Abstract
Introduction:
Chikungunya fever remains a growing global health challenge, with a subset of infected individuals developing long-lasting and severe disease manifestations. Although significant progress has been made in chikungunya virus (CHIKV) antiviral research, no licensed therapeutic options are currently available. In this context, drug repurposing offers a strategic approach to accelerate the identification and development of effective CHIKV treatments.
Methods:
In this study, we conducted a high-throughput phenotypic screening assay based on a reporter CHIKV to evaluate 1,600 clinically approved or well-characterized compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and initiated the characterization of the in vitro antiviral profile of the most promising candidate.
Results:
The developed high-throughput screening platform effectively identified 13 candidate antivirals. Farudodstat, a human dihydroorotate dehydrogenase (DHODH) inhibitor, emerged as the most promising candidate. It demonstrated robust inhibitory activity against epidemic CHIKV strains and efficacy that varied across cell lines of distinct origins, consistent with a host-dependent mechanism of action. As expected from the role of DHODH in the de novo pyrimidine biosynthesis pathway, supplementation with exogenous uridine restored viral replication in the presence of Farudodstat, indicating that blockade of DHODH underlies its anti-CHIKV activity.
Discussion:
The potency and outstanding selectivity of Farudodstat as a CHIKV inhibitor in vitro, together with its favorable safety and pharmacokinetic properties, support the potential of Farudodstat as a promising oral antiviral candidate for the treatment of CHIKV fever.
Introduction
Chikungunya virus (CHIKV) belongs to the family Togaviridae, genus Alphavirus. Alphaviruses are divided into viruses of the Old World and New World. Old World alphaviruses, including Chikungunya (CHIKV) and Ross River (RRV) viruses, primarily cause arthritogenic diseases, while New World alphaviruses, such as Western, Venezuelan, and Eastern equine encephalitis viruses (WEEV, VEEV and EEEV, respectively), are associated with fatal encephalitis (Strauss and Strauss, 1994). CHIKV is a re-emergent arbovirus (Thaikruea et al., 1997; ; ; Weaver, 2014) which has diverged in three major lineages defined by their geographic distribution: West African, East/Central/South African (ECSA), and Asian. The ECSA and Asian strains have disseminated globally during the last decade, while the West African remained confined to a sylvatic circulation in sparsely populated regions of Africa. Since its re-emergence, both autochthonous and imported CHIKV cases have been documented in the Americas and Europe (Wahid et al., 2017; World Health Organization, 2025; ).
CHIKV is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The main feature of CHIKV disease is joint pain, which is frequently bilateral and symmetric, affecting hands, feet, knees, and wrists. Other symptoms include headache, fatigue, and muscle pain (myalgia), all of which can be both debilitating and distressing (Taubitz et al., 2007; Nkoghe et al., 2012; ). Although the disease is generally self-limiting, a substantial proportion of patients develop chronic and debilitating sequelae, most notably persistent joint pain and other long-term complications that can severely impair quality of life (Paixão et al., 2018; Puntasecca et al., 2021). In some patients, CHIKV infection can trigger severe atypical, multisystem involvement—particularly affecting the cardiovascular and central nervous systems—leading to potentially life-threatening complications or death (Puntasecca et al., 2021).
Preventive measures include vector control and recently approved vaccines. Ixchiq (Valneva) (Valneva SE, 2025), a live-attenuated vaccine that has received regulatory approval in the United States, Canada, Europe, and Brazil. However, on August 2025, the U.S. Food and Drug Administration (FDA) Center for Biologics Evaluation and Research has suspended the biologics license of Ixchiq based on serious safety concerns (Food and Drug Administration, 2025b). In parallel, Vimkunya (Bavarian Nordic), a virus-like particle vaccine, recently achieved accelerated approval from major regulatory bodies, including the FDA, the European Medicines Agency (EMA), and the UK Medicines and Healthcare products Regulatory Agency (MHRA) (Vimkunyatm, 2025). Geographically restricted approval of vaccines for travelers in non-endemic regions, underscore the persistent challenges in achieving broad and effective immunization coverage.
Treatment of CHIKV disease is currently limited to supportive care, focusing on symptomatic relief through analgesics and anti-inflammatory agents. Despite significant progress in antiviral drug discovery for CHIKV over the past decade (Wang et al., 2024), no licensed antiviral agents currently exist for CHIKV infection (Nizi et al., 2025), underscoring the urgent need for targeted antiviral compounds and immunomodulatory strategies.
