Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a positive-sense, single-stranded-RNA virus of the Coronaviridae family that shares 79.5% sequence identity with SARS-CoV-1 (Huang et al., 2020; Lu et al., 2020; Wu et al., 2020a; Wu et al., 2020b; Zhou et al., 2020b; Zhu et al., 2020) . While both viruses are likely descendants of J o u r n a l P r e -p r o o f bat coronaviruses, the proximal source of SARS-CoV-2 zoonotic virus is unknown (Zhou et al., 2020b) . Since its first description at the end of 2019, SARS-CoV-2 has spread around the globe, infecting millions of people, and killing hundreds of thousands (https://coronavirus.jhu.edu). An urgent medical need exists for effective treatments for this disease.
In the early stages of COVID-19, the virus proliferates rapidly, and in some cases, triggers a cytokine storm -an excessive production of inflammatory cytokines (Quartuccio et al., 2020; Wiersinga et al., 2020) . This uncontrolled inflammation can result in hyperpermeability of the vasculature, multi-organ failure, acute respiratory distress syndrome (ARDS), and death (Gupta et al., 2020; Jose and Manuel, 2020; Quartuccio et al., 2020; Wang et al., 2020a; Zhou et al., 2020a) . Acute respiratory distress syndrome is one of the leading causes of mortality in COVID-19 (Moore and June, 2020) . In addition, elevated levels of interleukin (IL)-6 and IL-17 are reported to be associated with severe pulmonary complications and death (Pacha et al., 2020; Ruan et al., 2020; Russell et al., 2020) .
A therapeutic that can inhibit SARS-CoV-2 replication while attenuating the cytokine storm would be highly beneficial for both the early and late stages of COVID-19. Targeting the cellular de novo pyrimidine nucleotide biosynthesis pathway is one potential approach to treat both phases of COVID-19 as viral replication and over-production of a subset of inflammatory cytokines are controlled by pyrimidine nucleotide levels (Cheung et al., 2017; Xiong et al., 2020a) . Dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in this pathway, is located on the inner membrane of mitochondria and catalyzes the dehydrogenation of dihydroorotate (DHO) to orotic acid, ultimately resulting in the production of uridine and cytidine triphosphates (UTP and CTP) ( Fig 1A) (Munier-Lehmann et al., 2013) . Pyrimidine nucleotides are also supplied by the salvage pathway. The salvage pathway recycles pre-existing J o u r n a l P r e -p r o o f nucleotides from food or other extracellular sources and does not appear to be sufficient to support viral RNA replication. The salvage pathway does not seem to play a major role in replication of SARS-CoV-2 (Xiong et al., 2020b) .
The de novo pyrimidine biosynthesis pathway is also critical for the excessive production of a subset of inflammatory cytokines (including interferon-gamma, monocyte chemoattractant protein-1 [MCP-1], IL-5, and IL-6) in both cultured cells and animal models of viral infection (Cheung et al., 2017; Xiong et al., 2020a) . Consistent with DHODH being mechanistically central to COVID-19, a recent multi-omics study of drug targets for viral infections prioritized DHODH inhibition as one of the top three mechanisms to consider for SARS-CoV-2 treatment (Zheng et al., 2020) .
PTC299 is an orally bioavailable potent inhibitor of DHODH (see Fig 1B for chemical structure) (Cao et al., 2019) . Treatment of cultured cells with PTC299 results in inhibition of DHODH activity, leading to increased levels of DHO, the substrate for the DHODH enzyme (Cao et al., 2019) . Similar findings were documented in PTC299-treated cancer patients, where administration of PTC299 resulted in increased blood levels of DHO, indicating successful inhibition of DHODH in these patients (Cao et al., 2019) . These results are consistent with increased DHO levels observed in Miller syndrome patients who carry mutations in the DHODH gene that reduce DHODH activity (Duley et al., 2016) and with studies showing that PTC299 not only inhibits vascular endothelial growth factor (VEGF) levels in cultured cells, but also normalized VEGF levels in cancer patients (Cao et al., 2019) . VEGF is a stress-regulated cytokine the levels of which are increased in these cancer patients (Plotkin et al., 2009 ).
