key: cord-345499-hq5um68k
authors: Xiong, Rui; Zhang, Leike; Li, Shiliang; Sun, Yuan; Ding, Minyi; Wang, Yong; Zhao, Yongliang; Wu, Yan; Shang, Weijuan; Jiang, Xiaming; Shan, Jiwei; Shen, Zihao; Tong, Yi; Xu, Liuxin; Yu, Chen; Liu, Yingle; Zou, Gang; Lavillete, Dimitri; Zhao, Zhenjiang; Wang, Rui; Zhu, Lili; Xiao, Gengfu; Lan, Ke; Li, Honglin; Xu, Ke
title: Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2
date: 2020-03-12
journal: bioRxiv
DOI: 10.1101/2020.03.11.983056
sha: 
doc_id: 345499
cord_uid: hq5um68k

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of coronavirus SARS-CoV-2. Existing direct-acting antiviral (DAA) drugs cannot be applied immediately to new viruses because of virus-specificity, and the development of new DAA drugs from the beginning is not timely for outbreaks. Thus, host-targeting antiviral (HTA) drugs have many advantages to fight against a broad spectrum of viruses, by blocking the viral replication and overcoming the potential viral mutagenesis simultaneously. Herein, we identified two potent inhibitors of DHODH, S312 and S416, with favorable drug-like and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus (H1N1, H3N2, H9N2), Zika virus, Ebola virus, and particularly against the recent novel coronavirus SARS-CoV-2. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knocking-out cells. We also proposed the drug combination of DAA and HTA was a promising strategy for anti-virus treatment and proved that S312 showed more advantageous than Oseltamivir to treat advanced influenza diseases in severely infected animals. Notably, S416 is reported to be the most potent inhibitor with an EC50 of 17nM and SI value >5882 in SARS-CoV-2-infected cells so far. This work demonstrates that both our self-designed candidates and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-repression may have clinical potentials not only to influenza but also to COVID-19 circulating worldwide, no matter such viruses mutate or not.

Acute viral infections, such as influenza virus, SARS-CoV, MERS-CoV, Ebola virus, Zika virus, and the very recent SARS-CoV-2 are an increasing and probably lasting global threat 1 . Broad-spectrum antivirals (BSA) are clinically needed for the effective control of emerging and re-emerging viral infectious diseases. However, although great efforts have been made by the research community to discover therapeutic antiviral agents for coping with such emergencies, specific and effective drugs or vaccines with low toxicity have been rarely reported 2 . Thus, unfortunately, there is still no effective drugs for the infection of the novel coronavirus SARS-CoV-2 at present, which outbreak in December 2019 firstly identified by several Chinese groups [3] [4] [5] , and now has quickly spread throughout China and to more than 90 other countries, infecting 101,923 patients and killing 3486 ones by March 7, 2020 6 .

Discovery of nucleoside or nucleotide analogs and host-targeting antivirals (HTAs) are two main strategies for developing BSA [7] [8] [9] . With the former drug class usually causing drug resistance and toxicity, the discovery of HTAs has attracted much attention 10 . Several independent studies searching for HTAs collectively end up to compounds targeting the host's pyrimidine synthesis pathway to inhibit virus infections, which indicates that the replication of viruses is widely dependent on the host pyrimidine synthesis [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . However, most of these compounds lack verified drug targets making subsequent drug optimization and further application impossible 11, [13] [14] [15] [17] [18] [19] 21, 25 .

There are only a few inhibitors against pyrimidine synthesis that can be carried forward to animal studies, however, their antiviral efficacies were unsatisfactory or even ineffective at all 12, 16, 17, 21, [23] [24] [25] . For example, a pyrimidine synthesis inhibitor FA-613 without a specific target protected only 30.7% of mice from lethal influenza A virus infection when compared to the DAA drug Zanamivir (100%) in parallel 23 . Another two compounds, Cmp1 24 and FK778 25 , which target DHODH, a rate-limiting enzyme in the fourth step of the de novo pyrimidine synthesis pathway, could only inhibit the DNA virus (CMV) replication in RAG -/mice, but their therapeutic effects on the upcoming diseases were unexplored. Therefore, more potent pyrimidine synthesis inhibitors, especially ones with the specific drug target, are urgent to be developed to prove whether such an HTA drug is valuable towards clinical use or has any advantages over DAA drugs in antiviral treatment.

