Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants

We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wild type (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 μM, 8.5 μM, 24.1 μM, 8.2 μM and 13.6 μM, respectively. A separate experiment focusing on a combination of 10 μM stenoparib and 0.5 μM remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.


Introduction
As of June 2022, the coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2, has caused over 534 million infections and over 6.3 million deaths worldwide [1]. Although protective vaccines are available, the pandemic continues, and both old and new SARS-CoV-2 variants exhibit varying degrees of disease severity and resistance to vaccination. Additional effective therapeutics are urgently needed. Here we describe the antiviral activity of a small molecule, stenoparib, an inhibitor of mammalian poly (ADP-ribose) polymerases (PARPs). We show that stenoparib effectively inhibits replication of SARS-CoV-2 wild-type (wt) and variant strains in vitro.
SARS-CoV-2 has undergone adaptation and mutation since the beginning of the COVID-19 pandemic, resulting in new variants of the virus. Only two antiviral drugs, remdesivir and molnupiravir, or treatment with monoclonal antibodies, have been approved by the United States Food and Drug Administration as COVID-19 therapies under the Emergency Use Authorization [2,3]. Remdesivir and molnupiravir are nucleoside analogs that affect the activity of the RNA-dependent RNA polymerase (RdRp). After incorporation into viral RNA, remdesivir stalls the RdRp during elongation [4], while molnupiravir results in the incorporation of mutations that can be functionally deleterious [5].
We recently published a study on the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host [6] as opposed to targeting viral replication. ADP-ribosylation (ADPR) pathways may have either anti-or pro-viral properties, and their importance in host-virus interactions is becoming increasingly recognized [7]. Unlike remdesivir, which inhibits viral replication downstream of entry into the cell, stenoparib inhibits virus entry and postentry processes [6]. Stenoparib inhibited the SARS-CoV-2 USA-WA1/2020 virus and the HCoV-NL63 human seasonal respiratory coronavirus in vitro, exhibiting dose-dependent suppression of virus multiplication and cell-cell spread in cell culture [6]. Stenoparib exhibits a unique dual activity against the PARP1, 2 and PARP5a, 5b (tankyrase 1, 2) enzymes, which are important intermediates in the Wnt/β-catenin immune checkpoint [8,9]. As a host-targeting therapeutic that does not directly select for resistance in viruses, we hypothesize that stenoparib should be able to inhibit all SARS-CoV-2 variant strains.
For the cytotoxicity assays and plaque assays (performed in the United States), the cytotoxicity and antiviral activity of stenoparib on SARS-CoV-2 was assessed in vitro using Vero E6

ViroSpot reduction assay
The activities of stenoparib and remdesivir (catalog # 30354, Cayman Chemical Company, Ann Arbor, MI, USA) were assessed in a BSL3 facility against a wt SARS-CoV-2 strain (BavPat1/ 2020), and four additional SARS-CoV-2 variants of concern; alpha (B.1.1.7), beta (B.1.351) delta (B.1.617.2), and gamma (P1), hereafter referred to using Greek nomenclature (Table 1). We performed ten, serial 2-fold dilutions of compound, mixed with 100 plaque-forming units (pfu) of virus, and added the mixture to 80% confluent Vero E6 cells growing in multi-well plates. The Vero E6 cells were fixed 20 hours (h) after infection and stained with a SARS-CoV/ SARS-CoV-2 Nucleocapsid monoclonal antibody (Sino Biological, Wayne, PA, USA) followed by a horseradish peroxidase (HRP)-labeled Goat anti-Mouse IgG (catalog # G-21040, experiments were supported by service fees paid by Allarity Therapeutics. SK received funding in the form of salary from the commercial organization Allarity Therapeutics. PMN and ASLT received funding in the form of salary from the commercial organization Viroclinics-DDL. The funders had no additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.
Competing interests: SK is employed by and holds a financial interest in Allarity Therapeutics, which stands to potentially benefit from these results. PMN and ASLT are employed by Viroclinics-DDL. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.
ThermoFisher, Waltham, MA, USA) and TrueBlue Peroxidase Substrate (catalog # 5510-0030, Seracare, Milford, MA, USA). Spots were counted using a CTL ImmunoSpot Image Analyzer (Cleveland, OH, USA) as previously described [10]. Percent inhibition was calculated as percent of the well surface area covered by virus-positive immunostaining, and was normalized by taking the average fraction of the wells that showed positive staining compared to the control wells with the highest positive staining [10]. The EC 50 values were approximated and compared in a pair-wise fashion using package 'drc 3.0-1' in R version 4.1.1 [11][12][13]. We calculated means and standard deviations using package 'matrixStats' version 0.62.0 and 'psych' version 2.2.5 in R version 4.1.1 [13][14][15]. Mean normalized percent inhibition and dose-response curves were plotted using Hill coefficient, minimum, maximum, and EC 50 parameter estimates from package 'drc' version 3.0-1 and the Hill equation in R version 4.1.1 [11][12][13]16].

