Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an unprecedented pandemic disease (COVID-19) (The Lancet, 2020). Since its first case was reported in China, in December 2019, the virus has resulted in more than 100 million human infections in more than 200 countries, territories, and areas with an almost 2.16% case-fatality rate (based on the data of World Health Organization COVID-19 Dashboard, as of January 21, 2021) (WHO, 2020a). Needless to say, COVID-19 has been socially and economically paralyzing our global community (Van Lancker and Parolin, 2020; Jones
Drug repositioning uses an approved drug beyond its original targeted purpose. At the time of the pandemic caused by an unprecedented novel virus, it may be a time-saving strategy compared to developing a completely new drug. In this regard, several approved drugs have been investigated for their potential effects on COVID-19 (WHO, 2020b). Given the interim results of the WHO Solidarity Trial (Consortium
Vero, Calu-3, and Madin-Darby canine kidney (MDCK) cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Serana, Pessin, Germany) and penicillin-streptomycin (Gibco). SARS-CoV-2 (BetaCoV/korea/KCDC03/2020, NCCP no. 43326) and A/Korea/01/2009 (2009 pandemic influenza strain, H1N1 subtype), which were provided by the Korea Disease Control and Prevention Agency (KDCA; Osong, Korea), were prepared by propagation in Vero cells and embryonated chicken eggs, respectively, after plaque purification. Other seasonal influenza viruses, such as A/Perth/16/2009 (H3N2 subtype), B/Brisbane/60/2008 (Victoria lineage), and B/Wisconsin/01/2010 (Yamagata lineage), were provided by Il Yang Pharmaceutical Co. (Seoul, Korea) and prepared by propagation in embryonated chicken eggs after plaque purification. All the viruses were confirmed by commercial sequencing before use.
Pralatrexate was purchased from Selleckchem Chemicals Llc (Houston, TX, USA). Hydroxychloroquine sulfate was purchased from Sigma-Aldrich (St. Louis, MO, USA). These chemicals were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) or deionized water to achieve a final concentration of 10 mM.
A plaque assay was performed to determine infectious viral titers of SARS-CoV-2. Briefly, a confluent monolayer of Vero cells was prepared in advance and inoculated with diluted viruses. After 1 h of infection, the inoculum was discarded, and the cells were overlaid with DMEM-F12 (Sigma-Aldrich) containing 2% agarose. After 72 h at 37°C and 5% CO2, the cells were stained with crystal violet (Georgia Chemicals Inc., Norcross, GA, USA).
A confluent monolayer of MDCK cells was prepared in advance, and the cells were inoculated with 102 plaque-forming units (pfu) of influenza viruses. After 1 h of infection, the inoculum was discarded, and the cells were overlaid with DMEM-F12 (Sigma-Aldrich) containing 2% agarose and serially twofold diluted pralatrexate. After 72 h at 37°C and 5% CO2, the cells were stained with crystal violet (Georgia Chemicals Inc.). Control wells were treated with phosphate-buffered saline (PBS).
The cytotoxicity of the chemicals was determined using the cell proliferation reagent WST-1 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 2×104 cells/well (Vero) or 7×105 cells/well (Calu-3) in a 96-well clear flat-bottom TC-treated culture microplate (Thermo Fisher Scientific) and incubated with DMEM (Gibco) at 37°C and 5% CO2. The next day, 2% FBS cell culture media were discarded and washed once with PBS. Then, serial twofold dilutions of the chemicals were added to each well. DMSO (Sigma-Aldrich) was used as a control. At 24 and 48 h postinfection (hpi), 10 μL WST-1 was added to each well, followed by 2 h of incubation at 37°C and 5% CO2. Subsequently, cell viability was determined using a microplate reader. The 50% cytotoxic concentration (CC50) was calculated using GraphPad Prism 9 (GraphPad, San Diego, CA, USA).
