Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity Against Mammarenaviruses in Vitro

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Article Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity against Mammarenaviruses In Vitro

Yu-Jin Kim 1, Beatrice Cubitt 1 , Yingyun Cai 2 , Jens H. Kuhn 2 , Daniel Vitt 3, Hella Kohlhof 3 and Juan C. de la Torre 1,* 1 Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; [email protected] (Y.-J.K.); [email protected] (B.C.) 2 Integrated Research Facility at Fort Detrick (IRF-Frederick), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), B-8200 Research Plaza, Fort Detrick, MD 21702, USA; [email protected] (Y.C.); [email protected] (J.H.K.) 3 Immunic Therapeutics, New York City, NY 10036, USA; [email protected] (D.V.); [email protected] (H.K.) * Correspondence: [email protected]

Received: 27 June 2020; Accepted: 24 July 2020; Published: 29 July 2020

Abstract: Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently,no FDA-licensedmammarenavirusvaccines areavailable, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe sideeffects. Dihydroorotatedehydrogenase(DHODH)inhibitors, whichblockdenovopyrimidinebiosynthesis, have antiviral activity against viruses from different families, including Arenaviridae, the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC50] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

Keywords: mammarenavirus; DHODH; pyrimidine biosynthesis; interferon; antiviral

1. Introduction Mammarenaviruses (Bunyavirales: Arenaviridae: Mammarenavirus) cause chronic asymptomatic infections in their natural reservoir hosts, which are predominantly muroid rodents. Some of these viruses are public health threats because they can be transmitted to humans through aerosol transmission or by direct contact of abraded skin with contaminated material [1]. Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentinian Pampas, can cause severe disease in humans (Lassa fever and Argentinian hemorrhagic fever, respectively), and pose important public health problems in their endemic regions. LASV is highly prevalent in Western Africa, where the virus infects hundreds of thousands of individuals yearly resulting in a high number of Lassa fever cases that are associated with high morbidity and a high case fatality rate among hospitalized patients [2]. Notably, increased travel has resulted in imported cases of Lassa fever into non-endemic metropolitan regions, including the United States (U.S.) [3,4]. Evidence indicates that LASV-endemic regions are expanding [5], and the identification of novel zoonotic mammarenaviruses, such as Lujo virus (LUJV), responsible for a cluster of severe infections in humans in Southern Africa in 2009 [6], has raised

Viruses 2020, 12, 821; doi:10.3390/v12080821 www.mdpi.com/journal/viruses Viruses 2020, 12, 821 2 of 19 concerns about the emergence of novel arenaviruse hemorrhagic fevers. In addition to JUNV, several other New World mammarenaviruses are responsible for small recurrent outbreaks of high-consequence diseases in humans in South America, including Chapare virus (CHAPV) and Machupo virus (MACV) in Bolivia, Guanarito virus (GTOV) in Venezuela, and Sabiá virus (SBAV) in Brazil [7–11]. Moreover, mounting evidence suggests that the widely distributed lymphocytic choriomeningitis virus (LCMV) may be a neglected human pathogen, contributing to cerebral abnormalities associated with congenital infections [12–15], and LCMV poses a particular threat to immunocompromised individuals, as illustrated by fatal cases of LCMV infection associated with organ transplants [16]. The JUNV Candid#1 strain is largely considered a safe and effective live attenuated vaccine to prevent cases of Argentinian hemorrhagic fever [17] and was licensed in 2006 by the regulatory agency of Argentina for use exclusively in Argentina. In the U.S., Candid#1 is considered an investigational new drug (IND). No FDA-licensed mammarenavirus vaccines are available, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects [18–20]. Several small molecules, including the broad-spectrum mammarenavirus RNA-directed RNA polymerase inhibitor favipiravir and the mammarenavirus glycoprotein GP2-mediated fusion inhibitor ST-193, have promising anti-mammarenaviral effects in various animal models of mammarenavirus-induced human diseases [21–24]. Furthermore, recent drug repurposing strategies have identified different types of anti-mammarenaviral compounds, targeting distinct host cell factors and pathways, including de novo pyrimidine biosynthesis [25]. Inhibitors of de novo pyrimidine biosynthesis have broad-spectrum antiviral activity [25–29]. Dihydroorotate dehydrogenase (DHODH) is a key enzyme involved in de novo pyrimidine biosynthesis, converting dihydroorotate (DHO) into orotate, and DHODH has been validated as one of the targets of pyrimidine biosynthesis inhibitors exhibiting antiviral activity [30]. We investigated the antiviral activity of a series of novel DHODH inhibitors (i.e., Compound (Cmp) 1, Cmp 2, Cmp 3, Cmp 4, and Cmp 5) against diverse mammarenaviruses. Cmp 1, Cmp 2, Cmp 3, and Cmp 5 are new molecular entities with undisclosed structures. In addition to their inhibitory effect on DHODH, these compounds were also active against retinoic acid-related orphan receptor C (RORC, also known as RORg) with half-maximal inhibitory concentration (IC50) values ranging from 5 to 50 nM. Cmp 4 is IM90838 (IMU-838), the Ca2+ salt of vidofludimus (Figure 8A), which is currently under testing in phase 2b clinical trials against multiple sclerosis, ulcerative colitis, and primary sclerosing cholangitis. Both vidofludimus and Cmp 4 depend on the same active ingredient in blood (vidofludimus) for their mechanism of action, toxicology, and pharmacology [31]. All DHODH inhibitors tested in this study had an IC50 lower than 1 µM for human DHODH (hDHODH) and were potently active against LCMV (half-maximal eﬀective concentrations [EC50] in the low nanomolar range, selectivity index [SI] > 1000). Importantly, these inhibitors were also active against LASV and JUNV. The DHODH inhibitors did not aﬀect virus cell entry or budding, but rather targeted replication and transcription of the mammarenavirus genome, i.e., the biosynthetic processes directed by the mammarenavirus ribonucleoprotein complex (vRNP). The antiviral activity of some DHODH inhibitors has been linked to the activation of the type I interferon (IFN-I) system and the expression of IFN-stimulated genes (ISGs) [32]. However, the novel DHODH inhibitors exhibited potent IFN-I-independent anti-mammarenaviral activity. Non-limiting exogenous uridine supply promoted the pyrimidine salvage pathway and restored normal levels of virus multiplication in the presence of DHODH inhibitors, which could be partially prevented by inhibiting uridine-cytidine kinase 2 (UCK2), a key enzyme of the pyrimidine salvage pathway. The inhibition of DHODH in combination with UCK2 inhibitors might open a new avenue for combination therapy to target rapidly replicating RNA viruses, including human pathogenic mammarenaviruses. Viruses 2020, 12, 821 3 of 19

2. Materials and Methods

2.1. Cells and Viruses Grivet (Chlorocebus aethiops) Vero E6 (ATCC CRL-1586) and Homo sapiens A549 (ATCC CCL-185) and 293T (ATCC CRL-3216) cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (ThermoFisher Scientific, Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM of l-glutamine, 100 µg/mL of streptomycin, and 100 U/mL of penicillin. The tri-segmented form of the live attenuated vaccine Candid#1 strain of JUNV expressing green fluorescent protein (GFP, r3Can/GFP) [33], recombinant LCMV expressing Zoanthus sp. green fluorescent protein (ZsG) fused to nucleoprotein (NP) via a P2A ribosomal skipping sequence (rLCMV/ZsG-P2A-NP, referred to as rLCMV/ZsG) [34], a single-cycle infectious rLCMV expressing ZsG (rLCMV∆GPC/ZsG-P2A-NP, here referred to as rLCMV∆GPC/ZsG) [34], an immunosuppressive strain of LCMV, clone 13 (CL-13) expressing ZsG (rCL-13/ZsG), and an LASV-expressing GFP (rLASV/GFP) [35] have been described previously.

2.2. Compounds The in vitro inhibition of hDHODH was measured using an N-terminally truncated recombinant hDHODH enzyme as described previously [36]. Briefly, the hDHODH concentration was adjusted in a way that an average slope of approximately 0.2 AU/min served as the positive control (e.g., without inhibitor). The standard assay mixture contained 60 µM 2,6-dichloroindophenol (Sigma, St. Louis, MO, USA), 50 µM decylubiquinone (Sigma), and 100 µM dihydroorotate (Sigma). The hDHODH enzyme with or without at least six different concentrations of the compounds was added, and measurements were performed in 50 mM TrisHCl, 150 mM potassium chloride (KCl) (Merck), and 0.1% Triton X-100 (Sigma) at pH 8.0 and at 30 ◦C[37]. The reaction was started by adding dihydroorotate and measuring the absorption at 600 nm for 2 min. For the determination of the IC50 values, each data point was recorded in triplicate. DHODH inhibitors Cmp 1, Cmp 2, Cmp 3, Cmp 4, and Cmp 5 were provided by Immunic Therapeutics. Brequinar, uridine, 20-deoxyuridine, cytidine, and 20-deoxycytidine were purchased from Sigma. UCK2 inhibitor, UCK2-IN-20874830 was obtained from ChemDiv (San Diego, CA, USA).

2.3. Cell Cytotoxicity Assay and Half-Maximal Cytotoxic Concentration (CC50) Determination

Cell viability was assessed using CellTiter 96 AQueous One Solution reagent and cell proliferation assay (Promega, Madison). This method determined the number of viable cells based on conversion of formazan product from 3-(4,5-dimethylthazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolim by nicotinamide adenine dinucleotide phosphate (NADPH) or reduced NADPH (NADH) generated in living cells. A549 cells were plated on 96-well clear-bottom plates (2.0 104 cells/well). × Serial dilutions (three-fold) of each compound were added to cells and, 48 h after drug treatment, the viability reagent was added and incubated for 15 min (37 ◦C and 5% carbon dioxide [CO2]). Absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax ABS Plus, Molecular Devices, Sunnyvale, CA, USA). The resulting optical densities were normalized with dimethylsulfoxide (DMSO); the vehicle control group was adjusted to 100%. CC50 was determined using Prism (GraphPad, San Diego, CA, USA).