CHIKV possesses a ∼12-kb, positive-sense RNA genome encoding four non-structural proteins (nsP1–4) and five structural proteins (C, E3, E2, 6K, and E1). The virion surface displays 80 trimeric spikes composed of E1–E2 heterodimers. Viral entry begins when E2 engages its cellular receptor, followed by clathrin-mediated endocytosis. Endosomal acidification then induces conformational rearrangements in E1 that drive membrane fusion and release of the nucleocapsid into the cytoplasm. Translation of the incoming genome yields the polyproteins P123 and P1234, which are cleaved by the nsP2 protease to generate the mature replication complex. NsP1 catalyzes 5′RNA capping, nsP2 carries NTPase, helicase, triphosphatase, and protease activities, nsP3 uses its macrodomain and disordered C-terminus to interact with host factors, and nsP4 functions as the RNA-dependent RNA polymerase. Structural proteins are synthesized on membrane-associated ribosomes, processed by host enzymes, and trafficked for virion assembly and budding at the plasma membrane (). Targeting of both structural and non-structural proteins by small molecule inhibitors and monoclonal antibodies exerts antiviral effects interfering with the viral life cycle including binding, endocytosis, membrane fusion, replication, packaging, and release (Wang et al., 2024). However, most targeted candidates with reported anti-CHIKV activity remain limited to in vitro efficacy and are still at early developmental stages.
Given the lengthy process of de novo drug discovery, repurposing approved or clinically tested compounds represents a practical and increasingly valuable strategy to accelerate CHIKV therapeutic development (Wang et al., 2024; ). Multiple strategies have been employed to identify candidate antivirals for CHIKV repurposing, including in silico screening of viral (; Parigger et al., 2024; ) and host targets (Roa-Linares et al., 2023), in vitro enzymatic assays (Tripathi et al., 2020; ), and cell-based phenotypic assays (; ; ; ). Nevertheless, only a small subset of these compounds has progressed to in vivo preclinical testing (; ; ), and none has demonstrated clinical efficacy to date, underscoring the continued need for research dedicated to discovering effective therapeutics for CHIKV.
The Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library comprises nearly 12,000 small-molecule compounds with established suitability for human use, offering a powerful platform for systematic drug repurposing efforts (). This collection provides an extensive resource for identifying candidates with potential antifungal (Wall et al., 2020), antiparasitic (; ; ) and antiviral activity (; Redhead et al., 2021; ; Sake et al., 2024), thereby accelerating the discovery of effective and well-tolerated therapeutics. Here, we report the identification of potent anti-CHIKV candidates following a cell-based screening of 1,600 compounds from the ReFRAME library. Among the confirmed hits, Farudodstat (CBR-050-264-248-0; CAS 1035688-66-4), a selective human dihydroorotate dehydrogenase (DHODH) inhibitor, emerged as the most promising due to its robust potency, selectivity and novelty. Other DHODH inhibitors have been proposed as broad-spectrum antivirals against various RNA and DNA viruses in vitro, including CHIKV (). Here we demonstrate for the first time the antiviral activity of Farudodstat against CHIKV. We further describe the in vitro antiviral profile and delineate its mechanism of action, adding evidence of its antiviral activity which, together with the safety profiles and previous pharmacokinetic studies, position Farudodstat as a promising antiviral for the treatment of CHIKV fever.
Materials and methods
Cells and virus
Cells were cultured at 37 °C in a 5% CO2 atmosphere. Vero (Cercopithecus aethiops kidney, ATCC CCL-81), HEK-293T (human embryonic kidney cells expressing SV40 T antigen, ATCC CRL-3216), and NIH-3T3 (murine fibroblast, ATCC CRL-1658) cells were grown in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) (Internegocios, Mercedes, Buenos Aires, Argentina), 100 IU/mL penicillin, and 100 mg/mL streptomycin (Gibco) (DMEM-10). Huh-7 cells (human hepatoma cells, provided by Apath LLC) were grown in DMEM-10 containing 2 mM glutamine and 10 mM HEPES. BHK cells (Mesocricetus auratus hamster kidney, ATCC CCL-10) were grown in MEM alpha medium (Gibco) containing 10% FBS 100 IU/mL penicillin, and 100 mg/mL streptomycin (Gibco) (aMEM-10). For infections, cells were cultured in supplemented DMEM or aMEM with 2% FBS (DMEM-2 or aMEM-2, respectively).