Plaque assay
Vero E6 cells were infected with the SARS-CoV-2 alpha variant in a BSL3 facility using a multiplicity of infection (MOI) of 0.1, and the activity of stenoparib was assessed with and without remdesivir using a plaque reduction assay. Following viral infection and treatment with stenoparib and/or remdesivir, cells were overlaid with low melting point agarose (catalog # 1613112, BioRad, Hercules, CA, USA), fixed at 120 h post infection with 4.0% paraformaldehyde (catalog # AAJ19943K2, Fisher Scientific, Waltham, MA, USA), stained with crystal violet (catalog # V5265, Sigma Aldrich, St. Louis, MO, USA), and plaques were manually counted. We used Welch's Two Sample t-tests in R version 4.1.1 [13] to perform pairwise t-tests between the untreated wells and wells under all other treatment conditions. Standard deviations and error bars were plotted using packages 'matrixStats' and 'psych' in in R version 4.1.1 [13][14][15].
In an additional set of experiments, we focused on the SARS-CoV-2 alpha variant. Vero E6 cells were infected with the alpha variant using a multiplicity of infection of 0.1, and the activity of stenoparib was assessed with and without remdesivir using the plaque reduction assay. Plaques are areas of dead or destroyed cells and appear as small, clear regions in an infected cell monolayer after fixation and staining with crystal violet. We combined 2.5, 5.0, and 10 μM doses of stenoparib with the previously reported EC 50 of remdesivir (0.5 μM) [6]. The three control types were 1) infected and untreated cells, 2) uninfected and untreated cells, and 3) infected cells treated with a combination of camostat mesylate and aloxistatin (E64d) (combination hereafter referred to as Camostat-E64d, or 'CE'), which are protease inhibitors that prevent Spike (S) protein cleavage and virus entry into the cell [12].
As shown in Fig 2A and 2B, neither 10 μM stenoparib nor 0.5 μM remdesivir achieved greater than a 50% reduction in plaquing efficiency compared to the infected, untreated cells. When combined, however, plaque formation was reduced by over 80% compared to infected, untreated cells. This reduction was superior to what was achievable with stenoparib (p = 3.03×10 −5 ) or remdesivir (p = 1.99×10 −4 ) alone at these doses. Notably, cytotoxicity remained near baseline levels (Fig 2C). The cytotoxicity experiments were run in triplicate and cells were treated with CE, dimethylsulfoxide (DMSO), lysis buffer, or left untreated (no compound) as controls. When combined, the activity of two or more drugs with different mechanisms of activity may synergize, with the potential benefit of reducing individual doses of each drug and minimizing undesirable side effects in the patient.

Discussion
According to a recent report, the beta variant appears to exhibit a significantly reduced eclipse period (length of time between initial infection and the production of virus by a cell) and more rapid replication in vitro compared to the alpha variant [17]. This could explain the relatively higher stenoparib and remdesivir EC 50 estimates for the beta variant. In addition to numerous changes to the S protein, the beta variant carries some unique mutations in the N protein and in the Nsp3 polyprotein (ORF1a) that are not found in the other variants [18], and may be involved in viral degradation pathways including ADPR following infection [19]. Differential susceptibility to ADPR may be related to the altered replication kinetics of the beta variant, although verifying this requires further investigation.
In this study, the activity of remdesivir and stenoparib against SARS-CoV-2 was examined in Vero E6 African green monkey kidney cells, which are highly permissive to infection with SARS-CoV-2 [20]. Our previous stenoparib study utilized Vero E6 and Calu-3 human lung adenocarcinoma cells, which were less sensitive than Vero E6 to the toxic effects of stenoparib at higher concentrations and the extended incubation times (120 h) required for the plaque assay [6]. Host cell lines vary in their susceptibility to infection and in their response to treatment with antivirals. Additional studies using other host cell lineages, including primary respiratory epithelial cells, should be instructive regarding the efficacy of PARP inhibitors as hosttargeting antiviral therapeutics.
Besides the known roles for the PARP1 and PARP2 enzymes in DNA repair [21,22], members of the PARP family have numerous additional functions [23]. The 18 known human PARPs appear to differentially affect viral replication; some exhibit proviral activity and others exhibit antiviral roles [7,[24][25][26][27][28]. In coronavirus, the N protein is mono (ADP) ribosylated (MARylated) during infection. Stability of the N protein is critical for viral genome replication, translation, packaging, and modulation of the host cell cycle [29]. The N protein is the only ADPR target definitively identified in coronavirus thus far [24], and the prevalence of this modification across multiple coronavirus families suggests an important role in virus stability [24]. Despite this, the role of N protein MARylation, and of ADPR in general with regards to coronavirus stability, is not well characterized. Possibly ADPR has a role in regulating virus genome structure, like ADPR of histones or the adenovirus core proteins [30,31]. While Here, we showed that stenoparib is broadly inhibitory to SARS CoV-2, including emerging variants of concern. We expect these results to apply for additional, emergent variants as well, and recent preliminary data with stenoparib and the omicron variant supports this. A host-targeting therapeutic like stenoparib could be a significant benefit for COVID-19 patients as a standalone therapy, or as part of a combinatorial COVID-19 treatment strategy with an antiviral drug such as remdesivir or molnupiravir. Combinations of two or more drugs may lead to synergism through different mechanisms of action, which has the potential benefit of reducing individual doses of each drug and minimizing undesirable side effects.