To identify potent and low-toxicity DHODH inhibitors (DHODHi), we previously conducted a hierarchal structure-based virtual screening (Fig. 1A) against ~280,000 compounds library towards the ubiquinone-binding site of DHODH 26 . We finally obtained two highly potent DHODHi S312 and S416 with IC50s of 29.2 nM and 7.5 nM through structural optimization 27, 28 , which are > 10-folds potent than the FDA approved DHODHi Teriflunomide (IC50 of 307.1 nM). By using these two potent inhibitors, we could fully evaluate DHODH as a valuable host target both in infected cells and in vivo in infected animals. We identified that targeting DHODH offers broad-spectrum antiviral efficacies against various RNA viruses, including the DAA-resistant influenza virus and the newly emerged coronavirus SARS-CoV-2. Especially, our potent DHODHi can protect 100% mice from lethal influenza challenge, which is as good as the DAA drugs, and is even effective in the late phase of infection when DAA drug is no longer responding.

By determination of the X-ray crystal structure of DHODH in complex with S416

( Supplementary Data Fig. 1A, Table S1 ), we verified the binding mode of S416 at the ubiquinone-binding site of DHODH that similar to S312. The binding free energies for S312 and S416 were -45.06 and -46.74 kJ/mol, and the binding equilibrium dissociation constants (KD) were 20.3 and 1.69 nM, respectively ( Fig. 1B and 1C) .

Additionally, the two inhibitors exhibited a clear trait of fast-associating (kon) and slowdissociating (koff) inhibition (Table S2) , providing themselves as ideal drug candidates with a high level of target occupancy. Moreover, S312 and S416 showed proper halflives (8.20 and 9.12 h, respectively) ( Table S3) , which are much shorter than that of Teriflunomide and Leflunomide, indicating that they may have less possibility to bring side effects from drug accumulation in the body (Supplementary Data Fig. 1B and   1C ).

To examine the antiviral activities of these DHODHi, we use the influenza A virus as a model virus. A labor stain of A/WSN/33(H1N1, WSN) with 20 TCID50 was applied to infect MDCK cells, and serial dilutions of drugs (DMSO as controls) were added at the same time when cells were infected. Drug efficacies were evaluated by quantification of cell viability in both infected and non-infected cells, and the halfmaximal effective concentration (EC50) and the half-cytotoxic concentration (CC50) of the indicated drug were obtained accordingly. The selectivity index (SI) was calculated by CC50/EC50. As shown in Fig. 1D , the antiviral effect of Leflunomide is hardly detectable at the cell culture level (EC50>25 μ M). However, Teriflunomide, the active metabolite of Leflunomide, exhibited a clear antiviral effect against the WSN virus (EC50=29.33μM, CC50=178.50μM，SI=6.08). As compared to Teriflunomide, the potent DHODHi S312 is ~12-fold stronger (EC50=2.36μM) and S416 is ~480-fold stronger (EC50=0.061μM) than Teriflunomide in their antiviral efficacies. We also tested different influenza A virus subtypes of H3N2 and H9N2. The antiviral efficacies of DHODHi followed the same pattern as they were against H1N1, which is S416>S312>Teriflunomide>Leflunomide in viral inhibitory efficacies (summarized in Table 1 ). The drug effective curve of S416 to H3N2 (EC50=0.013μM) and H9N2 (EC50=0.020μM) is shown in Fig. 1E and 1F . When we compared the drug efficacy by virus plaque assay, the results in Fig. 1G showed that the positive control DAA drug Oseltamivir (Osel) could reduce the plaque size to needlepoint size. However, the virus plaque in equivalent S312-treatment was not observable at all indicating that S312 is more efficient in inhibiting virus replication than Osel.