2.4. Viral Growth Kinetics and EC50 Determination For growth kinetics, virus was added to cells (200 µL/well in a 24-well plate) at the indicated multiplicity of infection (MOI). After 90 min of adsorption, virus inocula were removed, cells were washed twice with DMEM and 2% FBS, and fresh media containing the indicated compounds and concentrations were added. At the indicated hours post-infection (pi), tissue culture supernatants (TCSs) were collected and viral titers were determined using a focus-forming assay (FFA) [38]. For EC50 determination, cells were plated on 96-well clear-bottom black plates (2.0 104 cells/well) and incubated × for 20 h at 37 ◦C and 5% CO2. Cells were pre-treated for 2 h before infection with three-fold serial Viruses 2020, 12, 821 4 of 19 dilutions of each compound. Cells were infected (MOI = 0.01) with rLCMV/ZsG-P2A-NP in the presence of compounds. At 48 h pi, cells were fixed with 4% paraformaldehyde, nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI), and ZsG expression was determined by fluorescence using a fluorescent plate reader (Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). Mean relative fluorescence units were normalized with the vehicle control group (DMSO), which was adjusted to 100%. ZsG expression was normalized for total cell protein in lysate (Pierce BCA Protein Assay Kit, ThermoFisher Scientific). EC50 was determined using Prism. SIs for hit compounds were determined using the ratio CC50:EC50.

2.5. Time-of-Addition Assay A549 cells were seeded (2 104 cells/well) on 96-well clear-bottom black plates and incubated for × 20 h at 37 ◦C and 5% CO2. Cells were treated with each compound (5 µM) at either 2 h prior to or 2 h following infection. Cells were infected (MOI = 0.5) with single-cycle infectious rLCMV∆GPC/ZsG to prevent the confounding factors caused by multiple rounds of infection. At 48 h pi, cells were fixed with 4% paraformaldehyde and nuclei were stained with DAPI. ZsG expression levels were determined using a fluorescent plate reader (Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). Mean relative fluorescence units were normalized to the vehicle control (DMSO) group, which was adjusted to 100%.

2.6. LCMV Minigenome (MG) Assay The LCMV MG assay was performed as described [39]. Briefly, 293T cells were cultured on poly-l-lysine-coated 12-well plates (4.5 105 cells/well). Cells were transfected using lipofectamine × 2000 (2.5 µL/µg of DNA) (ThermoFisher Scientific), with Pol II-based expression plasmids (pCAGGS) for T7 RNA polymerase (pC-T7, 0.5 µg), LCMV (NP) (pC-NP, 0.3 µg), and LCMV RNA-directed RNA polymerase (L) (pC-L, 0.3 µg), together with a plasmid directing intracellular synthesis of an LCMV MG expressing the chloramphenicol acetyl transferase (CAT) reporter gene (pT7-MG/CAT, 0.5 µg). After 5 h, the transfection mixture was replaced with fresh media and incubated for 72 h at 37 ◦C and 5% CO2. At 72 h post-transfection, whole-cell lysates (WCLs) were harvested to determine expression levels of CAT using a CAT ELISA kit (Roche, Sydney, Australia). Briefly, WCLs were prepared with 0.5 mL of lysis buffer, and 10 µL of each sample were used for the reaction. Diluted samples were added onto CAT-ELISA plates and incubated for 1 h at 37 ◦C. After incubation with samples, plates were washed, and primary antibody (anti-CAT-digoxigenin) and secondary antibody (anti-CAT-digoxigenin-peroxidase) were added sequentially, followed by the substrate. After 20 min, absorbance was measured using the ELISA reader at 405 nm for samples and 490 nm for the reference.

2.7. Budding Assay The luciferase-based budding assay was performed as described [40]. Briefly, 293T cells were seeded on poly-L-lysine-coated 12-well plates (3.5 105 cells/well). After overnight incubation, 2 µg × of DNA of either pC-LASV-Z-Gaussia luciferase (GLuc) or pC-LASV-mutant Z[G2A]-GLuc were transfected using Lipofectamine 2000 (2.5 µL/µg of DNA). After 5 h, transfection mixtures were replaced with fresh media containing the indicated hit compounds. After 48 h, TCSs containing virion-like particles (VLPs) were harvested and clarified by low-speed centrifugation to remove cell debris. Aliquots (20 µL each) from TCS samples were added to 96-well black plates (VWR, West Chester, PA, USA), and 50 µL of SteadyGlo luciferase reagent (Promega) were added to each well. WCLs from the same samples were processed to determine cell-associated activity of GLuc. The luminescence signal was measured using the Berthold Centro LB 960 luminometer (Berthold Technologies, Oak Ridge, TN, USA). The activity (relative light units) of GLuc in TCSs and WCLs was used as a surrogate of Z expression. Budding efficiency was defined as the ratio ZVLP:(ZVLP+ZWCL). Viruses 2020, 12, 821 5 of 19

2.8. Virus Titration Virus titers were determined by a focus-forming assay [38]. Serial dilutions of samples (10-fold) were used to infect Vero E6 cell monolayers in 96-well plates (2 104 cells/well). At 20 h pi, cells were fixed × with 4% paraformaldehyde in phosphate-buffered saline. The foci of cells infected with rLCMV/ZsG, rLCMV∆GPC/ZsG, or rLASV/GFP were determined by the epifluorescence of fluorescent reporter gene expression. The foci of cells infected with wild-type LCMV were identified by rat monoclonal antibody VL4 against NP (Bio X Cell, West Lebanon, NH, USA) conjugated with Alexa Fluor 488.

2.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR) A549 cells were infected with rLCMV/ZsG-P2A-NP in the presence (5 µM) of the indicated compounds or vehicle control (DMSO). Total cellular RNA was isolated using TriReagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (1~3.5 µg) was reverse- transcribed to cDNA using a SuperScript IV First-Strand Synthesis System (ThermoFisher Scientiﬁc). Target sequences were ampliﬁed by PCR with primer sets listed in Section 2.10. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.

2.10. Primers LCMV NP-speciﬁc primers •

forward: 50-ATGCAGTCCATGAGTGCACAGT-30 reverse: 50-GGTGAAGGATGGCCATACATAG-30 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-speciﬁc primers •

forward: 50-TGACATCAAGAAGGTGGTGAAGCAG-30 reverse: 50-ATTGTCATACCAGGAAATGAGCTTGAC-30 IFNB-speciﬁc primers •

forward: 50-TCAGTGTCAGAAGCTCCTGT-30 reverse: 50-ACAGCATCTGCTGGTTGAAG-30 Interferon-stimulated gene 15 (ISG15)-speciﬁc primers •

forward: 50-TGAGAGGCAGCGAACTCATCT-30 reverse: 50-AAGGTCAGCCAGAACAGGTCGT-30 Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1)-speciﬁc primers •

forward: 50- GCCTTGCTGAAGTGTGGAGGAA-30 reverse: 50-GCTTTTCTCTGTTCTGCCCTCT-30 DExD/H-box helicase 58 (DDX58)-speciﬁc primers •

forward: 50-CACCTCAGTTGCTGATGAAGGC-30 reverse: 50- CGGGCACAGAATATCTTTGCTC-30 Signal transducer and activator of transcription 1 (STAT1)-speciﬁc primers •

forward: 50-ATGGCAGTCTGGCGGCTGAATT-30 reverse: 50-GATGCACCCATCATTCCAGAGA-30 Interferon alpha inducible protein 27 (IFI27)-speciﬁc primers •

forward: 50-TAAGACGGTGAGGTCAGCTTCA-30 reverse: 50-ACCCAATGGAGCCCAGGATGAA-30 Viruses 2020, 12, 821 6 of 19

Interferon regulatory factor 1 (IRF1)-speciﬁc primers •

forward: 50-GAGGAGGTGAAAGACCAGAGCA-30 reverse: 50-CCAGGTTCATTGAGTAGGTACC-30 IRF9-speciﬁc primers •

forward: 50-TACTCACTGCTGCTCACCTTCA-30 reverse: 50-AGTCTGCTCCAGCAAGTATCGG-30 IFIT2-speciﬁc primers •

forward: 50-GGAGCAGATTCTGAGGCTTTGC-30 reverse: 50-GCAGGACTAACCTCTATGGGAT-30 IRF7-speciﬁc primers •

forward: 50-ACCATCTGCTGACAGCGTCAT-30 reverse: 50-GCTGCTATCCAGGGAAGACACA-30 C-X-C motif chemokine 10 (CXCL10)-speciﬁc primers •

forward: 50-GGTGAGAAGAGATGTCTGAATCC-30 reverse: 50-GGCAGTGGAAGTCCATGAAGTA-30 Interferon-induced GTP-binding protein Mx1 (MX1)-speciﬁc primers •

forward: 50-GGCTGTTTACCAGACTCCGACA-30 reverse: 50-GATCTCCTCCATGGAAGAGTCT-30

2.11. Animal Studies Adult (6-week-old) C57BL/6 inbred laboratory mice (Scripps Research breeding colony) were inoculated intravenously (IV) with LCMV CL-13 (2 106 FFU) and treated with Cmp 4 (150 mg/kg/d) × or vehicle control, administered orally. Treatment was administered daily from Day 1 to Day 17 pi. Mice were monitored daily for the development of clinical signs, weight loss, and survival. All animal experiments were conducted under Protocol 09-0137-4, approved by The Scripps Research Institute Institutional Animal Care and Use Committee (IACUC).

2.12. Biosafety All experiments involving the use of rLASV/GFP were performed under maximum containment, biosafety level 4 (BSL-4), conditions in the BSL-4 suites of the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility at Fort Detrick (IRF-Frederick) [41] following approved standard operating procedures [42,43]. All other experiments were performed under BSL-2 conditions.

