sigma-Aldrich Inc. RIPA buffer (Cell Signaling Technologies Inc. Beverly, MA, United States), Bradford reagent (Bio-Rad Laboratory, Hercules, CA, United States), nitrocellulose membrane (Merck Millipore, United States), and the enhanced chemiluminescence reagents (Invitrogen, Paisley, Scotland, United Kingdom) were also used in this study. For cell culture, DMEM, fetal bovine serum (FBS), penicillin-streptomycin solution, phosphate buffer saline (PBS), and 0.25% trypsin-EDTA solution were obtained from GIBCO (Grand Island, NY, United States).

The stable isotope labeled adenosine-¹³C₁₀,¹⁵N₅-triphosphate (ATP-¹³C₁₀,¹⁵N₅) and adenosine-¹³C₁₀,¹⁵N₅-monophosphate (AMP-¹³C₁₀,¹⁵N₅), other nucleotide standards and ammonium acetate (NH₄OAc) were purchased from Sigma-Aldrich Inc (St. Louis, MO, United States). TMSD and tetrafluoroboric acid (HBF₄) were obtained from Alfa Aesar Co. (Ward Hill, MA, United States). The methanol (LC-MS grade) and acetonitrile used for the HPLC-MS/MS analysis were bought from Anaqua Chemical Supply (Houston, TX, United States). Formic acid was bought from Fisher Scientific Co. (Fair Lawn, NJ, United States) and diethyl ether was obtained from Tedia Co. (Fairfield, OH, United States), while acetic acid (AcOH) and 30% ammonium hydroxide aqueous solution (NH₄OH) were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ, United States). The solid phase extraction (SPE) cartridges (WAX, 3 cm³; 30 mg, 60 μm) was bought from Waters Co. (Milford, MA, United States), and the chromatographic column Sepax GP-C₁₈ (2.1 × 150 mm, 1.8 μm) from Sepax Technologies (Newark, DE, United States) was also used. Ultrapure water was obtained on the basis of a Milli-Q Gradient water system (Millipore, Bedford, MA, United States).

Normal human bronchial epithelial cells BEAS-2B were purchased from the ATCC (Manassas, VA, United States). They were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin-streptomycin in a humidified incubator at 37°C, and 5% CO₂. Cell viability was determined by a modified colorimetric MTT assay (van Meerloo et al., 2011). Briefly, BEAS-2B cells in the exponential phase were seeded in a 96-well plate for 24 h at 37°C, then treated with RDV at different concentrations (0–100 μM) for 24, 48, and 72 h, respectively. After the appropriate incubation time, 10 μL MTT solution (5 mg/ml) was added for another 4 h incubation and 100 μL DMSO was dispensed to dissolve formazan crystals before spectrophotometric measurement at 570 nm using a microplate ultraviolet-visible spectrophotometer. Cell viability was calculated as follows: cell viability (%) = (absorbance of the test group/absorbance of the control group) × 100. The IC₅₀ value was taken as the concentration that caused 50% inhibition of cell viability and was calculated using GraphPad Prism (Graph-Pad Software, Inc., La Jolla, CA, United States).

In order to label and visualize specifically newly synthesized DNA and RNA, the click chemistry method was used. The experiments were conducted based on previous publications with a slight modification (Jao and Salic, 2008; Akbalik et al., 2017). In short, BEAS-2B cells were grown in 6-well plates at 2.0 × 10⁵ cells/well for 24 h and then incubated for 14 h with 1.0 mM EU or 10 μM EdU in the presence or absence of 10 μM RDV. After the labeling, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. The fixed cells were neutralized with 2 mg/ml glycine, rinsed with PBS, and stained for 30 min at room temperature with a click reaction buffer including 100 mM Tris, 1 mM CuSO4, 10 μM Alexa594-azide, and 100 mM ascorbic acid. After staining, cells were washed several times by using PBS with 0.5 mM EDTA, 1% TWEEN^®20, and 0.1% Triton™ X-100, and then stained with 0.5 μg/ml DAPI for 30 min. Finally, the cells were imaged by IncuCyte ZOOM Live-Cell Analysis Platform.

BEAS-2B cells were seeded in 6-well plates at 2.0 × 10⁵ cells/well, cultured for 24 h, and then treated with/without RDV for 12, 24, and 48 h, respectively. Cells were then harvested, resuspended in ice-cold PBS, and fixed with 70% ethanol at −20°C overnight. The fixed cells were washed again using ice-cold PBS and incubated with 500 μL PI containing 0.05% RNase A for 30 min at room temperature in the dark. Finally, a cell cycle distribution profile was accessed by flow cytometry after the staining treatment. The percentages of cells in G0/G1, S, and G2/M phases were analyzed using ModFit LT software (Verity Sofware House, Topsham, ME, United States).