CHIKV-ZsGreen was derived from infectious cDNA clone as previously reported (Noval et al., 2019). Viral stock was obtained in Vero cells and titrated by fluorescent Foci Forming Units (FFU) assays in the cell lines indicated above. CHIKV La Reunion (ECSA genotype, IOL lineage) () and Caribbean (Asian genotype) (Stapleford et al., 2016) were obtained from infectious clones as previously described and titrated by Plaque Forming Units (PFU) in Vero cells. Viral stocks were stored at −70 °C until use.
Compounds preparation
Antiviral set from the ReFRAME Library was kindly provided by The Scripps Research Institute, Calibr. For screening assays, compounds were supplied in DMSO in the form of pre-spotted single-use 96-well plates (0.1 µL of 10 mM for primary screening and 8 serial dilutions 1/3 starting from 20 mM for antiviral potency determination). Finally, for confirmation, solid samples of the selected compounds were dissolved in DMSO at 100 mM for cytotoxicity assays and 20 mM for antiviral assays.
Primary screening
In the present work, we performed a primary screening of 1,600 compounds using a fully infectious CHIKV-ZsGreen reporter virus, which reproduces the complete viral cycle and generates a productive infection in cultured Vero cells (Noval et al., 2019).
Vero cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well in DMEM-10 and incubated at 37 °C and 5% CO2. After 24 h, the culture medium was removed, monolayers were washed twice with phosphate-buffered saline (PBS) and infected with 700-800 FFUs of CHIKV-ZsGreen (MOI 0.05 FFU/cell). Simultaneously, pre-spotted compounds were resuspended in DMEM-2 and then transferred to the assay plate to be tested at a single concentration of 10 µM. Untreated infected cells and uninfected cells were included as controls. After 20 h at 37 °C and 5% CO2, cultures were fixed with paraformaldehyde (PFA) 4% at 4 °C for 1 h. The fluorescent protein ZsGreen is expressed because of viral replication in infected cells, and fluorescent infectious foci (fFFU) can be quantified. Automated counting of fFFUs was performed using the EliSpot equipment and the ImmunoSpot 7.0.30.2 software (ImmunoSpot C.T.L, Germany). Using this methodology, antiviral activity is detected as a decrease in the number of infectious foci respecting untreated infected cells. After foci quantification, fixed cultures were stained with crystal violet solution in methanol, and plates were scanned for determination of mean absorbance at 585 nm to estimate compound’s cytotoxicity as the ratio between mean absorbance of the treated well and the untreated cells ().
Antiviral potency determination of selected compounds
Sixteen compounds selected after primary screening were tested at 8 concentrations from 20 μM to 9 nM in two independent assays for antiviral potency determination. Antiviral assays were performed by FFU reduction assay against CHIKV-ZsGreen as described above. The focus count data for each antiviral concentration and the untreated infected cells were used to plot dose–response curves that were fitted by nonlinear regression using GraphPad Prism 8 software. Effective concentration 50 (EC50) values, which are defined as the concentration of compound that reduces fFFU number by 50% with respect to untreated infected cells, were calculated from dose–response curves. Cytotoxicity estimates with crystal violet were performed as described above. The cytotoxic concentration 50 (CC50), which is defined as the concentration of compound that reduces absorbance at 585 nm by 50% with respect to mock treated controls, were estimated from dose–response curves. Finally, the EC50 and CC50 values were used to calculate the Selectivity Index (SI = CC50/EC50).
Hits reconfirmation
Three compounds were selected after antiviral potency determination and cytotoxicity estimation: N-hydroxycytidine (CBR-001-856-178-9; CAS 3258-02-4), Gemcitabine Elaidate (CBR-001-573-415-3; CAS 210829-30-4) and Farudodstat (CBR-050-264-248-0; CAS 1035688-66-4). To confirm their anti-CHIKV activity, newly provided compounds were tested in eight serial dilutions in triplicate from 20 μM to 9 nM against CHIKV-ZsGreen. Antiviral assays were performed as described above and cytotoxicity was evaluated by MTS/PMS method as described below. EC50 and CC50 values were obtained from three independent experiments, and SI values were calculated.
Cytotoxicity assays
To determine the cytotoxicity of the aforementioned three compounds, Vero cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well in DMEM-10 and incubated at 37 °C and 5% CO2. After 24 h, the culture medium was removed, monolayers were washed twice with PBS, and serial dilutions of the test compounds were added. Compounds were tested from 500 to 3.9 µM in triplicate. Cultures were incubated 24 h at 37 °C, and then, cell viability was determined by the MTS/PMS method according to the manufacturer’s instructions (Promega). After 3 h at 37 °C and 5% CO2, the absorbance was determined at 490 nm. Media absorbance obtained from each antiviral concentration and the mock treated cells were used to plot dose–response curves that were fitted by nonlinear regression using GraphPad Prism 8 software. The cytotoxic concentration 50 (CC50) values, which are defined as the concentration of compound that reduces absorbance at 490 nm by 50% with respect to mock treated cells, were estimated from dose–response curves.