3. Results

3.1. Eﬀect of DHODH Inhibitors on Mammarenavirus Multiplication We investigated the dose-dependent effects of five novel DHODH inhibitors (i.e., Cmp 1, Cmp 2, Cmp 3, Cmp 4, and Cmp 5) on rLCMV/ZsG multiplication, cell viability, and in vitro hDHODH inhibition (Figure1 and Figure 8). A549 cells were treated with three-fold serial dilutions of each compound. At 2 h post-treatment, cells were infected with rLCMV/ZsG (MOI = 0.01) and at 48 h pi, ZsG expression levels were determined, as described in the Materials and Methods section. Normalized ZsG expression levels were used to determine EC50 values. Cell viability was determined by formazan production, and values Viruses 2020, 12, 821 7 of 19

were normalized for CC50 calculation. Brequinar, a known DHODH inhibitor [44–46], was used as a control (Figure1F). All tested DHODH inhibitors also strongly inhibited rLASV /GFP and rCan/GFP multiplicationViruses 2020, 12, withx FOR high PEER SIREVIEW values (Figure1G). 7 of 19

A Compound 1 B Compound 2 C Compound 3

150 120 150 120 150 120 100 100 100 100 100 100 80 80 80 50 60 50 60 50 60 40 40 40 0 0 0 -8 -7 -6 -5 -4 20 -8 -7 -6 -5 -4 20 -8 -7 -6 -5 -4 20 Cell viability (%) Cellviability (%) Cellviability

-50 0 -50 0 (%) Cell viability Concentration (log M) Concentration (log M) -50 0 ZsG expression (%) expression ZsG (%) expression ZsG

ZsG expression (%) Concentration (log M)

D Compound 4 E Compound 5 F Brequinar

150 120 150 120 150 120 100 100 100 100 100 100 80 80 80 50 60 50 60 50 60 40 40 40 0 0 0 -8 -7 -6 -5 -4 20 -8 -7 -6 -5 -4 20 -8 -7 -6 -5 -4 20 Cell viability (%) Cell viability -50 0 -50 0 Cell viability (%) -50 0 Cell viability (%)

ZsG expression (%) expression ZsG Concentration (log M) Concentration (log M) Concentration (log M) ZsG expression (%) ZsG expression (%) expression ZsG

CC50 (∝M) rLCMV (A549 cell) rLASV (A549 cell) r3Can (Vero cell) Compound A549 cell Vero cell EC50 (∝M) SI EC50 (∝M) SI EC50 (∝M) SI

Compound 1 69.66 37.40 <0.008 >8,707.50 0.017 4,160.86 <0.008 >4,675.00 Compound 2 166.20 46.42 <0.008 >20,775.00 0.007 22,237.45 <0.008 >5,802.50 Compound 3 133.00 18.05 <0.008 >16,625.00 0.001 89,620.83 <0.008 >2,256.25 Compound 4 54.41 5.92 1.284 42.38 3.016 18.04 1.681 3.52 Compound 5 116.10 52.21 <0.008 >14,512.50 0.007 15,969.78 <0.008 >6,526.25 Brequinar (Control) 79.04 47.21 0.041 1,921.71 0.063 1,249.65 0.040 1,180.25 FigureFigure 1. E1.ffect Effect of dihydroorotate of dihydroorotate dehydrogenase dehydrogenas (DHODH)e (DHODH) inhibitors oninhibitors lymphocytic on choriomeningitislymphocytic viruschoriomeningitis (LCMV) multiplication. virus (LCMV) (A– Fmultiplication.)Effect on LCMV (A– propagationF) Effect on LCMV (green line)propagation and cell (green viability line) (blue and line) ofcell the viability indicated (blue compounds. line) of the A549 indicated cells werecompounds. treated A549 with serialcells were dilutions treated of with each serial compound dilutions starting of 2 heach prior compound to infection starting (multiplicity 2 h prior of to infection infection [MOI] (multiplicity= 0.01) withof infection clone 13 [MOI] (CL-13) = 0.01) expressing with cloneZoanthus 13 sp.(CL-13) green fluorescentexpressing proteinZoanthus (ZsG) sp. (rCL-13green fluorescent/ZsG). Levels protein of ZsG (ZsG) expression (rCL-13/ZsG). were determined Levels of at ZsG 48 h pi (fourexpression replicates). were Cell determined viability at (six 48 replicates)h pi (four replicates). was evaluated Cell viability using the (six CellTiter-Glo replicates) was assay evaluated after 48 h ofusing treatment. the CellTiter-Glo Data were assay normalized after 48 withh of treatment. respect to Data the were vehicle normalized control group. with respect Error to bars the vehicle represent control group. Error bars represent mean +/- SEM. (G) CC50, EC50, and selectivity index (SI = CC50/EC50) mean +/ SEM. (G) CC50, EC50, and selectivity index (SI = CC50/EC50) values for recombinant LCMV values− for recombinant LCMV (rLCMV), Lassa virus (rLASV), and r3Can. (rLCMV), Lassa virus (rLASV), and r3Can.

To further characterize the antiviral activity of the newly tested DHODH inhibitors, we selected Cmp 1 and Cmp 3 as representative compounds with medium (8.7 103) and high (1.6 104) SI × × values, respectively. These compounds were examined for their eﬀects on virus cell-to-cell propagation, viral RNA synthesis, and the production of infectious progeny over time (Figure2). A549 cells were infected with rLCMV/ZsG (MOI = 0.01) in the presence of each compound (5 µM). At the indicated time points, the number of virus-infected cells (Figure2A), concentrations of viral RNA (Figure2B), and the production of infectious viral progeny (Figure2C) were determined. Both Cmp 1 and Cmp 3 induced strong inhibition of the cell-to-cell propagation of rLCMV/ZsG that correlated with reduced levels of viral RNA synthesis and production of infectious progeny.

Viruses 2020, 12, 821 8 of 19 Viruses 2020, 12, x FOR PEER REVIEW 8 of 19

FigureFigure 2. Effect 2. Effect of selected of selected DHODH DHODH inhibitors inhibitors on LCMV on LCMVmultiplication. multiplication. A549 cells A549 were cells infected were (MOI infected = 0.01)(MOI with= rCL-13/ZsG0.01) with in rCL-13 the presence/ZsG in ofthe the presenceindicated ofDHODH the indicated inhibitors DHODH (5 µM) or inhibitors dimethylsulfoxide (5 µM) or (DMSO)dimethylsulfoxide vehicle control (DMSO) (VC). (A vehicle) At thecontrol indicated (VC). hour (A (h)) Atpost-infection the indicated (pi), hour numbers (h) post-infection of ZsG-positive (pi), cellsnumbers were determined of ZsG-positive by epifluorescence. cells were determined (B) At the indicated by epifluorescence. time point, total (B) Atcellular the indicated RNA was timepurified point, andtotal equal cellular amounts RNA (1 wasµg) were purified used and in reverse equal amounts transcription (1 µg) reactions, were used using in reverserandom transcription hexamer primers reactions, to generateusing randomcomplementary hexamer primersDNAs (cDNAs) to generate that complementary were used in DNAspolymerase (cDNAs) chain that reactions were used (PCRs) in polymerase with specificchain primers reactions to (PCRs) amplify with a segment specific of primers 644 bp to of amplify the viral a segmentnucleoprotein of 644 (NP) bp of gene. the viral PCR nucleoprotein products were (NP) resolvedgene. PCRby agarose products gel wereelectrophoresis resolved byand agarose visualized gel electrophoresisby ethidium bromide and visualized staining. ( byC) ethidiumAt the indicated bromide h pi,staining. tissue culture (C) At supernatants the indicated we hre pi, harvested, tissue culture and virus supernatants titers were were determined. harvested, Values and virusfor VC-treated titers were samplesdetermined. correspond Values to two for VC-treatedreplicates. Error samples bars correspond represent mean to two +/− replicates. standard error Error of bars the representmean (SEM). mean The+/ − numberstandard below error each of theband mean indicates (SEM). the The normalized number below fold each change band on indicates band intensity the normalized compared fold to change mock- on infectedband intensityand vehicle-treated compared control to mock-infected cells. and vehicle-treated control cells.

Viruses 2020, 12, 821 9 of 19 Viruses 2020, 12, x FOR PEER REVIEW 9 of 19

3.2. Broad-Spectrum Antiviral Effects Effects of DHODH Inhibitors We examinedexamined the antiviralthe antiviral activity activity of the DHODH of the inhibitors DHODH against inhibitors two other against mammarenaviruses, two other mammarenaviruses,LASV and JUNV (Figure LASV3). and A549 JUNV cells (Figure (top row) 3). A549 were cells exposed (top (MOIrow) were= 0.1) exposed to rLASV (MOI/GFP = 0.1) in the to presencerLASV/GFP of each in the compound presence (5of µeachM), compound and IFN-deficient (5 µM), Vero and E6IFN-deficient cells (bottom Vero row) E6 were cells exposed (bottom to row) the werehighly exposed attenuated to the r3Can highly/GFP attenuated (MOI = 0.5).r3Can/GFP All five (MOI novel = 0.5). DHODH All five inhibitors, novel DHODH as well inhibitors, as brequinar, as wellstrongly as brequinar, inhibited rLASVstrongly/GFP inhibited and r3Can rLASV/GFP/GFP propagation and r3Can/GFP (Figure propagation3). (Figure 3).