In studies of over 300 human subjects, including healthy volunteers and oncology patients, PTC299 has manifested a favorable pharmacokinetic (PK) profile and has been generally well J o u r n a l P r e -p r o o f 7 tolerated. The mechanism of action and the favorable PK and safety profiles of PTC299 support its investigation for use as a therapeutic for COVID-19. Here, we evaluated the in vitro antiviral activity of PTC299 against a panel of RNA viruses, with a specific focus on SARS-CoV-2, as well as the drug's ability to attenuate the excessive production of inflammatory cytokines. Our results indicate that that PTC299 has considerable potential as a COVID-19 therapeutic.
J o u r n a l P r e -p r o o f 2. Poliovirus (CCL-2) was obtained from ATCC. The received virus was amplified in HeLa cells, grown in DMEM supplemented with 5% FBS and 1% penicillin and streptomycin solution and the titer evaluated by plaque assay on HeLa cell monolayers. EBOV-GFP was provided by the CDC. RVFV-GFP was provided by the University of Texas Medical Branch (UTMB).
Vero cells were pretreated with increasing concentrations (1 nM to 1 µM) of PTC299 or PTC299 with 100 µM uridine. After incubation at 37°C overnight, the medium was removed, and cells were inoculated with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.05. After 1 hour of incubation at 37°C in CO2, wells were washed 3 times with dilution medium and 1 mL of medium containing the indicated compound doses was added back to each well. Cells were incubated at 37°C and samples collected at 0, 16, and 24 hours post-infection. Collected timepoint medium was replaced with an equal amount of 1x compound or dilution medium.
Samples were stored at -80°C until the day of analysis. The SARS-CoV-2 titer in Vero cells via 50% tissue culture infectious dose assay (TCID50) was performed for each sample collected at 0, Data management was performed using the OMERO.web client (v5.4.6-ice36-b87) and the OMERO server v5.4.6 (https://www.openmicroscopy.org/omero/. inhibition of viral genomic RNA compared to mock treated control (EC50) was reported as the concentration of test compound required to cause a 50% reduction in polio virus RNA and was normalized to GAPDH levels in the same well. The concentration required for 50% inhibition of cellular GAPDH RNA compared to mock treated control (CC50) was reported as the concentration of test compound that caused a 50% reduction in GAPDH mRNA. The experiment was repeated 3 times for this study and average EC50 and CC50 values are reported.
Hepatitis C virus (HCV) replicon studies were performed as previously described. Huh-7
(hepatocarcinoma) cells harboring the subgenomic HCV genotype 1b (Con1) 
J o u r n a l P r e -p r o o f
The activity of PT299 was evaluated in complex primary culture human cell systems in the PTC299 was prepared in DMSO (final concentration ≤0.1%) and was added at the specified concentrations one hour before stimulation and remain in culture for 24 hours (sAg system), 48hours (HDFSAg system), 72-hours (BT system, soluble cytokines), or144 hours (BT system, secreted IgG)). J o u r n a l P r e -p r o o f BT system: The BT system is a co-culture of CD19+ B cells and PBMC that utilizes B cell receptor (BCR) stimulation and sub-mitogenic TCR stimulation (defined as 1/1000th TCR ligand strength) to capture the T cell dependent B cell activation and class switching that occurs in germinal centers. The BT system models diseases and chronic inflammatory conditions driven by B cell activation and antibody production.
HDFSAg system: The HDFSAg system is a co-culture of human primary dermal fibroblasts and PBMC that is stimulated with sub-mitogenic TCR levels (defined as 1/1000th TCR ligand strength) to model chronic T cell activation and inflammation responses within a tissue setting. 