Viral yield assay against epidemic CHIKV strains
To confirm the antiviral activity of the selected hit Farudodstat (CBR-050-264-248-0) against CHIKV La Reunion (IOL) and Caribbean (Asian) lineages we evaluated the compound effect on viral yield. To this end, Vero cells were seeded in 24-well plates at a density of 1.5 × 105 cells per well in DMEM-10 and incubated at 37 °C and 5% CO2. After 24 h, the culture medium was removed, monolayers were washed twice with PBS and infected with CHIKV (MOI 0.05 PFU/cell). Infected cells were incubated for 1 h at 37 °C and 5% CO2 after which, inoculum was removed and monolayers were washed twice with PBS. Infected cells were treated with 20, 2 and 0.2 µM Farudodstat in DMEM-2 and incubated at 37 °C and 5% CO2. Untreated infected cells and uninfected cells were included as controls. At 24 h post infection (p.i.), culture supernatants were collected and progeny virus were titrated in Vero cells by the PFU method.
Antiviral activity of Farudodstat in different cell lines
The effect of Farudodstat on CHIKV replication was evaluated in different cell lines: human Huh-7 and HEK293; mouse fibroblasts NIH-3t3 and hamster BHK. First, FFU reduction assays were performed as described above for Vero cells. To this end, cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well in their corresponding culture medium and incubated at 37 °C and 5% CO2. After 24 h, cells were infected with CHIKV-ZsGreen (MOI 0.05 FFU/cell) and simultaneously treated with Farudodstat from 20 μM to 9 nM in triplicate. Three independent assays were performed. The focus count data for each antiviral concentration and the untreated infected cells were used to plot dose–response curves that were fitted by nonlinear regression using GraphPad Prism 8 software. Effective concentration 50 (EC50) values were calculated from dose–response curves and cytotoxicity estimate with crystal violet was performed as described above.
Second, to verify the effect of Farudodstat on viral yield in each culture, infected cells were treated with 20, 2 and 0.2 µM Farudodstat and incubated at 37 °C and 5% CO2. Untreated infected cells and uninfected cells were included as controls. At 24 h p.i., culture supernatants were collected and progeny virus were titrated by FFU method in Vero cells.
Rescue of CHIKV replication by exogenous uridine
To test whether addition of exogenous uridine, a pyrimidine ribonucleoside, could revert the antiviral activity of Farudodstat and rescue viral replication, Vero cells were infected with CHIKV-ZsGreen (MOI 0.05 FFU/cell) and treated with Farudodstat (20 and 2 µM) in the presence of increasing concentrations of Uridine (Sigma) (10; 50 and 100 µM). The antiviral activity was evaluated by FFU reduction assays and viral yield assays as described above.
Statistical analysis
The data analysis was performed using GraphPad Prism 8 software. The results are presented as mean ± SD (standard deviation). Each experiment, except for primary and secondary screening, was run in triplicate and repeated independently at least two times. Statistical significance was determined using the Unpaired t-test (Mann-Whitney test) for comparisons between two groups. P value of less than 0.05 was considered statistically significant.
Results
Primary screening identified 11 active compounds with high selectivity index
To identify potentially active compounds against CHIKV, we carried out reporter-based High Throughput Screening (HTS) antiviral assay using infectious CHIKV expressing ZsGreen (CHIKV-ZsGreen) under the control of the virus subgenomic promoter (). The primary screening comprised assessment of the antiviral activity of compounds in the ReFRAME library at 10 µM in Vero cells using automated numbering of fluorescent foci as a readout (mean z’ score 0.76). Then, staining of fixed cells with crystal violet allowed to estimate cytotoxicity. One hundred and forty-eight compounds reduced >85% Abs595nm in comparison with mock-treated cells (0.1% DMSO) (>15% cytotoxicity) and were considered cytotoxic. Among the remaining non-cytotoxic compounds (n = 1,452), we retested 16 candidates that had showed more than 60% reduction in the number of fluorescent foci forming units (fFFU) relative to control (0.1% DMSO) in dose-dependent assays (Figure 1A). Dose–response curves were performed starting at 20 µM to calculate the EC50 values for each compound, and cytotoxicity was estimated with crystal violet staining. The approach confirmed antiviral activity of 13 compounds: 2 presented EC50 values in the low µM range but displayed low selectivity index (SI ∼ 3), 4 presented EC50 values in the low µM range with SI > 3, and 7 compounds showed EC50 values in the nM range and SI > 25 (Figure 1B; Table 1). In turn, among the compounds that proved active, CBR-001-584-962-4 (Chloroquine) was previously shown to have anti CHIKV activity (; ), indicating that the screening method effectively identified active compounds against CHIKV. Finally, we prioritized 3 hits for confirmation with distinct mechanisms of action: CBR-001-856-178-9 (N-Hydroxycytidine); CBR-001-573-415-3 (Gemcitabine Elaidate), and CBR-050-264-248-0 (Farudodstat).