Figure 3.3. InhibitionInhibition of of Lassa Lassa virus virus (LASV) (LASV) and Junandín virusJunín (JUNV)virus (JUNV) multiplication multiplication by DHODH by inhibitors.DHODH A549inhibitors. cells wereA549 infectedcells were with infected rLASV with/GFP rLASV/GFP (MOI = 0.01) (MOI and = Vero 0.01) cells and were Vero infected cells were with infected r3Can /withGFP (MOIr3Can/GFP= 0.5) (MOI in the = presence 0.5) in the of thepresence indicated of the inhibitors indicated (5 µinhibitorsM). At 72 (5 h (forµM). rLASV) At 72 h or (for 96 hrLASV) (for r3Can) or 96 pi, h (forcells r3Can) were ﬁxed, pi, cells and thewere numbers fixed, and of green the numbers ﬂuorescent of proteingreen fluorescent (GFP) -positive protein cells (GFP) were determined-positive cells by

wereepifluorescence. determined Nuclei by epifluorescence. were visualized byNuclei 40,6-diamidino-2-phenylindole were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. (DAPI) staining. 3.3. Effect of DHODH Inhibitors on Different Steps of LCMV Lifecycle 3.3. EffectTo investigate of DHODH the Inhibitors mechanism on by Different which theseSteps novelof LCMV DHODH Lifecycle inhibitors exerted anti-mammarenavirus activity,To weinvestigate evaluated the the effectsmechanism of Cmp by 1, Cmpwhich 2, Cmpthese 3,novel Cmp 4,DHODH and Cmp inhibitors 5 on distinct exerted steps ofanti- the mammarenavirusLCMV lifecycle (Figure activity,4). Towe examine evaluated whether the effects DHODH of Cmp inhibitors 1, Cmp affected 2, Cmp virus 3, Cmp cell 4, entry, and Cmp a time-of- 5 on distinctaddition steps experiment of the LCMV was conducted, lifecycle (Figure in which 4). eachTo examine compound whether was addedDHODH 2 h inhibitors prior to infection affectedor virus 2 h picell (Figure entry,4 aA). time-of-addition For this experiment, experiment the single-cycle was conducted, infectious in rLCMVwhich each∆GPC compound/ZsG was usedwas added to prevent 2 h priorthe confounding to infection factors or introduced2 h pi (Figure by multiple 4A). rounds For this of infection. experiment, All five the DHODH single-cycle inhibitors infectious strongly rLCMVinhibited∆GPC/ZsG virus-mediated was ZsGused expression to prevent regardless the confounding of the time factors of addition, introduced indicating by multiple that virus rounds cell entry of wasinfection. not targeted All five by DHODH the tested inhibitors DHODH inhibitors.strongly inhibited F3406, aknown virus-mediated inhibitor of ZsG LCMV expression cell entry, regardless was used ofas thethe controltime of [ 47addition,]. To investigate indicating whether that virus the inhibition cell entry of viruswas not multiplication targeted by by the DHODH tested inhibitorsDHODH wasinhibitors. mediated F3406, by reduceda known viralinhibitor RNA of synthesis, LCMV cell we entry, examined was used the expression as the control of CAT [47]. from To investigate a reporter whethergene driven the ininhibition the LCMV of MGvirus (Figure multiplication4B). Normalized by DHODH CAT expressioninhibitors was significantlymediated by reduced reduced in viral the presenceRNA synthesis, of all tested we examined DHODH the inhibitors, expression suggesting of CAT that from the a blockadereporter ofgenede novodrivenpyrimidine in the LCMV synthesis MG (Figureby DHODH 4B). inhibitorsNormalized interfered CAT expression with viral RNAwas synthesissignificantly directed reduced by the in intracellularlythe presence reconstitutedof all tested DHODHLCMV vRNP. inhibitors, Finally, tosuggesting study the effectthat the of DHODHblockade inhibitors of de novo on virionpyrimidine budding, synthesis a process by directed DHODH by inhibitorsthe mammarenavirus interfered matrixwith viral protein RNA (Z) synthesis (Figure4C), directed 293T cells by were the intracellularly transfected with reconstituted plasmids expressing LCMV vRNP.LASV-Z-GLuc Finally, into thestudy presence the effect of the of DHODH indicated inhibi inhibitors.tors on At virion 48 h post-transfection, budding, a process GLuc directed activity by was the mammarenavirusdetermined in clarified matrix TCSs protein and WCLs (Z) as(Figure surrogates 4C), of 293T VLP-associated cells were (Z VLPtransfected) and intracellular with plasmids (ZWCL) expressingZ levels, respectively. LASV-Z-GLuc Cmp in 1 andthe presence 4 had a very of the modest, indicated but statisticallyinhibitors. At significant, 48 h post-transfection, effect on Z budding GLuc activityefficiency was as determineddetermined by in the clarified ratio ZVLP TCSs:(ZVLP and+Z WCLsWCL). as surrogates of VLP-associated (ZVLP) and intracellular (ZWCL) Z levels, respectively. Cmp 1 and 4 had a very modest, but statistically significant, effect on Z budding efficiency as determined by the ratio ZVLP:(ZVLP+ZWCL).

Viruses 2020, 12, 821 10 of 19 Viruses 2020, 12, x FOR PEER REVIEW 10 of 19

A 2h Pre-infection 2h Post-infection 150 ****

100

* 50 * **

ZsG expression (%) expression ZsG O 4 5 6 S d 3 d d rin 0 n n inar i M u 34 D o oun arv F pou p p ib m m Brequ R o Compound Compound1 C2 Com Co B

1.2 1.0 0.8 0.6 0.20 **** 0.15 0.10 Normalized 0.05 CAT expression CAT 0.00 1 2 4 5 ar L SO d d d o n n in irin M u und 3 un N D o ound arv p po po requ ib m mpou B R o o C Comp C Com Com C

1.2 1.0 * 0.8 ** 0.6 0.4 *** 0.2 Normalized 0.0 r

budding efficiency O 1 2 S d d d 4 d 5 2A n n M un un uina .G D o ou o ou q Z p p p m m Bre omp o om C C Compound 3C Co FigureFigure 4. 4.Effect Effect of of DHODH DHODH inhibitors inhibitors on on different different steps steps of of the the virus virus lifecycle. lifecycle. (A )(A Time-of-addition) Time-of-addition assay. A549assay. cells A549 were cells treated were treated with the with indicated the indicated compounds compounds (5 µM) (5 2µM) h before 2 h before or after or after infection infection (MOI (MOI= 0.5) with= 0.5) the with single-cycle the single-c infectiousycle infectious rLCMV∆ rLCMVGPC/ZsG.∆GPC/ZsG. At 48 h pi,At ZsG48 h expressionpi, ZsG expression levels were levels determined. were Valuesdetermined. were normalized Values were to vehiclenormalized control to cells.vehicle (B )control Effect ofcells. DHODH (B) Effect inhibitors of DHODH on LCMV inhibitors minigenome on (MG)-derivedLCMV minigenome reporter (MG)-derived gene expression. reporter 293T gene cells expression. were transfected 293T cells with were pC-T7, transfected pMG-chloramphenicol with pC-T7, acetyltransferasepMG-chloramphenicol (CAT), pC-NP, acetyltransferase and pC-L. At(CAT), 5 h after pC-N transfection,P, and pC-L. fresh At medium 5 h after containing transfection, the indicatedfresh compoundmedium containing (5 µM) was the added. indicated Forty-eight compound hours (5 later, µM) cell was lysates added. were Forty-eight prepared, hours and CAT later, expression cell lysates levels werewere determined prepared, byand CAT-ELISA. CAT expression Values levels were were normalized determined to the by vehicle CAT-ELISA. control group.Values Aswere a negative normalized control, anto empty the vehicle vector control was substituted group. As fora negative pC-L (No control, L). (C an) Effect empty of vector the indicated was substituted compounds for pC-L on Z-mediated (No L). budding.(C) Effect 293T of the cells indicated were transfectedcompounds withon Z-mediated pC-LASV-Z-GLuc. budding. 293T At 48 cells hpost-transfection, were transfected with Z budding pC- efficiencyLASV-Z-GLuc. (BE) was At determined 48 h post-transfection, by measuring Z budding GLuc activity efficiency associated (BE) was with determined virion-like by particles measuring (VLPs) GLuc activity associated with virion-like particles (VLPs) in tissue culture supernatants and in whole- in tissue culture supernatants and in whole-cell lysates (WCLs) (BE = GLucVLP/GLucVLP + GLucWCL). cell lysates (WCLs) (BE = GLucVLP/GLucVLP + GLucWCL). Budding-deficient mutant Z-G2A was Budding-deficient mutant Z-G2A was used as control. Values correspond to four independent replicates. Error bars represent mean +/ SEM. Statistical significance was calculated by analysis of variance (ANOVA) − (* p < 0.05, ** p < 0.002, *** p < 0.0002, and **** p < 0.00001). Viruses 2020, 12, x FOR PEER REVIEW 11 of 19

used as control. Values correspond to four independent replicates. Error bars represent mean +/− SEM. Statistical significance was calculated by analysis of variance (ANOVA) (* p < 0.05, ** p < 0.002, *** p < 0.0002, and **** p < 0.00001). Viruses 2020, 12, 821 11 of 19 3.4. Effect of Pyrimidine Supplementation on the Antiviral Activity of DHODH Inhibitors 3.4. EDHODHﬀect of Pyrimidine is a key Supplementation enzyme in the on de the novo Antiviral pyrimidine Activity biosynthesis of DHODH Inhibitors pathway, and DHODH inhibitorsDHODH exert is atheir key enzyme antiviral in theactivityde novo viapyrimidine the depletion biosynthesis of intracellular pathway, and pyrimidine DHODH inhibitors levels. Accordingly,exert their antiviral the antiviral activity via activity the depletion of DHODH of intracellular inhibitors pyrimidine can be levels. reversed Accordingly, by promoting the antiviral the pyrimidineactivity of DHODHsalvage inhibitorspathway via can supplementation be reversed by promoting with exogenous the pyrimidine pyrimidine salvage ribonucleosides. pathway via Treatmentsupplementation of LCMV-infected with exogenous A549 pyrimidine cells with ribonucleosides.non-limiting exogenous Treatment uridine of LCMV-infected (Figure 5A) or A549 cytidine cells (Figurewith non-limiting 5B) supplementation exogenous uridinereverted (Figure completely5A) or or cytidine partially, (Figure respectively,5B) supplementation the antiviral revertedeffects of DHODHcompletely inhibitors. or partially, We respectively, used 2′-deoxy the antiviral analogs effects as negative of DHODH controls inhibitors. dueWe to usedtheir 2 0inability-deoxy analogs to be metabolizedas negative controls for ribonucleotide due to their synthesis. inability to be metabolized for ribonucleotide synthesis.