The results in all indicate that DHODHi, especially S312 and S416 exhibited direct antiviral activities to different subtypes of influenza A viruses by shutting off virus multiplication more thoroughly than Osel.

As all actuate infectious viruses rely on cellular pyrimidine synthesis process to replicate, it is reasonable to speculate that DHODHi have broad-spectrum antiviral efficacies. We, therefore, tested several highly impacted acute infectious RNA viruses.

All compounds of Teriflunomide, Brequinar, S312, and S416, showed inhibitory effects against Ebola virus (EBOV) mini-replicon, with EC50 of 6.43, 0.102, 14.96 and 0.018μM, respectively ( Fig. 2A) . To our supersize, S416 showing relatively high cytotoxicity in MDCK cells (CC50=1.64μM in Fig.1D ) was less toxic to EBOV-minireplicon supporting BSR-T7/5 cells (CC50=85.84μM). Thus, a significantly high SI=4746.11 was achieved by S416. We subsequently tested the inhibitory effects of DHODHi against Zika virus (Fig. 2B) . EC50 values were 17.72, 0.268, 2.29 and 0.021μM for Teriflunomide, Brequinar, S312 and S416, respectively. Again, the selective index of S416 reached the top of SI=1087.62.

When we prepared the manuscript, a severe outbreak of SARS-CoV-2 occurred in Wuhan in December 2019, we responded quickly to examine the antiviral activity of DHODHi against this new coronavirus. The data in Fig. 2C showed that all the DHODHi tested is low toxic to SARS-CoV-2 susceptible Vero E6 cells. Teriflunomide had a solid antiviral efficacy of EC50=26.06uM (at MOI=0.05, ~2.4-fold stronger than Favipiravir [EC50=61.88μM] 29 ) (Fig. 2C upper right) , whereas its pro-drug Leflunomide showed less inhibition of EC50=63.56uM (data not shown). We therefore further did immuno-florescent assay to visualize the drug efficacy. To determine more carefully the efficacy of Teriflunomide, which can be transferred to clinical treatment of SARS-CoV-2 immediately as an approved drug, a bit low MOI of 0.03 (Fig. 2C upper left) were applied. In this condition, the EC50 of Teriflunomide could reach 6μM with SI>33, indicating that Teriflunomide with effective EC50 and SI values have all the potentials to treat SARS-CoV-2-induced COVID-19 disease as an 'old drug in new use' option.

Additionally, S312 and S416 exhibited ideal antiviral efficacies of EC50=1.55μM (SI>64.62) and EC50=0.017μM (extensively high SI>5882.4), respectively ( Fig. 2C lower panel). Compared with our previous publication of Remdesivir (EC50=0.77μM, SI>129.87) and Chloroquine (EC50=1.13μM, SI>88.5) 29 , which are currently used in clinical trials against SARS-CoV-2, S416 had much greater EC50 and SI values (66.5fold stronger than Chloroquine in EC50) against SARS-CoV-2. The data in Fig. 2D clearly showed that as little as 4.6nM (0.3EC50) of S416 can dramatically inhibit SARS-CoV-2 infections, while, increased drug concentration of 370nM (22EC50) could further eliminate viral infected cells. Thus, S416 turns to be the best efficient chemical so far against SARS-CoV-2 at the cellular level.

In all the previous studies, inhibitors to DHODH or pyrimidine synthesis pathway were Fig. 3A ). The data in Fig. 3B showed that the bodyweights of mice from the DMSO-treated 'virus group' all dropped to less than 75% and died at D8 p.i.. DAA drug of Osel could indeed totally rescue all the mice from bodyweight loss and death. Equivalently, S312 (5mg/kg, red line) was also able to confer 100% protection and little bodyweight loss similar to Osel. Even S312 of 2.5mg/kg and 10mg/kg cold confer 75% protection and 50% protection, respectively.