The ability of PTC299 to inhibit SARS-CoV-2 replication was evaluated in vitro using two virus cell culture models. In the first model, Vero E6 cells were preincubated for 30 minutes with increasing concentrations of PTC299 and cells were subsequently infected with SARS-CoV-2 at a MOI of 0.1. Forty-eight hours after infection, viral protein was quantified by staining with antibody to the nucleocapsid protein of SARS-CoV-2 and with DAPI, which stains the cells nuclei, to determine cell number.
The results demonstrated that PTC299 treatment led to a dose-dependent reduction in the levels of SARS-CoV-2 nucleocapsid protein (green) with an EC50 of 1.96 nM (Figs 2A and 2B) .
DAPI staining of cell nuclei (blue) showed that PTC299 did not reduce cell number (Fig 2A) .
These results indicate thatPTC299 reduces viral load in the absence of cytotoxicity. The initial concentration range of PTC299 explored did not enable the determination of EC50
and EC90 for PTC299 against SARS-CoV-2. Therefore, a range of lower concentrations of PTC299 (from 1 pM to 1 µM) was evaluated. The EC50 and EC90 values were determined to be 2.6 nM and 53 nM, respectively (Fig 3C) , which is consistent with the findings of the immunofluorescence experiments described in Fig 2. The EC50, determined as the midpoint of the logarithmic curve, was 31.6 nM. Using a similar calculation based on the logarithmic curve, the EC90 was determined to be 80.3 nM. At the highest concentration of PTC299 tested (10 µM), the CC50 was determined to be >10 µM (Fig 3D) , indicating that, similar to the immunofluorescence experiments described above, PTC299 reduces viral load with little cell death.
The results in Figs 2 and 3 indicate that PTC299 is a potent inhibitor of SARS-CoV-2 replication. Considering its mechanism of inhibiting cellular de novo pyrimidine synthesis and the established role of pyrimidine nucleotides in viral replication, we investigated the spectrum of activity of PTC299 against a panel of RNA viruses of both positive and negative genome J o u r n a l P r e -p r o o f polarity. These studies were not designed to directly compare the potency of PTC299 across the different viruses but were designed to assess anti-viral activity for each virus.
PTC299 was found to have broad-spectrum antiviral activity against the RNA viruses tested ( 
PTC299 was originally identified as a compound that modulates expression of stressregulated proteins like VEGF and was only subsequently found to be a DHODH inhibitor (Cao et al., 2019; Weetall et al., 2016) . DHODH inhibitors have been shown to suppress virus-induced inflammatory cytokine production (Xiong et al., 2020a) . The effect of PTC299 on cytokine production and release into the cell culture medium was assessed in the BioMAP assay profiling platform. This system consists of co-cultures of PMBCs with other cell types, including B cells, fibroblasts, and endothelial cells, allowing for the measurement of physiologically relevant J o u r n a l P r e -p r o o f biomarker readouts of the activity of a test compound (Berg et al., 2006; Kunkel et al., 2004a; Kunkel et al., 2004b) . Each co-culture was stimulated to recapitulate relevant signaling networks that naturally occur in healthy human tissue and under certain disease states. For each system, cytotoxicity and cellular proliferation were assessed. PTC299 was added at increasing concentrations to each of the model systems one hour before cellular stimulation and remained present during the stimulation period for each co-culture. Cytokine production was subsequently assessed.
PTC299 was found to be a potent inhibitor of the production of a subset of immunomodulatory and inflammation-related cytokines that are associated with the stress response and poor prognosis in COVID-19 (Fig 4 and Table S1 ). In the SAg co-cell culture system, which models T-cell activation, PTC299 treatment resulted in significant decreases of MCP-1, CD40, and IL-8, as levels decreased after 24 hours of stimulation (Table S1 ). Compared with the control, 31% and 27% reductions in MCP-1 and IL-8, respectively, were observed when cells were incubated with 10 nM PTC299, and a 29% decrease in CD40 was detected with 100 nM PTC299 (all p values <0.01). A 23% reduction in endothelial cellular proliferation as measured by SRB (a measure of cell biomass) relative to control was seen following treatment with PTC299 at 100 nM (p<0.01) but not at 1 or 10 nM.