A Compound only + Uridine + 2'-Deoxyuridine

150 **** **** **** **** **** **** **** **** **** **** 100 **** ****

ZsG expression (%) expression ZsG 1 2 3 4 5 ar d d d n n i un und u un u DMSO o pound po p po po req m m m B o om Co C Co C Com

B Compound only + Cytidine + 2'-Deoxycytidine

150

100 **** **** **** **** **** **** 50 **** **** **** **** **** ****

ZsG expression (%) expression ZsG 1 2 3 4 5 ar d d d d n in un un un DMSO o mpo mpound mp Brequ o o o ompo C C C Compou C Figure 5. PyrimidinePyrimidine addition addition counteracts counteracts the antiviral activity of DHODH inhibitors. A549 cells were treated with the indicated DHODH DHODH inhibitors inhibitors (5 (5 µM)µM) and with or without uridine ( AA)) or or cytidine cytidine ( (BB)) and infected (MOI = 0.01)0.01) with with rCL-13/ZsG. rCL-13/ZsG. At At 48 48 h h pi, pi, ZsG ZsG expression expression levels levels were were determined. determined. Values Values correspond to four independent replicates. Error bars represent mean +/ SEM. Statistical signiﬁcance correspond to four independent replicates. Error bars represent mean +/− SEM. Statistical significance was calculated by ANOVA (****(**** pp << 0.00001). 3.5. Contribution of IFN-I to the Mammarenavirus Antiviral Activity of DHODH Inhibitors

Type I and Type III IFN responses could be enhanced by pyrimidine biosynthesis inhibition, which can further contribute to reduced virus multiplication [48]. However, we previously demonstrated that pyrimidine biosynthesis inhibitors exhibited similar antiviral anti-LCMV activity in IFN-competent (A549) Viruses 2020, 12, 821 12 of 19 and IFN-deficient (Vero E6) cells [25]. Consistent with our previous findings, all five DHODH inhibitors had a similarly potent anti-LCMV activity in both IFN-competent (A549) and IFN-deficient (Vero E6) cells (Figure6). Some pyrimidine biosynthesis inhibitors have been shown to promote an IFN-independent antiviral cellular state [28,49]. To examine this issue, we selected Cmp 1 as a representative member of the newly tested DHODH inhibitors and used brequinar as a positive control to examine their effects on the expression of ISGs previously shown to be upregulated by pyrimidine biosynthesis inhibitors in an IFN-independent manner (Figure7). In non-infected A549 cells, ISG15 and IFIT1 were moderately upregulated by treatment with Cmp 1 or brequinar, and the increased expression of ISG15 and IFIT1 was reversed by uridine supplementation (Figure7, left panel). Neither Cmp 1 nor brequinar treatment significantly affected the expression of IFNB, indicating that upregulated ISG15 and IFIT1 expression was IFNB-independent. LCMV infection appreciably upregulated the messenger RNA (mRNA) levels of IFNB and all of the tested ISGs (Figure7, right panel). Treatment with Cmp 1 or brequinar significantly decreased the mRNA levels of IFNB and ISGs in LCMV-infected cells, an effect that was reversed by uridine supplementation.

3.6. Assessment of Cmp 4 (IM90838) Anti-LCMV Activity In Vivo

VirusesTo 2020 assess, 12, thex FOR antiviral PEER REVIEW activity in vivo of the novel DHODH inhibitors, we used the C57BL12/6 of mouse 19 model of infection with the immunosuppressive CL-13 variant of the Armstrong strain of LCMV [50]. In3.5. this Contribution model, IV of infection IFN-I to ofthe adult Mammarena C57BLvirus/6 mice Antiviral with aActivity high dose of DHODH (2 10 6Inhibitorspfu) of CL-13 resulted in × transientType weight I and loss Type and III the IFN establishment responses could of viral be enha persistencenced by lasting pyrimidine over 90 bi days.osynthesis We selected inhibition, Cmp 4 (Figurewhich8 A)can for further this experiment contribute because to reduced it is currently virus multiplication under testing in[48]. phase However, 2 clinical we trials, previously with more detaileddemonstratedin vivo datathat available,pyrimidine including biosynthesis toxicology inhibito andrs exhibited pharmacology. similar Following antiviral anti-LCMV infection, we activity observed lessin bodyIFN-competent weight loss (A549) and fasterand IFN-deficient recovery (Figure (Vero8B) E6) in micecells treated[25]. Consistent with Cmp with 4 than our in previous the vehicle controlfindings, group; all but,five treatmentDHODH withinhibi Comptors had 4 did anot similarly causea potent significant anti-LCMV reduction activity in viral in load both (Figure IFN-8C). competent (A549) and IFN-deficient (Vero E6) cells (Figure 6). Some pyrimidine biosynthesis 3.7.inhibitors Eﬀect of have Inhibiting been the shown Uridine to/ Cytidinepromote Kinase an IFN-independent 2 (UCK2) on the Antiviralantiviral Activitycellular ofstate Cmp [28,49]. 4 in the To Presenceexamine of this Unlimited issue, Uridinewe selected Supply Cmp 1 as a representative member of the newly tested DHODH inhibitorsSeveral and DHODH used brequinar inhibitors as with a positive potent control antiviral to examine activity their in cultured effects cellson the have expression been shown of ISGs to be ineffectivepreviouslyin shown vivo due to tobe the upregulated efficient salvage by pyrimidine of exogenous biosynthesis uridine, inhibitors which could in an have IFN-independent accounted for the lackmanner of Cmp (Figure 4 antiviral 7). In efficacy non-infected in the mouseA549 cells, model ISG15 of CL-13 and IFIT1 infection. were The moderately requirement upregulated of UCK2 forby the treatment with Cmp 1 or brequinar, and the increased expression of ISG15 and IFIT1 was reversed pyrimidine nucleotide salvage pathway led us to examine whether the inhibition of UCK2 enhanced by uridine supplementation (Figure 7, left panel). Neither Cmp 1 nor brequinar treatment the antiviral activity of the DHODH inhibitor Cmp 4 under conditions of an unlimited uridine supply. significantly affected the expression of IFNB, indicating that upregulated ISG15 and IFIT1 expression For this, we examined the effect of UCK2 inhibitor 20874830 in the pyrimidine supplementation-mediated was IFNB-independent. LCMV infection appreciably upregulated the messenger RNA (mRNA) reversion of the antiviral activity of Cmp 4 (Figure9). Consistent with our previous results (Figure5A), levels of IFNB and all of the tested ISGs (Figure 7, right panel). Treatment with Cmp 1 or brequinar supplementationsignificantly decreased with exogenous the mRNA uridine levels of reverted IFNB and the ISGs Cmp in LCMV-infected 4-induced antiviral cells, effect, an effect and that treatment was withreversed UCK2 by inhibitor uridine 20874830supplementation. resulted in partial recovery of the antiviral effect of Cmp 4.

DMSO Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Brequinar

Vero E6

A549

FigureFigure 6. 6.Effect Effect of of DHODH DHODH inhibitors inhibitors onon LCMVLCMV multiplicationmultiplication inin interferoninterferon (IFN) -deficient (Vero E6) andE6) IFN-competent and IFN-competent (A549) (A549) cells. cells. IFN-deficient IFN-deficient Vero Vero E6 cells E6 andcellsIFN-competent and IFN-competent A549 A549 cells werecells were infected infected (MOI = 0.01) with rCL-13/ZsG in the presence of the indicated DHODH inhibitors (5 µM). At (MOI = 0.01) with rCL-13/ZsG in the presence of the indicated DHODH inhibitors (5 µM). At 48 h pi, 48 h pi, cells were fixed and stained with DAPI. ZsG-expressing cells were detected by cells were fixed and stained with DAPI. ZsG-expressing cells were detected by epifluorescence. epifluorescence.

Viruses 2020, 12, 821 13 of 19 Viruses 2020, 12, x FOR PEER REVIEW 13 of 19

Mock infection LCMV.Cl13 48 h pi

+ + - - - - + + - - - - DMSO - - + + - - - - + + - - Compound 1 - - - - + + ----+ + Brequinar - + - + - + - + - + - + Uridine

IFNB 1.00 1.43 1.94 1.67 2.02 2.04 3.96 4.23 2.71 4.14 2.80 4.41

ISG15 1.00 0.95 1.45 0.97 1.52 1.01 2.35 2.39 1.76 2.43 1.89 2.30

IFIT1 1.00 1.73 4.06 1.78 4.98 2.83 20.23 20.65 5.29 17.67 3.22 15.86 DDX58 1.00 1.17 1.44 1.17 1.43 1.23 1.98 2.00 1.63 2.02 1.68 2.02

STAT1

1.00 0.89 0.90 0.89 0.81 0.76 1.25 1.24 0.77 1.20 0.68 0.88 IFI27 1.00 0.93 1.12 1.04 1.21 1.00 3.86 3.61 1.18 3.36 1.07 2.67 IRF1 1.00 0.92 0.95 0.86 0.92 0.89 1.55 1.62 1.03 1.62 1.02 1.38 IRF9 1.00 0.88 1.09 0.86 0.88 0.71 1.24 1.21 0.34 1.22 0.94 0.99 IFIT2 1.00 1.02 1.25 1.06 1.22 1.05 2.05 2.03 1.19 2.08 1.28 2.09 IRF7 1.00 0.86 1.03 0.92 0.95 0.90 3.50 3.60 1.01 3.25 0.69 1.54 CXCL10 1.00 1.03 1.07 1.13 1.14 1.26 3.82 3.64 0.72 2.71 0.44 1.33