FIGURE 1
TABLE 1
| Hit compound | Primary screening | Antiviral potency determination | ||||
|---|---|---|---|---|---|---|
| FFU reduction 10 µM (%)1 | Cytotoxicity 10 µM (%)2 | EC50 (µM)3 | CC50 (µM)4 | SI | Conclusion | |
| CBR-001-586-214-3 | 68 | 3 | >6.7 | 15 | - | Inactive |
| CBR-001-846-979-9 | 91 | 10 | 0.6 | >20 | >33 | Active |
| CBR-001-586-825-4 | 99 | 12 | 0.1 | >20 | >200 | Active |
| CBR-050-264-248-0 | 100 | 0 | 0.3 | >20 | >67 | Active |
| CBR-001-851-585-0 | 74 | 12 | >20 | 16 | - | Inactive |
| CBR-001-154-313-8 | 79 | 0 | 5.7 | 18 | 3 | Active Low SI |
| CBR-050-127-705-0 | 87 | 15 | 0.7 | >20 | >29 | Active |
| CBR-001-572-426-2 | 82 | 0 | 4.2 | >20 | >5 | Active |
| CBR-001-586-193-5 | 83 | 4 | 6.2 | 20 | 3 | Active Low SI |
| CBR-001-573-606-8 | 83 | 0 | 0.1 | >20 | >200 | Active |
| CBR-001-584-962-4 | 100 | 0 | 2.1 | >20 | >10 | Active |
| CBR-001-573-415-3 | 96 | 11 | 0.7 | >20 | >29 | Active |
| CBR-001-856-178-9 | 88 | 11 | 2.1 | >20 | >10 | Active |
| CBR-050-353-072-1 | 100 | 15 | 0.8 | >20 | >25 | Active |
| CBR-001-584-587-1 | 63 | 0 | >6.7 | 19 | - | Inactive |
| CBR-001-844-872-1 | 79 | 5 | 5.9 | >20 | >3 | Active |
Antiviral activity and cytotoxicity of the 16 selected compounds. Primary screening and antiviral potency determination.
Reduction of the number of CHIKV-ZsGreen Foci Forming Units (FFU) relative to mock treated control at 10 µM of compound.
Cytotoxicity respecting mock infected control estimated after crystal violet staining.
Effective concentration 50 (EC50) is defined as the concentration of compound that reduces CHIKV-ZsGreen FFU, number by 50% with respect to mock treated control.
Cytotoxic concentration 50 (CC50) is defined as the concentration of compound that reduces crystal violet absorbance at 585 nm by 50% with respect to mock treated control. SI: Selectivity Index = CC50/EC50. Means of two independent experiments are shown. Compound selected for confirmation of activity are marked in bold.
Antiviral potency and cytotoxicity determinations identified novel Anti-CHIKV candidate drugs
First, to re-evaluate antiviral potency and selectivity of N-Hydroxycytidine; Gemcitabine Elaidate, and Farudodstat, dose–response curves against CHIKV-ZsGreen were performed with newly provided compounds in triplicate, and the cytotoxicity in Vero cells was assessed by MTS/PMS method. As expected, these compounds showed EC50 values in the low µM and nM range, and CC50 > 500 μM, resulting in high SI (SI > 200) (Figure 2).
FIGURE 2
N-Hydroxycytidine, a cytidine analog that is the active molecule of the oral prodrug Molnupiravir, showed an EC50 = 2.17 ± 0.97 µM and SI > 231. Gemcitabine Elaidate, a derivative of gemcitabine, which is a deoxynucleoside analogue, presented an EC50 = 0.79 ± 0.24 µM and SI > 630.