Considering the Cmax of S312 (≈15μM), we used both 5mg/kg and 10mg/kg in the following experiments. The results suggest that S312 of a modest dose (5mg/kg) would achieve the equivalent 100% protection to DAA drug when used from the beginning of infection.

Except for broad active to different viruses, HTA drug such as DHODHi has another advantage over DAA drug to overcome drug-resistant. To prove this, we generated a current-circulating Oseltamivir-resistant NA H275Y mutant virus (in WSN backbone) by reverse genetics (Supplementary data Fig. 2A and 2B) . We found that the NA H275Y virus did not respond to Osel (20mg/kg/day)-treatment at all but 2.5mg/kg/day of S312 can rescue 50% of mice from lethal infection of NA H275Y virus (Supplementary data Fig. 2C ). When mice were infected with a natural-isolated pandemic strain SC09, which is less sensitive to either Osel (40% protection) or S312 (20% protection) ( Fig.   3C ), we further observed 100% protection in combined treatment of S312+Osel, indicating that HTA and DAA drug combination can augment therapeutic effects.

The data in all refresh DHODH as an attractive host target in treating viral disease with equivalent efficacy to DAA drug, and is advantageous when facing DAA-drugresistant viruses.

To elucidate the essential role of DHODH in the viral replication cycle, we generated a DHODH -/-A549 cell line by CRISPR-Cas9 gene knock-off (KO) technology (Fig. 4A) .

Unexpectedly, the cell proliferation rate was barely affected in DHODH -/cells indicating DHODH is not indispensable for cell growth at least for three days (72 hours) ( Supplementary Fig. 3 ). By contrast, virus growth was largely inhibited in DHODH -/cells as compared to wild-type (WT) A549 cells with almost 1000-fold reduction of infectious particles at 72 hours post-infection (h.p.i.) (Fig. 4B) . When S312 (5IC50) was added into the culture medium, dramatic reduction of virus growth only occurred in WT cells but not in DHODH -/cells ( Fig. 4C and 4D) . These results prove that virus growth but not the coincident cell growth requires DHODH activity, and antiviral action of S312 is implemented by targeting DHODH.

The general virus growth cycle includes virus entry, viral genome replication, and virus release. To further validate virus genome replication is the major target of DHODHi, we used the influenza-A-virus mini-replicon system to quantify viral genome replication. Brequinar, another potent inhibitor of DHODH was included as a positive control 30 , whereas, Osel targeting influenza NA protein served as a negative control. The results in Fig.4E showed no inhibition on viral genome replication in the Osel-treated group, but there were obvious inhibitions on viral genome replication in both S312-and S416-treated groups as well as Brequinar-treated group in dosedependent manners. Almost 90% of viral genome replication was suppressed by 10IC50 of S312 (24μM) and S416 (0.6μM). As DHODH catalyzes oxidation of dihydroorotate (DHO) to produce orotic acid (ORO) and finally forms UTP and CTP， we add four nucleotides (adenine nucleotide(A), guanine nucleotide(G), uracil nucleotide(U), and cytosine nucleotide(C)), DHO, ORO respectively to mini-replicon system to identify the target of S312 and S416. The results in Fig. 4F showed that the addition of 50μM either U or C could effectively rescue viral genome replication in S312-and S416-treated cells (as well as Brequinar-treated cells), whereas addition of neither A nor G changed the inhibitory effects. Moreover, supplement of DHODH substrate DHO cannot rescue viral genome replication (Fig. 4G) , but a supplement of DHODH product ORO can gradually reverse the inhibition effects of S312 and S416 (Fig. 4H) . The results further confirm that compounds S312 and S416 inhibit viral genome replication via targeting DHODH and interrupting the fourth step in de novo pyrimidine synthesis.