In the BT co-cell culture system, which models chronic inflammatory conditions driven by B cell activation and antibody production, incubation of cells with 10 nM PTC299 resulted in a significant reduction in the levels of soluble (s)IgG, sIL-17A, sIL-17F, sIL-6, and sTNFα released from the cells after 72 hours of stimulation (range, 49% to 68%) (all p values <0.01) (Fig 4 and Table S1 ). The production of sIgG was also significantly decreased by 97% following J o u r n a l P r e -p r o o f 144 hours of stimulation (p <0.01). Similar to results obtained in the SAg co-cell culture system, 100 nM PTC299 significantly inhibited proliferation (31%) compared with control (p<0.05).
In the HDFSAg co-cell culture system, which models chronic T cell activation and inflammation responses within a tissue setting, PTC299 was associated with significantly lower levels of secreted MCP-1, IL-8, MMP-1, sIL-10, sIL-17A, sIL-17F, sIL-2, sIL-6, sTNFα and sVEGF compared with control (all p values <0.01) (Fig 4 and Table S1 ). PTC299 inhibition of cytokine production was particularly apparent for sIL-17F, sIL-6, and sVEGF, where a significant reduction in the levels of these cytokines was observed at as low as 1 nM PTC299
(range 33% to 90%; all p values <0.01). No significant differences between co-cultures treated with PTC299 or the control were seen with regard to proliferation or cytotoxicity.
In the /TH2 co-cell culture system, a model of mixed vascular Th1 and Th2 type inflammation that creates a pro-angiogenic environment promoting vascular permeability and recruitment of mast cells, basophils, eosinophils and T and B cells, significant inhibition of MCP-1 and sIL-17A production was seen as relative to controls (all p values <0.01) (Fig 4   andTableS1 ). Significant inhibition of MCP-1 production of 21% was observed when cells were incubated with 10 nM PTC299 (p<0.01). Similar to results obtained with the HDFSAg co-culture system, treatment with PTC299 did not result in a significant decrease in cell proliferation or increased cytotoxicity in the /TH2 system.
IL-17A and IL-17F are cytokines produced by activated T-helper-17 (Th17) cells that mobilize and activate neutrophils. In COVID-19, disease severity is positively correlated with levels of IL-17A, and the levels of this cytokine are increased in patients in the intensive care unit (Megna et al., 2020; Pacha et al., 2020) . Results from the BioMap analysis described above J o u r n a l P r e -p r o o f using the BT and HDFSAg co-cell culture systems demonstrated that PTC299 significantly inhibits the production of IL-17A and IL-17F. The ability of PTC299 to decrease IL-17A and IL-17F levels was further verified by assessing the effects of PTC299 on the production of IL-17A and IL-17F by Th17 cells, a subset of CD4 T cells that produce these cytokines (Martinez et al., 2012) .
The ability of PTC299 to inhibit the production of secreted IL-17A and IL-17F was assessed in a model system in which PBMCs were stimulated with human T-activator CD3/CD28
Dynabeads in a culture containing cytokines and antibody to induce Th17 differentiation. The A different DHODH inhibitor, brequinar, also inhibited IL-17A and IL-17F production, but at substantially higher concentrations than PTC299. Addition of 100 µM uridine rescued the PTC299-dependent inhibition consistent with PTC299 acting on DHODH (Figs 5C and 5D ).
J o u r n a l P r e -p r o o f 4.