Finally, Farudodstat, a DHODH inhibitor, emerged as the most active compound (EC50 = 0.31 ± 0.02 µM, SI > 1,613). Given that Farudodstat was not previously studied as an antiviral agent and its clinical safety and efficacy in treatments with oral formulations was previously reported (ClinicalTrials.gov: NCT02342652, NCT01992367, NCT03451084 and NCT05865041), it was chosen for further characterizing its antiviral profile and mode of action against CHIKV.
Farudodstat is active against epidemic CHIKV strains and inhibits virus replication in different cell lines
To evaluate the antiviral activity of Farudodstat against epidemic CHIKV strains, viral yield assays against La Reunion (ECSA genotype, IOL lineage) and Caribbean (Asian genotype) strains were performed. Farudodstat inhibited infective virus production of both strains in a dose-dependent manner, reducing progeny viral titers by 2-log with comparable results (Figure 3).
FIGURE 3
Given that Farudodstat was initially developed as a DHODH inhibitor, we hypothesized that its antiviral activity is likely host targeted. Then, we first assessed the ability of Farudodstat to inhibit virus replication in FFU reduction assays in cell lines of different origin infected with CHIKV-ZsGreen: human epithelial Huh-7 and HEK293T cells; mouse fibroblasts NIH-3t3 and hamster BHK cells. Farudodstat was no cytotoxic or slightly toxic (i.e. 26% reduction of viability in HEK 293T cells) at the maximum concentration evaluated (20 µM). Potent antiviral activity was observed in Huh-7, HEK293 and NIH-3t3 with EC50 in the low µM and nM range, with higher antiviral potency in human cell lines compared to mouse NIH-3t3 cells (Table 2). However, Farudodstat was inactive in hamster BHK cells (EC50 > 20 µM), which expectedly suggests a host-dependent activity.
TABLE 2
| Cell line | Origin of cell line | Farudodstat EC50 (µM)1 |
|---|---|---|
| Vero | African green monkey | 0.31 ± 0.02 |
| Huh-7 | Human | 0.09 ± 0.02 |
| HEK293 | 0.09 ± 0.01 | |
| NIH-3t3 | Swiss mouse | 1.33 ± 0.82 |
| BHK | Hamster | >20 |
Antiviral activity of Farudodstat against CHIKV-ZsGreen in different cell lines.
Means of three independent experiments are shown.
Effective concentration 50 (EC50) is defined as the concentration of compound that reduces CHIKV-ZsGreen FFU, number by 50% with respect to mock treated control.
Second, to verify the effect of Farudodstat on viral yield, culture supernatants from treated infected cells (Vero, Huh-7, HEK293 or NIH-3t3) and mock-treated controls were collected at 20 h p.i. and infectious virus particles were titrated by the FFU method in Vero cells. Results showed that treatment with Farudodstat reduced the production of infectious progeny virus in a concentration-dependent manner (Figure 4). Farudodstat reduced viral yield by 2-Log in Vero and Huh-7 cells and 1-Log NIH 3T3 cells treated at 20 μM, and it reduced viral yield by 1.5-Log in HEK293 cells at 2 µM. As shown above, Farudodstat inhibited viral replication with a potency that varied depending on the species of origin of cell line used; it was more active in human and monkey cell lines and less active in mouse cells. Together, these results add new evidence on the anti-CHIKV activity of Farudodstat as a host-targeted antiviral agent.
FIGURE 4
The antiviral activity of Farudodstat is reversed by uridine addition
To validate the pyrimidine biosynthesis pathway as the target of Farudodstat for anti-CHIKV activity, we investigated whether addition of exogenous uridine, a pyrimidine ribonucleoside, could revert the antiviral activity of Farudodstat and rescue viral replication. To this end, antiviral activity was assessed by means of fFFU reduction (Figure 5A) and viral yield assays (Figure 5B) in cells infected with CHIKV-ZsGreen and treated with Farudodstat (20 and 2 µM) in the presence of increasing concentrations of Uridine (10; 50 and 100 µM). While Uridine alone did not affect CHIKV-ZsGreen replication, its addition to infected cells treated with Farudodstat rescued viral replication in a dose-dependent manner. Complete restoration of viral replication was observed with 50 µM of Uridine as assessed by FFU quantification and infectious progeny production. Notably, at 20 µM of Farudodstat and 50 µM of Uridine, viral replication appeared fully restored based on FFU counts, yet viral yield still showed an 82% reduction. Overall, these results support inhibition of pyrimidine biosynthesis as the mechanism of action of Farudodstat against CHIKV.