It is documented elsewhere that DAA drugs such as Osel is only completely effective in the early phase of infection, optimally within 48 hours of symptom onset 31 . And till now, there is no approved drug to treat advanced influenza disease at the late phase specifically. We suppose that S312 could be effective in the middle or late phase of disease because it targets a host pro-viral factor of DHODH not affected by viral replication cycle. To test this, we compared the therapeutic windows of S312 and Osel in early (D3-D7), middle & late (D5-D9)，severe late (D7-D11 or D6-D14) phases (workflow shown in Fig. 5A ). When drugs were given in the early phase, both Oseltreatment and 'Osel+S312'-combination-treatment conferred 100% protection (Fig.   5B ). When drugs were given at the middle & late phase (Fig. 5C) , single Osel-treatment wholly lost its antiviral effect with no surviving. However, S312-treatment could provide 50% protection, and drug combination reached to 100% protection. When drugs were given at severe late phase of disease that mice were starting dying (Fig. 5D) , neither single treatment of Osel nor S312 can rescue the mice from death but combined treatment still conferred to 25% survival. To really show the advantage of S312 in treating severe disease, we additionally treated the mice a bit early before dying around 80% of initial weights (D6-D14) with a more optimal dose of S312 (5mg/kg). The data in Fig. 5E showed that S312 rescued 50% of mice from severe body-weight losses, and combined treatment coffered additional 25% survival. These results once again highlight that S312 has remarkable advantages over Osel to treat severe diseases at the late phase, and its therapeutic effectiveness could even be improved when S312 was combined with DAA drug.

It is known that severe acute infections including influenza and COVID-19 always induce pathogenic immunity as cytokine/chemokine storms. Leflunomide and Teriflunomide are already clinically used in autoimmune disease to inhibit pathogenic cytokines and chemokines. We, therefore, suspect that DHODHi should also be anticytokine-storm in viral infectious disease. BALF from either Osel-or 'S312+Osel'treated mice were collected at D14 in an independently repeated experiment with a lower infection dose. A parallel body weights excluded differences in virus load (Supplementary Fig. 4A) . The data in Supplementary Fig. 4B showed that the pathogenic inflammatory cytokines in 'S312+Osel'-treated group was largely reduced as compared to Osel-treated mouse in the levels of IL6, MCP-1, IL5, KC/GRO(CXCL1), IL2, IFN-γ, IP-10, IL9, TNF-α, GM-CSF, EPO, IL12p70, MIP3α and IL17A/F (listed in the order of reduce significance).

The results in all provide striking information that DHODH inhibitors are effective in infected animals not only by inhibiting virus replication (Shown in Fig. 3 and Fig. 4) but also by eliminating excessive cytokine/chemokine storm. Usage of DHODHi could finally benefit to advanced disease in late infection. In this study, we applied DHODH inhibitors including a computer-aided designed compound S312 into viral infectious disease. We found that direct-targeting DHODHi are broad-spectrum antiviral both in cell culture and in vivo. The candidate S312 had further advantage to be used in infected animals with low toxicity and high efficiency.

Moreover, S312 can rescue severe influenza infection by limiting inflammatory cytokine storm in vivo.

DHODH is a rate-limiting enzyme catalyzing the fourth step in pyrimidine de novo synthesis. It catalyzes the dehydrogenation of dihydroorotate (DHO) to orotic acid (ORO) to finally generate Uridine (U) and Cytosine (C) to supply nucleotide resources in a cell. Under normal conditions, nucleotides are supplied via both de novo biosynthesis and salvage pathways, the latter of which is a way of recycling pre-existing nucleotides from food or other nutrition. However, in virus-infected cells, a large intracellular nucleotide pool is demanded by rapid viral replication. It is therefore reasonable that de novo nucleotides biosynthesis rather than salvage pathway is more critical for virus replication. Our data indeed show that virus replication is largely restricted when the DHODH gene was knocked off even with a complete culture medium. By contrast, cell growth was not affected by lacking DHODH at all, indicating that de novo nucleotides biosynthesis is not indispensable in normal cell growth without infection at least for days. More interestingly, we notice that compared with DNA viruses, RNA viruses need unique UMP but not TMP in their genomes. UMP is the particular nucleoside produced by DHODH, which means RNA viruses might be more sensitive to DHODH activity. SARS-CoV-2, for instance, has around 32% of UMP in its genome explaining why DHODHi are effective and superior to SARS-CoV-2.