MX1

1.00 1.03 1.17 1.07 1.22 1.17 4.38 4.30 1.53 4.33 1.47 4.38

GAPDH

1.00 1.13 1.10 1.17 1.16 1.13 1.06 1.03 1.01 0.98 0.94 0.89

FigureFigure 7.7. LimitedLimited role role of of pyrimidine pyrimidine depletion-mediated depletion-mediated IFN-stimulated IFN-stimulated genes genes (ISG) (ISG) expression expression on the on antiviralthe antiviral activity activity against against LCMV. LCMV. A549 A549 cells cells were were treated treated with with the indicated the indicated DHODH DHODH inhibitors inhibitors (5 µM) (5 andµM) uridine and uridine (100 (100µM), µM), and and then then incubated incubated for for 48 h48 with h with or or without without rCL-13 rCL-13 infection. infection. Total Total cellularcellular RNARNA waswas puriﬁedpurified andand equalequal amountsamounts (3.5(3.5 µµg)g) werewere usedused inin reversereverse transcriptiontranscription reactionsreactions usingusing randomrandom hexamerhexamer primers primers to to generate generate cDNAs cDNAs that that were were used used in in PCR PCR reactions reactions with with speciﬁc specific primers primers to amplifyto amplify the the cDNAs cDNAs of of indicated indicated genes. genes. PCR PCR products products were were resolved resolved by by agarose agarose gel gelelectrophoresis electrophoresis andand visualizedvisualized byby ethidiumethidium bromidebromide staining.staining. TheThe numbernumber belowbelow eacheach bandband indicatesindicates thethe normalizednormalized foldfold changechange ofof bandband intensityintensity comparedcompared toto mock-infectedmock-infected andand vehicle-treatedvehicle-treated controlcontrol cells. cells. EachEach bandband intensityintensity waswas normalized normalized to theto correspondingthe corresponding glyceraldehyde glyceraldehyde 3-phosphate 3-phosphate dehydrogenase dehydrogenase (GAPDH) band(GAPDH) intensity. band intensity.

Viruses 2020, 12, x FOR PEER REVIEW 14 of 19

3.6. Assessment of Cmp 4 (IM90838) Anti-LCMV Activity in Vivo To assess the antiviral activity in vivo of the novel DHODH inhibitors, we used the C57BL/6 mouse model of infection with the immunosuppressive CL-13 variant of the Armstrong strain of LCMV [50]. In this model, IV infection of adult C57BL/6 mice with a high dose (2 × 106 pfu) of CL-13 resulted in transient weight loss and the establishment of viral persistence lasting over 90 days. We selected Cmp 4 (Figure 8A) for this experiment because it is currently under testing in phase 2 clinical trials, with more detailed in vivo data available, including toxicology and pharmacology. Following infection, we observed less body weight loss and faster recovery (Figure 8B) in mice treated with VirusesCmp2020 4 ,than12, 821 in the vehicle control group; but, treatment with Comp 4 did not cause a significant14 of 19 reduction in viral load (Figure 8C).

A C Compound 4 8 VC 6 FFU/ml)

10 * 4

Virus titer Virus (log 0 I. P.I. P. P.I. 3 6 y y 17 a a D D ay D

B Compound 4 110 VC

100 90 **** * * ** 80

Body weight (%) weight Body 70

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Days post infection Viruses 2020, 12, x FOR PEER REVIEW 15 of 19 FigureFigure 8. 8.In In vivo vivoantiviral antiviral activity activity of Cmp of Cmp 4 against 4 against LCMV. LCMV. (A) Structural (A) Structural formula formula of Cmp of 4 (IMU90838,Cmp 4 pyrimidinealso(IMU90838, known supplementation-media as also vidoﬂudimus known as vi calcium).dofludimusted (Breversion calcium).and C) C57BL (Bof and the/ 6C mice) antiviralC57BL/6 (n = mice4)activity were (n = orally 4) of were Cmp treated orally 4 withtreated(Figure Cmp 9). Consistent4 (150with mgCmp with/kg 4/ (150d)our for mg/kg/d)previous 17 days for results or 17 with days (Figure VC. or with Both 5A), VC. groups supplementationBoth ofgroups mice of were mice infectedwith were exogenous infected (IV) with (IV) uridine 2with10 2 6 ×reverted pfu106 of × theCL-13. Cmppfu of 4-induced BodyCL-13. weight Body antiviral weight changes effect,changes (B) andand (B serum) treatmentand serum virus withvirus titers UCK2titers (C) were( Cinhibitor) were determined determined 20874830 at theat resulted the indicated indicated in partial time recoverypoints.time ofpoints. Statistical the antiviral Statistical signiﬁcance effect significance of was Cmp calculated was 4. calculated by ANOVA by ANOVA (* p < (*0.05, p < **0.05,p < **0.002, p < 0.002, and ******* pp << 0.0002,0.00001). and **** p < 0.00001). 140 3.7. Effect of Inhibiting the Uridine/Cytidine120 Kinase 2 (UCK2) on the Antiviral Activity of Cmp 4 in the Presence of Unlimited Uridine Supply100 * Several DHODH inhibitors with80 potent antiviral activity in cultured cells have been shown to be ineffective in vivo due to the efficient60 salvage of exogenous uridine, which could have accounted for the lack of Cmp 4 antiviral efficacy40 in the mouse model of CL-13 infection. The requirement of UCK2 for the pyrimidine nucleotide20 salvage pathway led us to examine whether the inhibition of UCK2 enhanced the antiviral activity0 of the DHODH inhibitor Cmp 4 under conditions of an

unlimited uridine supply. For (%) ZsG expression this,-20 we examined the effect of UCK2 inhibitor 20874830 in the DMSO Compound 4

Compound only + Uridine + Uridine + UCK2 inhibitor + UCK2 inhibitor

FigureFigure 9. 9.A549 A549 cells cells were were treatedtreated withwith Cmp 4 4 (5 (5 µM)µM) and and with with or or without without uridin uridinee or orUCK2 UCK2 inhibitor inhibitor andand infected infected (MOI (MOI= =0.01) 0.01) withwith rCL-13rCL-13/ZsG/ZsG (six (six rep replicates).licates). At At 48 48 h hpi, pi, ZsG ZsG expression expression levels levels were were determined.determined. Error Error barsbars represent mean mean +/+/− SEM.SEM. Statistical Statistical significance signiﬁcance waswas calculated calculated by ANOVA by ANOVA (* − (* p < 0.05).0.05).

Viruses 2020, 12, 821 15 of 19

4. Discussion In this study, a series of novel DHODH inhibitors were evaluated for their antiviral activities against three mammarenaviruses. The tested DHODH inhibitors exhibited broad-spectrum anti-mammarenavirus activity as they inhibited the multiplication of LCMV, LASV, and JUNV with high SI values in cultured cells. Our results also provide evidence that these DHODH inhibitors do not affect cell entry or budding, but rather the inhibition of RNA synthesis mediated by the vRNP (Figure4B). This finding is consistent with the well-established effect of DHODH inhibitors on cellular pyrimidine nucleoside pools. This mechanism of action was further supported by the results from pyrimidine supplementation experiments (Figure5), showing that uridine supplementation reversed the antiviral activity associated with pyrimidine depletion caused by treatment with DHODH inhibitors. Unexpectedly, supplementation with exogenous cytidine showed only partial reversion of the DHODH-mediated antiviral activity (Figure5B), which might reflect differences in the activity of enzymes in each uridine or cytidine salvage pathway. Thus, maintaining pyrimidine nucleoside pools is likely to be essential for virus propagation, suggesting that pyrimidine depletion is a key mechanism for antiviral effects mediated by DHODH inhibitors. The contribution of IFN-I to the antiviral effects mediated by inhibitors of pyrimidine biosynthesis remains controversial. Pyrimidine biosynthesis inhibitors promote enhanced IFN-I-mediated innate immune responses contributing to the inhibition of the multiplication of several viruses, including measles virus, chikungunya virus, and West Nile virus [48]. However, pyrimidine depletion was shown to trigger IFN-I-independent expression of ISGs that resulted in cellular antiviral states that inhibited the replication of vesicular stomatitis Indiana virus [49]. In addition, the IFN-independent expression of IRF1 contributed to the antiviral activity of a DHODH inhibitor (SW835) against Ebola virus (EBOV) [28]. For mammarenaviruses, different pyrimidine biosynthesis inhibitors exhibited similar antiviral activities in both IFN-competent and IFN-deficient cells [25]. Similarly, the DHODH inhibitors evaluated in this study are antivirally active in an IFN-independent way against mammarenaviruses (Figure6). We found that treatment with DHODH inhibitors moderately increased the mRNA levels of ISG15 and IFIT1 in uninfected cells, whereas other ISGs and IFNB were not affected (Figure7, left panel). Consistent with previous findings [51], LCMV infection increased the transcription of IFNB, which was suppressed by treatment with DHODH inhibitors. Inhibitors of pyrimidine biosynthesis did not affect ISG expression levels in IFN-I-deficient Vero E6 cells (data not shown). These findings suggest that the pyrimidine depletion-induced anti-mammarenavirus effect is IFN-I independent and that ISGs have a minimal contribution to this antiviral effect. Studies testing the in vivo efficacy of pyrimidine biosynthesis inhibitors as antiviral drug candidates have had only moderate success. In mice exposed to a typically lethal dose of influenza A virus, treatment with the DHODH inhibitor FA-613 increased survival [52]. Likewise, intranasal treatment with leflunomide’s active metabolite, A77-1726, reduced the pathophysiological signs associated with human respiratory syncytial virus (HRSV) infection in laboratory mice, but this clinical improvement was not associated with a reduction in lung viral load [53]. In this study, we found that CL-13-infected mice treated with Cmp 4 had less body weight loss and showed a faster recovery (Figure8). However, treatment with Cmp 4 did not reduce viral load. The pyrimidine salvage pathway can provide LCMV-infected cells with pyrimidine pools that could counteract the effect of DHODH inhibitors. Therefore, targeting the pyrimidine salvage pathway could be a strategy to enhance the antiviral effect of pyrimidine biosynthesis inhibitors in vivo. The feasibility of this approach is supported by knockdown mediated by short hairpin RNA (shRNA) and CRISPR-Cas9-mediated deletion of UCK2, a key enzyme of the pyrimidine salvage pathway that sensitized cells to GSK983, a known DHODH inhibitor [54]. Consistent with this observation, we found that co-treatment with Cmp 4 and UCK2 inhibitor 20874830 partially prevented the uridine supplementation-mediated reversion of the antiviral effect exerted by Cmp 4 in cultured cells (Figure9). Unfortunately, we were unable to test the in vivo efficacy of combination therapy with DHODH and UCK2 inhibitors due to a limited supply of UCK2 inhibitor 20874830. Viruses 2020, 12, 821 16 of 19

5. Conclusions A series of novel DHODH inhibitors exhibited strong broad-spectrum antiviral activity against mammarenaviruses in vitro. The anti-mammarenavirus activity of the tested DHODH inhibitors was mediated by pyrimidine depletion, which negatively impacted viral RNA synthesis and was independent of the IFN-I response. However, the IFN-I independent induction of some ISGs might have contributed to the anti-mammarenaviral activity of the tested DHODH inhibitors.

