Stenoparib, an Inhibitor of Cellular Poly(ADP-Ribose) Polymerase, Blocks Replication of the SARS-CoV-2 and HCoV-NL63 Human Coronaviruses In Vitro

Abstract

By late 2020, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new poly(ADP-ribose) polymerase (PARP) inhibitor, stenoparib, that recently advanced to phase II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the human seasonal respiratory coronavirus HCoV-NL63. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and postentry processes, as determined by time-of-addition (TOA) experiments. Moreover, a 10 μM dosage of stenoparib-below the approximated 25.5 μM half-maximally effective concentration (EC₅₀)-combined with 0.5 μM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a stand-alone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19.IMPORTANCE New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in phase II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans have already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and postentry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection.

Keywords: COVID-19; NL63; PARP; SARS-CoV-2; stenoparib.

FIG 1

Stenoparib exhibits dose-dependent inhibition of SARS-CoV-2 as measured by RT-qPCR. RT-qPCR was performed on viral RNA collected from cell culture supernatants at 48 h postinfection. Replicates within each run were averaged, and a total of three experiments were performed. Error bars were based on averaged standard deviations within runs. Cytotoxicity against Vero E6 cells was determined at 48 h using the Promega CytoTox 96 assay kit, and values are the averages from two independent experiments (reported in Fig. 2A).

FIG 2

Stenoparib is cytotoxic in Vero E6 cells at concentrations greater than 30 μM but not in Calu-3 cells. Cytotoxicity was determined using the Promega CytoTox 96 lactate dehydrogenase release assay kit by harvesting culture medium every 24 h up to 120 h postexposure. (A) Vero E6 cells; (B) Calu-3 cells. Stenoparib concentrations used were 10, 20, 30, and 60 μM. Measurements were normalized to cells treated with 1.0% Triton X-100 and compared to untreated controls. Biological replicates from two runs were averaged, and median values are plotted. Results are representative of two experiments, and error bars are based on the standard deviation.

FIG 3

Stenoparib exhibits dose-dependent inhibition of SARS-CoV-2 in Calu-3 cells. (A) Plaque-forming efficiency using SARS-CoV-2. Values are normalized as a percentage of inhibition compared to infected but untreated cells. Plaques were counted 120 h after infection, replicates from each run were averaged, and assays were performed three times. Error bars are based on the standard deviation across all runs. (B) RT-qPCR was performed on viral RNA collected from cell culture supernatants at 48 h postinfection, and replicate values within each run were averaged; a total of three runs were performed. Error bars are based on averaged standard deviations within runs. Cytotoxicity against Calu-3 cells was determined at 48 and 120 h, as appropriate, using the Promega CytoTox 96 assay kit, and values represent the average of the two independent experiments (reported in Fig. 2B).

FIG 4

Stenoparib exhibits dose-dependent inhibition of HCoV-NL63 in LLC-MK2 cells. (A) Plaquing efficiency values are normalized as a percentage of inhibition compared to infected but untreated cells. Plaques were counted 120 h after infection, and assays were performed three times. Error bars are based on the standard deviation across all runs. (B) RT-qPCR was performed on viral RNA collected from cell culture media at 120 h postinfection. Biological replicates from each run were averaged, and three independent runs were performed. Error bars were based on averaged standard deviations within runs. Cytotoxicity against LLC-MK2 cells was determined at 120 h using the Promega CytoTox 96 assay kit, and values are averages from the three independent experiments.

FIG 5

Stenoparib inhibits HCoV-NL63 entry and postentry events, while remdesivir inhibits postentry events. (A) Plaque assays were performed three times, and replicate PFU counts from each run were averaged. Error bars are based on standard deviation among runs. Brackets indicate the t test comparison and P value for the Entry group. No significant difference was observed between stenoparib and remdesivir under any treatment (N/S). “N/D” indicates that no plaques were detected. (B) RT-qPCR was performed on viral RNA collected from cell culture medium at 120 h postinfection, and replicate values within each run were averaged; a total of three runs were performed. Error bars were based on averaged standard deviations within runs. Brackets indicate the t test comparison and P value for the Entry group. Significant differences were observed between stenoparib and remdesivir for all treatments.

FIG 6

Stenoparib and remdesivir in combination is a potent inhibitor of NL63. Plaque assays were performed a minimum of two times, and replicate values from each run were averaged. Plaquing efficiency values are normalized as a percentage of inhibition compared to infected but untreated cells. Three data sets are plotted to illustrate the treatment of NL63 with stenoparib and remdesivir each as monotherapy and with both as combination therapy, whereby increasing concentrations of stenoparib are combined with 0.5 μM remdesivir; the EC₅₀ was computationally approximated at 0.46 μM. The stenoparib monotherapy data are the same as reported above (Fig. 4A). The synergistic activity threshold is defined as the sum of the mean values of 10 μM stenoparib and 0.5 μM remdesivir as monotherapies, while the gray highlighted area represents the minimum and maximum possible additive activity values based on the range of error for the same concentrations observed during these experiments.