BEAS-2B cells were treated with RDV at the indicated concentration for 12, 24, and 48 h, respectively. Then the cells were washed with cold PBS twice and lyzed with RIPA buffer on ice. The lysates were centrifuged at 12,000 g for 30 min at 4°C in order to acquire the protein samples. The concentration of cellular total protein was measured by using the Bradford reagent at 595 nm according to the manufacturer’s instructions. 30 μg protein samples were loaded on 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1.5 h, followed by the incubation of primary antibodies diluted in Tris-buffered saline with a Tween^® 20 (TBST) buffer (1:1,000 for β-tubulin, ribonucleotide reductase subunit M1 (RRM1), ribonucleotide reductase subunit M2 (RRM2), and p53-controlled ribonucleotide reductase ((p53R2), Cell Signaling Technologies, Danvers, MA, United States) overnight at 4°C. After that, the membranes were washed with TBST and incubated with secondary horseradish peroxidase-conjugated antirabbit IgG antibody (Cell Signaling Technologies, Inc. Danvers, MA, United States) for 1 h at room temperature. The immunoreactive protein bands were finally detected with an Amersham Imager 600 Western blotting system. Densitometry analysis of protein band was performed by Quantity One software (Version 4.6.2, Bio-Rad, United States).

BEAS-2B cells were plated in 10 cm Petri dishes and cultured with medium for 24 h before treatment with RDV. The seeded cell number for 12, 24, and 48 h RDV treatments were 2.5 × 10⁶, 2.0 × 10⁶ , and 1.5 × 10⁶ cells/dish, respectively. After that, the cells were resuspended with ice-cold PBS. The number of cells was counted before centrifugation at 1,200 rpm for 5 min, and the cell pellet was washed with 1.0 ml ice-cold PBS again and centrifuged at 1,200 rpm for 5 min. Subsequently, cell pellets were treated with 150 μL 80% methanol containing 4 µM AMP-¹³C₁₀,¹⁵N₅ and 2 µM ATP-¹³C₁₀,¹⁵N₅ as an internal standards (IS). The following sample preparation and the determination of endogenous RNs and dRNs were performed based on the method previously described (Li et al., 2019). The concentrations of cellular nucleotides were finally calculated according to dividing the absolute amount of each RN and dRN in each sample by the corresponding cell number.

Data analyses were performed using GraphPad Prism software and values were expressed as mean ± standard deviation (SD) from three independent replicate experiments. The statistical significance of the comparison between control and treated groups was determined by Kruskal–Wallis tests. Statistical significance is indicated as *p < 0.05 and **p < 0.01.

At the beginning of this study, we investigated the cytotoxicity of RDV on BEAS-2B cells using MTT assays. The cells were treated with RDV at various concentrations (0–100 μM) for 24, 48, and 72 h. Figure 1A shows that the cell number gradually decreased as the concentration of RDV increased in all the time points of incubation. The viability of cells presented a dose- and time-dependent reduction. The calculated IC₅₀ values in 48 and 72 h were 25.3 ± 2.6 and 9.6 ± 0.7 μM, respectively. 10 μM was chosen for the subsequent experiments.

Based on its significant inhibitory effect on cell viability and proliferation, we investigated the effect of RDV treatment on the distribution of cells in cell cycle at different time points. BEAS-2B cells were treated with or without 10 μM RDV for 12, 24, and 48 h and analyzed by flow cytometry. As shown in Figure 1B, an altered pattern of cell cycle was observed in BEAS-2B cells exposed to 10 μM RDV compared to control. With increase in incubation time, the proportion of cells in S phase significantly increased while the percentage of cells in G2/M phases obviously decreased in comparison to untreated cells. After incubation for 24 h, the percentage of cells in S phase was 35.3 ± 0.75% in control, which gradually increases to 48.69 ± 1.8% in the RDV group (p < 0.01). The number of cells in G2 phase decreased from control from 29.67 ± 1.59% to 20.81 ± 1.92% of RDV (p < 0.05). Similar results at 48 h were obtained. In summary, RDV could arrest the cells in S phase.

In order to detect the effects of RDV on RNA and DNA synthesis in proliferating cells, we performed EU and EdU staining based on click chemistry. EU and EdU are the structural analogues of uridine and deoxyuridine, respectively. Their triphosphate metabolites compete with UTP and TTP to incorporate into newly synthesized RNA and DNA and subsequently react with azide-modified fluorophores. The fluorescence intensity is proportional to the amount of the incorporated EU and EdU in nascent RNA and DNA. As shown in Figures 2A–B, after incubation with RDV for 14 h, the fluorescence intensity of Alexa594-azide decreased significantly compared to control group, indicating the reduction of RNA and DNA synthesis and the inhibition of proliferation of BEAS-2B cells. Interestingly, not all DAPI stained cells were labeled with EdU. The reason for this phenomenon is that the incorporation of EdU only occurs in S phase during DNA replicating, while DAPI is a nonspecific fluorescent dye with the strong binding ability to the existing or nascent DNA (Qu et al., 2011).