Nevertheless, the comparison between different viruses is worth to be studied in the future.

Although several DHODHi have been documented to be antiviral by high-throughput screening [34] [35] [36] [37] . Most of these compounds are still at cell culture level with unknown in vivo efficacy. Therefore, the development of broad-spectrum antiviral agents targeting DHODH is still an exciting avenue in antiviral research. S312 and S416 present more potent inhibition and favorable pharmacokinetic profiles, moreover, the half-lives of S312 and S416 (8.20 and 9.12 h, respectively) are much shorter and more appropriate than that of Teriflunomide, indicating that they may have less possibility to bring toxic side effects from drug accumulation in the body. Strikingly, S312 showed active effects in vivo in lethal dose infection of influenza A viruses not only when used from the beginning of infection but also in the late phase when DAA drug is not responding anymore. Another surprise is the high SI value of S416 to against Zika (SI=1087.62), Ebola (SI=4746.11), and the current SARS-CoV-2 (SI>5882). These data interpreted that S416 is highly promising to develop further as it should be to S312. The extremely high SI of S416 may be due to its high binding affinity and favorable occupation of the ubiquinone-binding site of DHODH with faster-associating characteristics (kon = 1.76×10 6 M -1 s -1 ) and slower dissociating binding characteristic (koff=2.97×10 -3 s -1 ), which will reduce the possibility of off-target in vivo.

Acute viral infections usually cause severe complications associated with hyper induction of pro-inflammatory cytokines, which is also known as "cytokine storm" firstly named in severe influenza disease 38, 39 . Several studies showed that lethal SARS patients expressed high serum levels of pro-inflammatory cytokines (IFN-γ, IL-1, IL-6, IL-12, and TGFβ) and chemokines (CCL2, CXCL10, CXCL9, and IL-8) compared to uncomplicated SARS patients [40] [41] [42] [43] . Similarly, in severe COVID-19 cases, ICU patients had higher plasma levels of IL-2, IL-7, IL-10, GSCF, MCP1, MIP1A, and TNFα compared to non-ICU patients 44 . Moreover, A clinical study of 123 patients with COVID-19 showed that the percentage of patients with IL-6 above normal is higher in severe group 45 . In terms of treatment, immunomodulatory agents can reduce mortality and organ injury of severe influenza. However, these immunomodulatory are mostly non-specific to viral infection but rather a systemic regulation, such as corticosteroid, intravenous immunoglobulin (IVIG) or angiotensin receptor blockers [46] [47] [48] [49] . Leflunomide and its active metabolite Teriflunomide have been approved for clinical treatment for excessive inflammatory diseases such as rheumatoid arthritis and multiple sclerosis 50 .

Our data once again proved that DHODHi could further reduce cytokine storm than DAA drugs when using influenza-A-virus infected animal as a model. We believe that a similar immune-regulating role of DHODHi will exist in COVID-19 patients. Thus, by targeting DHODH, the single key enzyme in viral genome replication and immuneregulation, a dual-action of DHODH can be realized in fighting against a broad spectrum of viruses and the corresponding pathogenic-inflammation in severe infections. We hope our study may quickly and finally benefit the patients now suffering from severe COVID-19 and other infectious diseases caused by emerging and reemerging viruses. 

ITC measurements were carried out at 25 °C on a MicroCal iTC200 (GE Healthcare).

For the titration of an inhibitor to DHODH, both were diluted using the buffer (20 mM HEPES, pH8.0, 150 mM KCl, 10% Glycerol and 10% DMSO). The concentration of DHODH in the cell was 20 µM, and the concentration of inhibitor in the syringe was 150 µM for S312 or 100 µM for S416. All titration experiments were performed by adding the inhibitor in steps of 2 µL. The data were analyzed using Microcal origin software by fitting to a one-site binding model.