Twelve-well plates (SPL Life Sciences, Pocheon, Korea) were seeded with 0.9×106 cells/well (Vero) or 1×106 cells/well (Calu-3) in advance. After 24 h, the cells were washed once with PBS and infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 or 0.1 for Vero or Calu-3 cells, respectively. After 1 h of incubation at 37°C and 5% CO2, the inoculum was discarded, and the cells were washed once with PBS. Subsequently, the cells were treated with serial twofold dilutions of the chemicals. At 24 and 48 hpi, viral RNAs extracted from the cell supernatants were quantified by qRT-PCR. Briefly, 130 μL cell supernatants were harvested for RNA extraction with a Maxwell RSV Viral Total Nucleic Acid Purification kit (Promega, Madison, WI, USA). A SARS-CoV-2 RdRp region was amplified using forward (5’-GTGARATGGTCATGTGTGGCGG) and reverse (5’-CARATGTTAAASACACTATTAGCATA) primers and a probe (5’-FAMCAGGTGGAACCTCATCAGGAGATGC – BHQI) (Corman
The replication kinetics of SARS-CoV-2 were analyzed in Vero and Calu-3 cells. Briefly, monolayered Vero or Calu-3 cells in 12-well plates were inoculated with 0.01 or 0.1 MOI, respectively, for 1 h. Then, the inoculum was discarded, and the cells were washed with DMEM (Gibco) three times and maintained with DMEM containing 2% FBS (Serana). Various concentrations of chemicals were added to the cell supernatants, and the cell supernatants were harvested at 24, 48, and 72 hpi for titration by the plaque assay in Vero cells.
Confluent Calu-3 cell monolayers in 12-well tissue culture plates were inoculated with 0.1 MOI (105 pfu/100 μL) of SARS-CoV-2 for 1 h. After infection, the inoculum was discarded, and the cells were washed with PBS once. DMEM (Gibco) containing 2% FBS (Serana) was added (nontreated group). Pralatrexate (12.5 µM) was added to the medium at 1, 3, 5, 7, 9, 11, 13, 25, and 37 hpi. The cell supernatants were harvested at 48 h later from each drug addition time, and virus titers were determined by the plaque assay in Vero cells.
The statistical significance of viral titer differences in the replication kinetics between the control (or virus-only) and drug-treated groups, two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was applied using GraphPad Prism 9 (GraphPad). The results of the time-to-addition assay between the virus-only and drug-treated groups were analyzed by Student’s
In our search of antiviral drug candidates (Kim
We then evaluated the anti-SARS-CoV-2 efficacy of pralatrexate in human lung epithelial Calu-3 cells by comparing it to that of hydroxychloroquine (Fig. 3). Both drugs exhibited no cytotoxicity in Calu-3 cells. However, only pralatrexate successfully inhibited the RNA synthesis of SARS-CoV-2, whereas hydroxychloroquine showed no efficacy (Fig. 3A and B). Given these results, the IC50 values of pralatrexate and hydroxychloroquine were determined to be 0.054 µM and 107.8 µM, respectively, and the selective index (SI) value of pralatrexate was more than 1,800-fold higher than that of hydroxychloroquine (Table 2). The effects of pralatrexate on the replication of SARS-CoV-2 were also investigated in Calu-3 cells. Compared to hydroxychloroquine (Fig. 3D), 1.56-50 µM pralatrexate showed significantly higher inhibitory effects against the replication of SARS-CoV-2 at 48 and 72 hpi, with statistical significance (***
Given the time and costs of new drug discovery, drug repurposing may have great advantages, and it might be the reason that FDA-approved remdesivir and hydroxychloroquine have been investigated to treat COVID-19 patients (FDA, 2020; WHO, 2020b). For a similar reason, we also investigated the
Notably, pralatrexate showed different inhibitory effects on the replication kinetics of SARS-CoV-2 in Vero and Calu-3 cells (Fig. 2, 3). In Vero cells, pralatrexate more reduced the replication of SARS-CoV-2 at 24 hpi than at 48 hpi. In the qRT-PCR assay in Vero cells, pralatrexate also exhibited better effects at 24 hpi than at 48 hpi. Given the half-life of pralatrexate (12-18 h) (Drugbank, 2021), these results might be acceptable. However, the antiviral effects of pralatrexate were maximized at 48 hpi in Calu-3 cells, not at as early as 24 hpi. This discrepancy suggests that the onset of pharmacological action of pralatrexate might be cell- or host-dependent, which should be considered in determining a medication administration protocol. Furthermore, as indicated in the cytotoxicity assay, the potential adverse effects of pralatrexate on normal cells and its exact MOA against SARS-CoV-2 must also be explored further in suitable animal models.
With therapeutic benefits and limits, the FDA-approved anticancer drug pralatrexate can be repurposed upon approval of its pharmacological effects to equip ourselves against COVID-19 in the influenza virus-circulating winter season, and it may also reduce health risks in cancer patients, providing dual beneficial effects.
This study is supported by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, Republic of Korea (Grant No. NRF-2018 M3A9H4056537).