Cell-Based High-Throughput Screening Assay Identifies 2′,2′-Difluoro-2′-deoxycytidine Gemcitabine as a Potential Antipoliovirus Agent

Abstract

As we approach the global eradication of circulating wild-type polioviruses (PV), vaccination with oral poliovirus vaccine (OPV) has led to the emergence of circulating vaccine-derived poliovirus (cVDPV) and vaccine-associated paralytic poliomyelitis (VAPP). Complete cessation of all poliovirus infections may require stopping use of OPV and formulating improved vaccines and new antiviral drugs. Currently, no licensed drugs are available to treat chronically infected poliovirus excretors. Here, we created a modified PV expressing Gaussia Luciferase (Sb-Gluc) and developed a cell-based high-throughput screening (HTS) antiviral assay. Using the validated HTS assay, we screened the FDA-approved drug library of compounds and identified candidate agents capable of inhibiting PV replication. We then characterized antipoliovirus activity for the best hit, gemcitabine, a nucleoside analogue used in tumor chemotherapy. We found that gemcitabine inhibited PV Mahoney replication with an IC50 of 0.3 μM. It completely protected HeLa cells from PV-induced cytopathic effects at 25 μM, without detectable toxicity for cell viability. Furthermore, a gemcitabine metabolite directly inhibited the ability of PV RNA polymerase to synthesize or elongate PV RNA. Because PV RNA polymerase is somehow conserved among species in the Picornaviridae family, gemcitabine may be further developed as an attractive broad-spectrum antiviral for PV and others.

Selected nucleos(t)ide-based prescribed drugs and their multi-target activity

“…Recently, experimental data on the antiviral activity of gemcitabine have been widely reported (Shin et al, 2018;Ianevski et al, 2019). The broad range of viruses against which gemcitabine has shown activity includes HIV-1 (Rawson et al, 2013;Clouser et al, 2010Clouser et al, , 2012, murine leukaemia virus (MuLV) (Clouser et al, 2012), HIV-2 (Beach et al, 2014), ZIKV (Kuivanen et al, 2017), enteroviruses such as poliovirus (Kang et al, 2015;Zhang et al, 2017), rhinoviruses (Song et al, 2017), HCV (Beran et al, 2012), coronaviruses (Dyall et al, 2014), Sindbis virus (SINV), HSV-1 and FLUAV (Denisova et al, 2012). The synergistic effect of gemcitabine combined with low doses of other nucleoside analogues was also reported.…”

“…Myelodysplastic syndrome (2004) AdV (Alexeeva et al (2001); (Alexeeva et al, 2015) HMPV (Bösl et al, 2019) HIV-1 (Dapp et al, 2009;Rawson et al, 2016a) HIV-1 -synergy in combination with resveratrol (Rawson et al, 2016b) HIV-2 (Beach et al, 2014) RVFV ( (Clouser et al, 2012) HIV-1 -synergy in combination with decitabine (Clouser et al, 2010) MuLV (Clouser et al, 2012) HIV-2 (Beach et al, 2014) ZIKV (Kuivanen et al, 2017) Enteroviruses including poliovirus (Kang et al, 2015;Zhang et al, 2017) Enteroviruses -synergy with ribavirin (Kang et al, 2015) Rhinoviruses (Song et al, 2017) HCV (Beran et al, 2012) Coronaviruses (Dyall et al, 2014) SINV, HSV-1, FLUAV (Denisova et al, 2012) Gram-positive bacteria (Jordheim et al, 2012;Sandrini et al, 2007;…”

Repurposing approved drugs on the pathway to novel therapies

“…Side effects may also limit the clinical usefulness of a variety of off‐the (drug‐)‐shelf compounds suggested as treatments for serious virus infections. The FDA‐approved cancer drug, gemcitabine, inhibits picornaviruses, and other viruses . The bioflavonoid rutin inhibits norovirus .…”

In vitro methods for testing antiviral drugs

“…Non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (Tino et al, 1993;Tisdale et al, 1993). The activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (Zhang et al, 2017). More straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (Coates et al, 1992;Hirashima et al, 2006;Joyce, 2010); these include HTS methods using homopolymeric polycytidylic acid and [ 3 H]-GTP (Amraiz et al, 2016).…”

“…Numerous kits for quantification of products of both DNA and RNA polymerases, including reverse transcriptase, are commercially available (Bustin, 2000). Quantitative real-time PCR is a preferred method to monitor the activity of DNA polymerases (cellular as well as viral) in the presence of inhibitors (Beadle et al, 2016;Zweitzig et al, 2012), and quantitative real-time reverse-transcription PCR has become standard for screening inhibitors of viral RNA polymerases Okon et al, 2017;Pelliccia et al, 2017;Zhang et al, 2017). White et al described a HTS method using a microfluidic apparatus combined with digital PCR for single-cell analysis (White et al, 2013).…”