Surface plasmon resonance experiments were performed with a BIAcore T200 (GE Healthcare) according to our previous work 53 All drug concentrations were performed at least three replicates. Data were processed with Graphpad prism software to calculate EC50 and CC50 values of the compounds.

Ebolavirus must be operated in the BSL-4 laboratory. To reduce the biological safety risks, the Ebolavirus replicon system was chosen for antiviral efficacy assay 54 

To detect viral protein expression in Vero E6 cells, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then incubated with the primary antibody (a polyclonal antibody against the NP of a bat SARS-related CoV) after blocking, followed by incubation with the secondary antibody (Alexa 488-labeled goat anti-rabbit, Abcam).

To 

The 293T cells were seeded into 24-well plates at 1×10 5 respectively. Diluted compounds were given by intraperitoneal (i.p) injection once a day. The drug treatment was initiated on days 0, 3, 5, 6, 7 post-infection respectively and continued for several days. Animal weight and survival were monitored daily, and mice were euthanized until the end of the experiment or when body-weight lost more than 25%.

The protein structure data has been uploaded to the Protein Data Bank with accession number 6M2B. (2.5, 5, 10mg/kg), Oseltamivir (20mg/kg) and S312+Oseltamivir (10mg/kg+20mg/kg) once per day from D1-D14 respectively. The body weight and survival were monitored for 14 days or until body weight reduced to 75% (n = 4 mice per group). (C) Mice were inoculated intranasally with 600 PFU of A/SC/09 (H1N1) and then i.p. with S312 (10mg/kg), Oseltamivir (20mg/kg) and S312+Oseltamivir (10mg/kg+20mg/kg) once per day from D1 to D14. The body weight and survival were monitored until 14 days post-infection or when the bodyweight reduced to 75%. The dotted line indicates endpoint for mortality (75% of initial weight). The body weights are present as the mean percentage of the initial weight ±SD of 4-5 mice per group and survival curve were shown. Tables   Table1   Table 2 Fig. 1. Discovery of novel DHODH inhibitors and their anti-influenza-A-virus activities.

Broad-spectrum antiviral activity of DHODH inhibitors. 

A"IV to "Z"IKV: Attacks from Emerging and Re-emerging Pathogens

Expanding the activity spectrum of antiviral agents

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Replication and/or Transcription

New low-viscosity overlay medium for viral plaque assays

This work was supported in part by the National Key 

/-) cells. (E) The 293T cells were co-transfected with the influenza virus minigenome plasmid system (PB1, PB2, PA, NP, pPoⅡ-NP-luc, and pRLSV40). After 12 h.p.i., cells were treated with 2-fold serial dilutions of Oseltamivir, S312, S416, and Brequinar respectively. The luciferase activities were measured 24 h of post-treatment. (F) Effects of nucleotides addition on the antiviral efficacies of S312 and S416. The 10-fold IC50 of S312 (24 μM) or S416 (0.6 μM) and 50μM four nucleotides (Adenosine, Uridine, Cytidine, Guanosine) were added at the same time on 293T cells. After 24 h of treatment, the luciferase activities were measured. (G and H) Effects of addition of dihydroorotate (DHO) or Orotic acid (ORO) on the antiviral efficacies of S312 and S416. The luciferase activities were detected as above after treating with indicated concentrations of DHO or ORO. All results are presented as a mean of three replicates ± SD. Statistical analysis, two-way ANOVA for B, C, and D. One-way ANOVA for E, F, G and H. NS, p >0.05; *, p <0.05; **, p <0.01; ***, p <0.001. D7-11 (D) . Another groups of S312 (5mg/kg) or S312+Oseltamivir (5mg/kg+20mg/kg) were given i.p. once per day from D6 to D13 in E. The green bars indicate the period of drug administration. The body weight and survival were monitored until 14 days post-infection or when the bodyweight reduced to 75%, respectively (n =4-5 mice per group). The dotted line indicates endpoint for mortality (75% of initial weight). The body weights are present as the mean percentage of the initial weight ±SD of 4-5 mice per group and survival curve were shown.