FIGURE 5
Discussion
To identify new bioactive compounds against CHIKV under a repositioning strategy, we implemented a cell-based high-throughput screening to evaluate 1,600 compounds from the ReFRAME library (), followed by hit confirmation. The approach yielded 11 CHIKV antiviral candidates that exhibited activity in the low µM to nM range with SI > 3. Based on previous data, 3 out of the 11 hits were selected for further evaluation of antiviral potency and selectivity.
First, N-Hydroxycytidine (CE50 = 2.17 ± 0.97 µM and SI > 231) is a cytidine analog and the active molecule of the oral prodrug Molnupiravir. This broad-spectrum antiviral was reported active against enterovirus (), Respiratory Syncytial Virus and influenza virus (Yoon et al., 2018), Coronaviruses (; Vangeel et al., 2022; Strizki et al., 2023) and VEEV (Urakova et al., 2018), among others. Its mechanism of action and pharmacokinetics were broadly studied (), and its safety and antiviral efficacy was assessed in different clinical trials (; ). In 2021, Molnupiravir received emergency use authorization in the USA for COVID (). N-Hydroxycytidine (EIDD-1931) was shown to have broad activity against alphaviruses, including CHIKV (Urakova et al., 2018; Rosales-Rosas et al., 2024). Furthermore, it displayed synergistic activity in combination with sofosbuvir, another direct-acting RNA virus replication inhibitor, both in vitro and in a mouse model of CHIKV infection (Verwimp et al., 2025).
Second, Gemcitabine Elaidate (CE50 = 0.79 ± 0.24 µM and SI > 630) showed a high potency against CHIKV. It is a lipophilic, unsaturated fatty acid ester derivative of gemcitabine, which is a deoxynucleoside analogue with broad-spectrum antitumor activity (). Gemcitabine was reported to have antiviral activity against influenza virus and SARS-CoV-2 in vitro (). In clinical trials, Gemcitabine Elaidate has been used for the treatment of different types of cancers (; Venugopal et al., 2015) by intravenous administrations. Given its poor absorption and the rapid pre-systemic metabolism (Stuurman et al., 2013) its oral formulation was not successful, hampering its further development as a CHIKV antiviral treatment.
Finally, Farudodstat (ASLAN 003) emerged as the most active compound with an outstanding SI (CE50 = 0.31 ± 0.02 µM, SI > 1613). Farudodstat is an inhibitor of the human dihydroorotate dehydrogenase (DHODH) (Zhou et al., 2020). This mitochondrial enzyme catalyzes the rate-limiting oxidation of dihydroorotate to orotate in the de novo pyrimidine biosynthesis pathway, which becomes essential when the salvage pathway, normally sufficient for quiescent or differentiated cells, cannot meet the heightened nucleotide demands of rapidly proliferating or virus-infected cells (Traut, 1994; Okesli et al., 2017; Reis et al., 2017). Thus, Farudodstat would likely act as an antiviral by targeting DHODH and thereby reducing the pyrimidine pool required for viral replication.
Farudodstat was primarily studied as an antitumor agent. Preclinical studies showed good bioavailability, efficacy and safety in mouse models of acute myeloid leukemia (AML) (Zhou et al., 2020). It also demonstrated safety in adults in Phase I (ClinicalTrials.gov: NCT02342652 and NCT01992367) and Phase II clinical trials for the treatment of AML and alopecia areata in oral formulations (ClinicalTrials.gov: NCT03451084 and NCT05865041, respectively). Importantly, pharmacokinetic data from clinical trials (ClinicalTrials.gov: NCT03451084) showed that at an optimal dose for oral administration it achieved maximum plasma concentration (Cmax) above Farudodstat EC90 (0.51 µM) against CHIKV.
While DHODH has been proposed as an antiviral target against distinct viruses () such as influenza (Sibille et al., 2022), RSV (), and SARS-CoV-2 (Stegmann et al., 2022), studies on the antiviral activity of Farudodstat remain limited and inhibition of CHIKV replication is novel. Given its clinical safety and efficacy in treatments with oral formulations, Farudodstat was chosen to further characterize its antiviral profile, and its mode of action against CHIKV.