Author Contributions: Conceptualization, Y.-J.K. and J.C.d.l.T.; methodology, Y.-J.K., B.C., Y.C., J.H.K., D.V., H.K., and J.C.d.l.T.; software, Y.-J.K., B.C., Y.C., and D.V.; validation, Y.-J.K., B.C., Y.C., and D.V.; formal analysis, Y.-J.K., B.C., Y.C., and D.V.; investigation, Y.-J.K., B.C., Y.C., and D.V.; resources, H.K. and J.C.d.l.T.; data curation, Y.-J.K., B.C., and Y.C.; writing—original draft preparation, Y.-J.K.; writing—review and editing, Y.-J.K., B.C., Y.C., J.H.K., D.V., H.K., J.C.d.l.T., L.B., and A.C.; visualization, Y.-J.K.; supervision, J.C.d.l.T., J.H.K., and H.K.; project administration, J.C.d.l.T.; funding acquisition, J.C.d.l.T., Y.C., and J.H.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part through Battelle Memorial Institute’s former prime contract with the National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I and Laulima Government Solutions’ current prime contract with NIAID under Contract No. HHSN272201800013C). Y.C. and J.H.K. performed this work as former employees of Battelle Memorial Institute and current employees of Tunnell Government Services (TGS), a subcontractor of Laulima Government Solutions, under Contract No. HHSN272201800013C. This research was also supported by NIAID R21 grants AI125626 and AI128556 (J.C.T.). This is manuscript #29945 from The Scripps Research Institute. Acknowledgments: We thank Laura Bollinger (IRF-Frederick) and Anya Crane (IRF-Frederick) for editing the manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services or the institutions and companies affiliated with the authors. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Grande-Pérez, A.; Martin, V.; Moreno, H.; de la Torre, J.C. Arenavirus quasispecies and their biological implications. Curr. Top. Microbiol. Immunol. 2016, 392, 231–276. [PubMed] 2. Happi, A.N.; Happi, C.T.; Schoepp, R.J. Lassa fever diagnostics: Past, present, and future. Curr. Opin. Virol. 2019, 37, 132–138. [CrossRef][PubMed] 3. Freedman, D.O.; Woodall, J. Emerging infectious diseases and risk to the traveler. Med. Clin. N. Am. 1999, 83, 865–883. [PubMed] 4. Isaäcson, M. Viral hemorrhagic fever hazards for travelers in Africa. Clin. Infect. Dis. 2001, 33, 1707–1712. [CrossRef][PubMed] 5. Sogoba, N.; Feldmann, H.; Safronetz, D. Lassa fever in West Africa: Evidence for an expanded region of endemicity. Zoonoses Public Health 2012, 59, 43–47. [CrossRef][PubMed] 6. Briese, T.; Paweska, J.T.; McMullan, L.K.; Hutchison, S.K.; Street, C.; Palacios, G.; Khristova, M.L.; Weyer, J.; Swanepoel, R.; Egholm, M.; et al. Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS Pathog. 2009, 5, e1000455. [CrossRef] 7. Patterson, M.; Grant, A.; Paessler, S. Epidemiology and pathogenesis of Bolivian hemorrhagic fever. Curr. Opin. Virol. 2014, 5, 82–90. [CrossRef] 8. Delgado, S.; Erickson, B.R.; Agudo, R.; Blair, P.J.; Vallejo, E.; Albariño, C.G.; Vargas, J.; Comer, J.A.; Rollin, P.E.; Ksiazek, T.G.; et al. Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia. PLoS Pathog. 2008, 4, e1000047. [CrossRef] 9. Ellwanger, J.H.; Chies, J.A. Keeping track of hidden dangers—The short history of the Sabia virus. Rev. Soc. Bras. Med. Trop. 2017, 50, 3–8. [CrossRef] 10. Escalera-Antezana, J.P.; Rodriguez-Villena, O.J.; Arancibia-Alba, A.W.; Alvarado-Arnez, L.E.; Bonilla-Aldana, D.K.; Rodríguez-Morales, A.J. Clinical features of fatal cases of Chapare virus hemorrhagic fever originating from rural La Paz, Bolivia, 2019: A cluster analysis. Travel Med. Infect. Dis. 2020, 101589. [CrossRef][PubMed] Viruses 2020, 12, 821 17 of 19

11. de Mello Malta, F.; Amgarten, D.; Nastri, A.C.d.S.S.; Ho, Y.-L.; Boas Casadio, L.V.; Basqueira, M.; Selegatto, G.; Cervato, M.C.; Duarte-Neto, A.N.; Higashino, H.R.; et al. Sabiá virus-like mammarenavirus in patient with fatal hemorrhagic fever, Brazil, 2020. Emerg. Infect. Dis. 2020, 26, 1332–1334. [CrossRef][PubMed] 12. Barton, L.L.; Mets, M.B. Lymphocytic choriomeningitis virus: Pediatric pathogen and fetal teratogen. Pediatric Infect. Dis. J. 1999, 18, 540–541. [CrossRef][PubMed] 13. Barton, L.L.; Mets, M.B. Congenital lymphocytic choriomeningitis virus infection: Decade of rediscovery. Clin. Infect. Dis. 2001, 33, 370–374. [CrossRef] 14. Barton, L.L.; Mets, M.B.; Beauchamp, C.L. Lymphocytic choriomeningitis virus: Emerging fetal teratogen. Am. J. Obstet. Gynecol. 2002, 187, 1715–1716. [CrossRef] 15. Jahrling, P.B.; Peters, C.J. Lymphocytic choriomeningitis virus. A neglected pathogen of man. Arch. Pathol. Lab. Med. 1992, 116, 486–488. 16. Peters, C.J. Lymphocytic choriomeningitis virus—An old enemy up to new tricks. N. Engl. J. Med. 2006, 354, 2208–2211. [CrossRef] 17. Maiztegui, J.I.; McKee, K.T., Jr.; Barrera Oro, J.G.; Harrison, L.H.; Gibbs, P.H.; Feuillade, M.R.; Enria, D.A.; Briggiler, A.M.; Levis, S.C.; Ambrosio, A.M.; et al. AHF Study Group, Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. J. Infect. Dis. 1998, 177, 277–283. [CrossRef][PubMed] 18. Damonte, E.B.; Coto, C.E. Treatment of arenavirus infections: From basic studies to the challenge of antiviral therapy. Adv. Virus Res. 2002, 58, 125–155. 19. Moreno, H.; Gallego, I.; Sevilla, N.; de la Torre, J.C.; Domingo, E.; Martín, V. Ribavirin can be mutagenic for arenaviruses. J. Virol. 2011, 85, 7246–7255. [CrossRef] 20. Parker, W.B. Metabolism and antiviral activity of ribavirin. Virus Res. 2005, 107, 165–171. [CrossRef] [PubMed] 21. Gowen, B.B.; Juelich, T.L.; Sefing, E.J.; Brasel, T.; Smith, J.K.; Zhang, L.; Tigabu, B.; Hill, T.E.; Yun, T.; Pietzsch, C.; et al. Favipiravir (T-705) inhibits Junín virus infection and reduces mortality in a guinea pig model of Argentine hemorrhagic fever. PLoS Negl. Trop. Dis. 2013, 7, e2614. [CrossRef][PubMed] 22. Mendenhall, M.; Russell, A.; Juelich, T.; Messina, E.L.; Smee, D.F.; Freiberg, A.N.; Holbrook, M.R.; Furuta, Y.; de la Torre, J.-C.; Nunberg, J.H.; et al. T-705 (favipiravir) inhibition of arenavirus replication in cell culture. Antimicrob. Agents Chemother. 2011, 55, 782–787. [CrossRef][PubMed] 23. Mendenhall, M.; Russell, A.; Smee, D.F.; Hall, J.O.; Skirpstunas, R.; Furuta, Y.; Gowen, B.B. Effective oral favipiravir (T-705) therapy initiated after the onset of clinical disease in a model of arenavirus hemorrhagic Fever. PLoS Negl. Trop. Dis. 2011, 5, e1342. [CrossRef][PubMed] 24. Safronetz, D.; Rosenke, K.; Westover, J.B.; Martellaro, C.; Okumura, A.; Furuta, Y.; Geisbert, J.; Saturday, G.; Komeno, T.; Geisbert, T.W.; et al. The broad-spectrum antiviral favipiravir protects guinea pigs from lethal Lassa virus infection post-disease onset. Sci. Rep. 2015, 5, 14775. [CrossRef] 25. Kim, Y.-J.; Cubitt, B.; Chen, E.; Hull, M.V.; Chatterjee, A.K.; Cai, Y.; Kuhn, J.H.; de la Torre, J.C. The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors. Antiviral Res. 2019, 169, 104558. [CrossRef] 26. Yang, C.-F.; Gopula, B.; Liang, J.-J.; Li, J.K.; Chen, S.-Y.; Lee, Y.-L.; Chen, C.S.; Lin, Y.-L. Novel AR-12 derivatives, P12-23 and P12-34, inhibit flavivirus replication by blocking host de novo pyrimidine biosynthesis. Emerg. Microbes Infect. 2018, 7, 187. [CrossRef] 27. Zhang, L.; Das, P.; Schmolke, M.; Manicassamy, B.; Wang, Y.; Deng, X.; Cai, L.; Tu, B.P.; Forst, C.V.; Roth, M.G.; et al. Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export. J. Cell Biol. 2012, 196, 315–326. [CrossRef] 28. Luthra, P.; Naidoo, J.; Pietzsch, C.A.; De, S.; Khadka, S.; Anantpadma, M.; Williams, C.G.; Edwards, M.R.; Davey, R.A.; Bukreyev, A.; et al. Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses. Antiviral Res. 2018, 158, 288–302. [CrossRef] 29. Hoffmann, H.-H.; Kunz, A.; Simon, V.A.; Palese, P.; Shaw, M.L. Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc. Natl. Acad. Sci. USA 2011, 108, 5777–5782. [CrossRef] 30. Boschi, D.; Pippione, A.C.; Sainas, S.; Lolli, M.L. Dihydroorotate dehydrogenase inhibitors in anti-infective drug research. Eur. J. Med. Chem. 2019, 183, 111681. [CrossRef] Viruses 2020, 12, 821 18 of 19