To examine metabolic reprogramming events that influence the cellular response to virus, we used targeted LC/MS-MS via selected reaction monitoring (SRM) to examine changes in the steady-state metabolomic profile of BEAS-2B cells after exposure to 10 μM RDV for 12, 24, and 48 h. The specific nucleotide levels are shown in Supplementary Tables S1, S2. The fold changes of the nucleotides were evaluated by comparison of their concentrations in cells treated with RDV and in the parallel controlled RDV-free cells at the same time points. Significant differences in the metabolite profiles of cells with or without RDV were observed. In general, RDV increased the abundance of the majority of RN and dRN species after 24 h incubation, including a greater than 2-fold increase in AMP, GTP, dAMP, dGDP, dGTP, dCTP, and TMP levels, and then decreased to the normal levels at 48 h (Figures 2C–F). A rational interpretation is that RDV significantly inhibited the synthesis of nascent RNA and DNA, and arrested the cell cycle in S phase, inevitably resulting in the accumulation of (deoxy)nucleoside triphosphates and subsequently the increase of their respective di- and monophosphates (Du et al., 2019). However, it was observed that most of the pyrimidine ribonucleotides remained unchanged or even reduced, among which the significant decrease of CTP was in stark contrast to the 3-fold increase of CMP after incubation for 24 h (Figure 2D).

The possible mechanism of remdesivir-induced CTP depletion and the imbalance of other nucleotides in de novo and salvage pathways is shown in Figure 3. CTP is synthesized from UTP by CTP synthase, which is the rate-limiting step of de novo CTP biosynthesis and probably a practical target just as in the treatment of leukemia (Verschuur et al., 1998) and parasitic infestations (Hofer et al., 2001; Fijolek et al., 2007; Tamborini et al., 2012). In this study, the ratio of CTP/UTP was calculated showing a significant decrease after 24 h incubation (Figure 4D), implying the inhibition effect of RDV on CTP synthase. Besides the de novo pathway, the salvage pathway plays an important role in metabolism of cellular nucleotides too. The relative low level of CTP might allosterically activate the recycle of free bases and nucleosides to promote the production of CMP, resulting in the abnormal elevation of CMP (Figure 3).

The alterations in nucleotide pools were also evaluated by comparing the percent of each NTP in the whole nucleotide pools. It showed that RDV exposure (10 μM) stimulates an increase in GTP and a decrease in CTP (Figure 4A). Consequently, a significant increment of GTP/CTP was observed (Figure 4C), indicating the huge disequilibrium in RN pools. Although there were no statistically significant differences, the ATP level reduced and UTP level increased slightly (Figure 4A), resulting in the elevated ratio of ATP/UTP (Figure 4C). From the aspect of drug disposition, RDV was hydrolyzed to RDV-MP in cell, and furtherly metabolized to RDV-TP. Due to the structural similarity of RDV-MP to AMP, the further phosphorylation of RDV-MP was achieved through the competitive inhibition of adenylate kinase, which inevitably resulted in the accumulation of AMP and the decrease in ADP and ATP. Meanwhile, the accumulation of AMP might inhibit the activity of adenylosuccinate synthase and the whole purine biosynthesis pathway in a negative feedback mode, which would simultaneously decrease the production of GMP, and ultimately GDP and GTP (Nelson et al., 2008). This speculation was proven by the relatively high AMP/GMP ratio and the reduced ATP/GTP and ADP/GDP ratios at 24 and 48 h (Figures 4C–D). The relative percent of dNTPs pools is shown in Figure 4B. The changes in dNTPs percent were contrary to that of NTPs, and there was obvious hysteresis, which was probably because of the allosteric regulation of NTPs to ribonucleotide reductase (RR). In summary, RDV exerted the antiviral activity partly via aggravating the imbalance of nucleotide pools, especially by reducing CTP.

The remarkably elevated dNTP pools in cells are probably related to the dNTP synthesis enzymes, especially RR that catalyzes the formation of dRNs from RNs (Mathews, 2006; Liu and Grosshans, 2019). Mammalian RR comprises three subunits including RRM1, RRM2, and p53R2, which are expressed in a cell cycle-dependent manner (Yousefi et al., 2014). In cycling cells, the RRM1 protein is metabolically stable throughout the cell cycle, while the expression and degradation of RRM2 protein limit the S-phase–dependent activity of RR complex, leading to the high cellular dNTPs pools at S phase and low dNTPs pools outside S phase (Engström et al., 1985). In quiescent cells, p53R2 substitutes for protein RRM2 to supply precursor deoxyribonucleotides, which is fundamental to mitochondrial DNA replication and DNA repair. To further investigate whether the growth inhibitory activity of RDV had resulted from the induction of RR, we determined the expression of RRM1, RRM2, and p53R2 by using the western blot assay. Figure 1 C shows that there was no obvious difference of RRM1 level after the BEAS-2B cells incubated with RDV for 12, 24, and 48 h. However, the expression of RRM2 was significantly increased after 24 h exposure to RDV at 10 μM (p < 0.05) caused by the S phase arrest. Simultaneously, the p53R2 level presented distinct down-regulated tendency (p < 0.05). In addition, there were also no changes in the levels of RRM2 and p53R2 after 48 h incubation with RDV. Taken together, it suggested that RDV inhibited the proliferation of BEAS-2B cells through the impact on RR expression.