In line with our results indicating that the magnitude of inhibition of Farudodstat differed across cell lines, with higher antiviral activity observed in human and nonhuman primate cells and reduced activity in murine cells, the antiviral potency of some hDHODH inhibitors was already shown to exhibit species-specific dependency using cells of different animal origin in vitro (), and suggests a host-dependent mechanism of action. As a matter of fact, supplementation of infected Vero cells with exogenous Uridine restored viral replication in the presence of Farudodstat, likely through replenishment of the intracellular pyrimidine pool. This metabolic rescue suggests that inhibition of the de novo pyrimidine biosynthesis pathway contributes, at least in part, to the compound’s anti-CHIKV activity. A well-characterized DHODH inhibitor, Brequinar (Schrell et al., 2025), was also active against CHIKV in our screening procedure, showing a CE50 similar to that obtained for Farudodstat (0.11 µM). Although off target activity cannot be ruled out, these data together with complete restoration of viral replication after uridine addition strongly suggest DHODH as the antiviral target. Together, these data establish Farudodstat as a Host-targeting antiviral (HTA) with robust activity against CHIKV. As for other DHODH inhibitors (Zheng et al., 2022; Zhang et al., 2025) and HTAs, Farudodstat is expected to display broad-spectrum antiviral activity.
A significant challenge for antivirals is the potential selection of resistant viral variants. While host-targeting antivirals generally present a higher genetic barrier to resistance compared to direct-acting antivirals, adaptive mutations have been observed under DHODH inhibitor pressure, such as NS5 mutations in dengue virus selected by Brequinar (). Combining DHODH inhibitors with nucleoside analogs offers a synergistic strategy to enhance potency and mitigate resistance (Stegmann et al., 2022; Schrell et al., 2025; Sirihongthong et al., 2025). Mechanistically, depletion of intracellular pyrimidine pools through DHODH inhibition, would increase nucleotide analog incorporation in viral RNA by reducing competition with native nucleotides. In this line, 4′-Fluorouridine (4′-FlU) was reported to have potent activity against CHIKV by targeting the nsP4 RdRp (Yin et al., 2024). Although CHIKV can develop nsP4 mutations that reduce 4′-FlU sensitivity, these variants typically suffer from decreased viral fitness (Yin et al., 2025). Based on the previous studies addressing synergy between DHODH inhibitors and nucleoside analogs, we speculate that DHODH inhibition can re-sensitize 4′-FlU-resistant mutants, then combination of Farudodstat and 4′FIU emerges as a robust therapeutic avenue for CHIKV antiviral development.
In conclusion, our screening strategy enabled the identification of new active hits against CHIKV. We defined the in vitro antiviral profile of Farudodstat and elucidated its mechanism of action. Its potency, selectivity, and favorable safety and pharmacokinetic properties support its potential as a promising oral antiviral candidate for the treatment of CHIKV fever. Future studies will assess the activity of Farudodstat against additional alphaviruses and arboviruses and evaluate its in vivo efficacy in a mouse model, thereby advancing its progression toward preclinical development.
Statements
Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Author contributions
EC: Data curation, Formal analysis, Writing – original draft, Writing – review and editing, Conceptualization, Investigation, Methodology, Visualization, Funding acquisition. DEA: Writing – original draft, Writing – review and editing, Conceptualization, Validation, Methodology, Project administration, Resources, Supervision.
Funding
The author(s) declared that financial support was received for this work and/or its publication. The work was supported by funding from Universidad Nacional de San Martin, Argentina (UNSAM investiga 2025 80020250100099SM, to EC).
Acknowledgments
The authors thank The Scripps Research Institute, Calibr for the ReFrame library and supplying the compounds. CHIKV La Reunion infectious clone expressing ZsGreen was a kind gift of Kenneth A. Stapleford (Department of Microbiology, New York University Grossman School of Medicine, New York, USA). CHIKV La Reunion (wt) and Caribe infectious clone were a kind gift of Marco Vignuzzi (Institut Pasteur, Viral Populations and Pathogenesis Unit, Paris, France).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Summary
Keywords
alphavirus, Farudodstat, phenotypic screening, ReFRAME library, reporter virus
Citation
Castro E and Alvarez DE (2026) High-throughput repurposing screen identifies a DHODH inhibitor as a potent antiviral candidate against chikungunya virus. Front. Drug Discov. 6:1846068. doi: 10.3389/fddsv.2026.1846068
Received
02 April 2026
Revised
05 May 2026
Accepted
11 May 2026
Published
29 May 2026
Volume
6 - 2026
Edited by
José L Medina-Franco, National Autonomous University of Mexico, Mexico
Reviewed by
Floriano Paes Silva-Jr, Oswaldo Cruz Foundation (Fiocruz), Brazil
Harish Kundaikar, DY Patil Deemed to be University, India
Updates
Copyright
© 2026 Castro and Alvarez.
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*Correspondence: Diego Ezequiel Alvarez, dalvarez@iibintech.com.ar; Eliana Castro,ecastro@iib.unsam.edu.ar
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