COVID-19 is characterized by an early stage of viral replication followed in some cases by overproduction of inflammatory cytokines. Both the viral replication and the cytokine response are dependent upon pyrimidine nucleotides produced by the de novo biosynthesis pathway (Cheung et al., 2017; Xiong et al., 2020a) . The results presented here demonstrate that PTC299 has a dual mechanism of action that inhibits viral replication and attenuates the production of a subset of inflammatory cytokines. PTC299 potently inhibited SARS-CoV-2 replication in vitro, with an EC50 of 2.6 nM (31.6 nM based on the logarithmic curve), and an antiviral selectivity of >3,800. Inhibition of SARS-CoV-2 replication by PTC299 was prevented by the addition of exogenous uridine, which obviated the requirement for the de novo pyrimidine nucleotide synthesis pathway, consistent with the drug functioning as a DHODH inhibitor and with the fact that many RNA viruses require high concentrations of pyrimidine nucleotides to transcribe or replicate the viral genomes (Xiong et al., 2020a) . Accordingly, PTC299 also demonstrated broad-spectrum antiviral activity.
A unique aspect of a DHODH inhibitor such as PTC299 is its ability to affect both viral replication and attenuate the cytokine storm (Breedveld and Dayer, 2000; Li et al., 2013; Xiong et al., 2020a) . The cytokine response associated with COVID-19 dramatically complicates the course of the infection in some patients and can result in ARDS with a high mortality rate.
Experience with other viral infections indicates that treating the excessive inflammatory immune events downstream of the infection is paramount to patient recovery (Quartuccio et al., 2020) .
Studies presented here demonstrated that PTC299 inhibited production of multiple cytokines, including MCP-1, IL-6, TNFα, VEGF, and IL-17.
Inhibition of IL-6, IL-17, VEGF, and IgG by PTC299 may be of particular importance for treating COVID-19. IL-6 appears to play a key role in excessive cytokine production resulting from viral infection and pulmonary complications in COVID-19 (Ruan et al., 2020; Russell et al., 2020) . Preliminary results from two small studies suggest inhibition of the IL-6 pathways by tocilizumab and siltuximab resulted in treatment benefit for COVID-19 patients (Gritti et al., 2020; Xu et al., 2020) . Comparable to what was found for MERS-CoV and SAR-CoV infections,
IL-17A has also been associated with disease severity and lung injury in COVID-19 (Pacha et al., 2020) . Similarly, increased VEGF levels promote vascular permeability and leakage, helping in the pathophysiology of hypotension and pulmonary dysfunction (Teuwen et al., 2020) . The attenuation of expression of these cytokines by PTC299 that result from SARS-CoV-2 infection may provide important and unique therapeutic benefits.
The reduction in the levels of IgG associated with PTC299 treatment may be clinically meaningful as an elevated antibody titer has been shown to be associated with COVID-19 disease severity (Tan et al., 2020; Zhao et al., 2020) . The latter findings may be evidence for antibody-dependent enhancement of disease, which was observed in SARS-CoV-1 (Fu et al., 2020; Liu et al., 2019; Zhao et al., 2020) , an infection in which the appearance of IgG is associated with acute respiratory disease in 80% of patients (Peiris et al., 2003) . It is hypothesized that antiviral antibodies may trigger an Fc receptor-mediated inflammatory response resulting in severe lung injury (Fu et al., 2020) .
The novel dual mechanism of action of PTC299 distinguishes it from most other therapeutics being investigated in the clinic for the treatment of COVID-19, as many of these target either viral-specific processes or the immune response, but not both. Due to its dual mechanism of action, PTC299 is expected to be effective in treating both early and later stages of COVID-19.
This contrasts with direct-acting antivirals that typically show their greatest effectiveness primarily in the early phase of the disease. Further, since PTC299 targets a cellular gene product (DHODH), and it is unlikely that viruses can overcome the need for pyrimidine nucleotides, the likelihood that the therapeutic effect of PTC299 will be compromised by the development of viral resistance is low. This may be particularly important for RNA viruses whose high mutation frequency often promotes evasion of direct-acting antivirals.