31. Muehler, A.; Kohlhof, H.; Groeppel, M.; Vitt, D. The selective oral immunomodulator vidofludimus in patients with active rheumatoid arthritis: Safety results from the COMPONENT study. Drugs R D 2019, 19, 351–366. [CrossRef][PubMed] 32. Lucas-Hourani, M.; Dauzonne, D.; Munier-Lehmann, H.; Khiar, S.; Nisole, S.; Dairou, J.; Helynck, O.; Afonso, P.V.; Tangy, F.; Vidalain, P.-O. Original chemical series of pyrimidine biosynthesis inhibitors that boost the antiviral interferon response. Antimicrob. Agents Chemother. 2017, 61, e00383-17. [CrossRef][PubMed] 33. Emonet, S.F.; Seregin, A.V.; Yun, N.E.; Poussard, A.L.; Walker, A.G.; de la Torre, J.C.; Paessler, S. Rescue from cloned cDNAs and In Vivo characterization of recombinant pathogenic Romero and live-attenuated Candid #1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease. J. Virol. 2011, 85, 1473–1483. [PubMed] 34. Iwasaki, M.; Minder, P.; Caì, Y.; Kuhn, J.H.; Yates, J.R., III; Torbett, B.E.; de la Torre, J.C. Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses. PLoS Pathog. 2018, 14, e1006892. [CrossRef] 35. Caì, Y.; Iwasaki, M.; Beitzel, B.F.; Yú, S.; Postnikova, E.N.; Cubitt, B.; DeWald, L.E.; Radoshitzky, S.R.; Bollinger, L.; Jahrling, P.B.; et al. Recombinant Lassa virus expressing green fluorescent protein as a tool for high-throughput drug screens and neutralizing antibody assays. Viruses 2018, 10, 655. [CrossRef] 36. Baumgartner, R.; Walloschek, M.; Kralik, M.; Gotschlich, A.; Tasler, S.; Mies, J.; Leban, J. Dual binding mode of a novel series of DHODH inhibitors. J. Med. Chem. 2006, 49, 1239–1247. [CrossRef] 37. Leban, J.; Kralik, M.; Mies, J.; Gassen, M.; Tentschert, K.; Baumgartner, R. SAR, species specificity, and cellular activity of cyclopentene dicarboxylic acid amides as DHODH inhibitors. Bioorganic Med. Chem. Lett. 2005, 15, 4854–4857. [CrossRef] 38. Battegay, M. Quantifizierung des Lympho-Choriomeningitis-Virus mit einer immunologischen Fokustechnik in 24- oder 96-Loch-Platten. ALTEX 1993, 10, 6–14. 39. Perez, M.; de la Torre, J.C. Characterization of the genomic promoter of the prototypic arenavirus lymphocytic choriomeningitis virus. J. Virol. 2003, 77, 1184–1194. [CrossRef] 40. Capul, A.A.; de la Torre, J.C. A cell-based luciferase assay amenable to high-throughput screening of inhibitors of arenavirus budding. Virology 2008, 382, 107–114. [CrossRef] 41. Jahrling, P.B.; Keith, L.; St Claire, M.; Johnson, R.F.; Bollinger, L.; Lackemeyer, M.G.; Hensley, L.E.; Kindrachuk, J.; Kuhn, J.H. The NIAID Integrated Research Facility at Frederick, Maryland: A unique international resource to facilitate medical countermeasure development for BSL-4 pathogens. Pathog. Dis. 2014, 71, 213–219. [CrossRef][PubMed] 42. Janosko, K.; Holbrook, M.R.; Adams, R.; Barr, J.; Bollinger, L.; Newton, J.T.; Ntiforo, C.; Coe, L.; Wada, J.; Pusl, D.; et al. Safety precautions and operating procedures in an (A)BSL-4 laboratory: 1. biosafety level 4 suit laboratory suite entry and exit procedures. J. Vis. Exp. 2016, 116, e52317. [CrossRef] 43. Mazur, S.; Holbrook, M.R.; Burdette, T.; Joselyn, N.; Barr, J.; Pusl, D.; Bollinger, L.; Coe, L.; Jahrling, P.B.; Lackemeyer, M.G.; et al. Safety precautions and operating procedures in an (A)BSL-4 laboratory: 2. general practices. J. Vis. Exp. 2016, 116, e53600. [CrossRef][PubMed] 44. Chen, S.-F.; Ruben, R.L.; Dexter, D.L. Mechanism of action of the novel anticancer agent 6-fluoro-2-

(20-ﬂuoro-1,10-biphenyl-4-yl)-3-methyl-4-quinolinecarbo xylic acid sodium salt (NSC 368390): Inhibition of De Novo pyrimidine nucleotide biosynthesis. Cancer Res. 1986, 46, 5014–5019. [PubMed] 45. Peters, G.J.; Schwartsmann, G.; Nadal, J.C.; Laurensse, E.J.; van Groeningen, C.J.; van der Vijgh, W.J.F.; Pinedo, H.M. In Vivo inhibition of the pyrimidine De Novo enzyme dihydroorotic acid dehydrogenase by brequinar sodium (DUP-785; NSC 368390) in mice and patients. Cancer Res. 1990, 50, 4644–4649. [PubMed] 46. Peters, G.J. Re-evaluation of brequinar sodium, a dihydroorotate dehydrogenase inhibitor. Nucleosides Nucleotides Nucleic Acids 2018, 37, 666–678. [CrossRef] 47. Ngo, N.; Henthorn, K.S.; Cisneros, M.I.; Cubitt, B.; Iwasaki, M.; de la Torre, J.C.; Lama, J. Identiﬁcation and mechanism of action of a novel small-molecule inhibitor of arenavirus multiplication. J. Virol. 2015, 89, 10924–10933. [CrossRef] 48. Lucas-Hourani, M.; Dauzonne, D.; Jorda, P.; Cousin, G.; Lupan, A.; Helynck, O.; Caignard, G.; Janvier, G.; André-Leroux, G.; Khiar, S.; et al. Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity. PLoS Pathog. 2013, 9, e1003678. [CrossRef] Viruses 2020, 12, 821 19 of 19

49. Chung, D.-H.; Golden, J.E.; Adcock, R.S.; Schroeder, C.E.; Chu, Y.-K.; Sotsky, J.B.; Cramer, D.E.; Chilton, P.M.; Song, C.; Anantpadma, M.; et al. Discovery of a broad-spectrum antiviral compound that inhibits pyrimidine biosynthesis and establishes a type 1 interferon-independent antiviral state. Antimicrob. Agents Chemother. 2016, 60, 4552–4562. [CrossRef] 50. Sullivan, B.M.; Emonet, S.F.; Welch, M.J.; Lee, A.M.; Campbell, K.P.; de la Torre, J.C.; Oldstone, M.B. Point mutation in the glycoprotein of lymphocytic choriomeningitis virus is necessary for receptor binding, dendritic cell infection, and long-term persistence. Proc. Natl. Acad. Sci. USA 2011, 108, 2969–2974. [CrossRef] 51. Pythoud, C.; Rodrigo, W.W.; Pasqual, G.; Rothenberger, S.; Martínez-Sobrido, L.; de la Torre, J.C.; Kunz, S. Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε. J. Virol. 2012, 86, 7728–7738. [CrossRef][PubMed] 52. Cheung, N.N.; Lai, K.K.; Dai, J.; Kok, K.H.; Chen, H.; Chan, K.-H.; Yuen, K.-Y.; Kao, R.Y.T. Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response. J. Gen. Virol. 2017, 98, 946–954. [CrossRef][PubMed] 53. Davis, I.C.; Lazarowski, E.R.; Chen, F.-P.; Hickman-Davis, J.M.; Sullender, W.M.; Matalon, S. Post-infection A77-1726 blocks pathophysiologic sequelae of respiratory syncytial virus infection. Am. J. Respir. Cell Mol. Biol. 2007, 37, 379–386. [CrossRef][PubMed] 54. Deans, R.M.; Morgens, D.W.; Ökesli, A.; Pillay, S.; Horlbeck, M.A.; Kampmann, M.; Gilbert, L.A.; Li, A.; Mateo, R.; Smith, M.; et al. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identiﬁcation. Nat. Chem. Biol. 2016, 12, 361–366. [CrossRef]

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