PTC299 is a highly potent inhibitor of SARS-CoV-2 replication, with an EC50 of 2.6 nM.
Several other DHODH inhibitors have also shown activity against SARS-CoV-2 (Xiong et al., 2020a) , including leflunomide, teriflunomide, brequinar, and two recently identified DHODH inhibitors, S312 and S416 (Xiong et al., 2020a) . Leflunomide and its active metabolite teriflunomide are FDA approved treatments for rheumatoid arthritis and relapsing forms of multiple sclerosis, respectively. Differences in experimental design make it difficult to directly compare the findings of PTC299 to those reported for these other DHODH inhibitors, but the results suggest that PTC299 is highly potent compared to the other molecules. At an MOI of 0.05, the EC50 values for SARS-CoV-2 were 41.5 µmol/L for leflunomide, 26.1 µmol/L for teriflunomide, 0.123 µmol/L for brequinar, 0.017 µmol/L for S416, and 1.56 µmol/L for S312 (Xiong et al., 2020a) and the respective selectivity indices ranged from 21 for leflunomide to 10505 for S416. Similar to PTC299, these other DHODH inhibitors showed broad spectrum antiviral activity (Xiong et al., 2020a) .
Several other compounds that are under evaluation in the clinic for the treatment of SARS-CoV-2 infection have EC50 values reported to be the micromolar range. While these compounds have different mechanisms of action than PTC299, it is notable that the reported EC50 of remdesivir is 770 nM, that of chloroquine is 1.1 to 5.5 µM, and that of hydroxycholorquine is J o u r n a l P r e -p r o o f 720 nM (Choy et al., 2020; Sanders et al., 2020; Wang et al., 2020b; Xiong et al., 2020a; Yao et al., 2020) .
The key to a successful COVID-19 treatment is to not only have a potent molecule, but to also have a dose that can be delivered safely and that will sustain exposure in the blood to inhibit viral replication or infection. PTC299 is currently being evaluated to treat COVID-19 in the phase 2/3 study PTC299-VIR-015-COV19 (referred to as FITE19)
(https://clinicaltrials.gov/ct2/show/NCT04439071?term=PTC299+AND+COVID&draw=2&ran k=1). The dose being used is based on a PK/pharmacodynamic relationship obtained in monkeys and in cancer patients with neurofibromatosis type 2 (NF2), and the well-characterized PK profile from healthy volunteer and oncology studies (Cao et al., 2019 )(data on file). The PTC299
dose in the FITE19 study is predicted to yield a Cave of 1371 ng/mL and will thus be approximately1100-fold higher than the drug's EC50 and about 55-fold greater than the EC90 values against SARS-CoV-2. The PTC299 levels would also be approximately 12-fold higher than the 250 nM concentration that resulted in the 3-log reduction in the titer of SARS-CoV-2 in cultured Vero cells.
PTC299 is a highly potent inhibitor of SARS-CoV-2 replication that also suppresses production of a subset of pro-inflammatory cytokines, suggesting it has the potential to act through this dual mechanism to treat the viral and immune components of COVID-19. Due to its ability to block viral replication and cytokine production, PTC299 may be effective in treating both early and later stages of the disease. This contrasts with direct-acting antivirals that typically show their greatest effectiveness primarily in the early phase of the disease (McNicholl and McNicholl, 2001; Xiong et al., 2020a) and with anti-inflammatory drugs that treat only the later phase of COVID-19 (Moore and June, 2020; Quartuccio et al., 2020) . It has been argued that it is important to combine antiviral therapy with immune suppressants as using only compounds that modify the immune response to treat the cytokine storms may make viral clearance more difficult (Quartuccio et al., 2020) . Importantly, PTC299 is orally bioavailable, has been extensively evaluated in human subjects, has well established PK and safety profiles and is generally well tolerated. These findings and prior clinical experience with PTC299 support the further development of this novel molecule for the treatment of COVID-19. 