Identification of IMP Dehydrogenase as a Potential Target for Anti-Mpox Virus Agents

ABSTRACT Mpox virus (formerly monkeypox virus [MPXV]) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.026 to 0.89 μM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the postentry step to reduce the intracellular production of virions. Knockdown of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. This evidence shows that IMPDH is a potential target for the development of anti-MPXV agents. IMPORTANCE Mpox is a zoonotic disease caused by infection with the mpox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against mpox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against mpox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-mpox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited mpox virus propagation and exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested IMP dehydrogenase as a potential target for the development of anti-mpox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-mpox virus activity than mycophenolic acid.

M pox (formerly monkeypox) is a zoonotic disease caused by infection with the mpox virus (MPXV). MPXV is an enveloped virus with a double-stranded DNA genome of approximately 190 kb in length. It belongs to the genus Orthopoxvirus of the family Poxviridae, which includes the smallpox, vaccinia, and cowpox viruses and other animal-associated poxviruses (1). The natural hosts of MPXV are most likely rodents, and MPXV is transmitted to humans by infected animals through bites or contact with the blood or body fluids. MPXV is also transmitted through human-to-human contact via droplets or body fluids (2). Starting in May 2022, wide-scale mpox cases were reported in multiple countries where this disease had not been previously endemic, and by November 2022, more than 80,000 cases of infection had been reported in over 110 countries, mainly in Europe and the United States, with most of these infections transmitted via sexual contact (3). Given the current status of MPXV and possible future outbreaks, medical countermeasures and further research on MPXV should be developed.
The smallpox vaccine has recently been approved for clinical use against mpox in the United States (4). Brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration (FDA) under the agency's animal rule (5). Brincidofovir is a lipid conjugate of cidofovir, a nucleoside analog active against cytomegalovirus, and it suppresses viral genome replication by inhibiting viral DNA polymerase (6)(7)(8)(9)(10). The efficacy of brincidofovir on mpox has not been established, and a recent clinical report showed no clinical benefit to mpox patients and rather indicated liver toxicity by brincidofovir (5,11). Tecovirimat is an FDA-approved anti-smallpox drug that inhibits virion maturation; however, its clinical efficacy against mpox is poorly documented because of the limited chance of clinical treatment (5,(11)(12)(13)(14). In cell culture studies, tecovirimat treatment has been reported to induce drug-resistant viruses (15,16), although the clinical drug resistance profile is not clear. Thus, development of a new anti-MPXV strategy would provide alternative therapeutic options.
In this study, we aimed to identify a new drug target for MPXV. We screened a chemical compound library using an MPXV infection cell culture assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV replication. An analysis of the anti-MPXV activity of MPA showed that IMP dehydrogenase (IMPDH) and the guanine nucleotide biosynthesis pathway have significant roles in regulating MPXV replication. By targeting IMPDH, we identified compounds with more potent anti-MPXV activity than MPA. Therefore, we propose IMPDH as a potential target for the development of anti-MPXV agents.

RESULTS
Anti-MPXV activity of gemcitabine, trifluridine, and mycophenolic acid. To identify compounds that inhibit MPXV propagation, we screened 121 compounds previously reported to have anti-vaccinia virus activity; however, most of their modes of action are unknown (17) (see Table S1 in the supplemental material). On the first screen, VeroE6 cells were infected with MPXV Zr-599 (Congo Basin strain, clade I) at a multiplicity of infection (MOI) of 0.1 for 72 h in the presence of 10 mM each compound, with the exception of two compounds treated at 2 mM (Table S1). The cytopathic effect induced by MPXV propagation was detected by observing cell morphology using a microscope (Fig. 1A) and quantifying cell viability using a high-content imaging analyzer (see Fig. S1 in the supplemental material) (18). Although cells remained confluent without virus inoculation, inoculation with MPXV induced extensive cell death after 72 h ( Fig. 1Aa and Ab). As positive controls, treatment with tecovirimat and brincidofovir protected cells from MPXV-induced cytopathic effects and augmented the number of surviving cells to 223and 103-fold, respectively ( Fig. 1Ac and Ad; see also Fig. S1). The screening revealed 74 compounds that increased the survival cell number by more than 50-fold relative to that of the dimethyl sulfoxide (DMSO)-treated control cells (Fig. S1).
Among the hit compounds, we focused on three compounds, gemcitabine, trifluridine, and mycophenolic acid (MPA) (Fig. 1Ae to Ag), because they have been reported to inhibit the replication of multiple virus species, such as adenovirus, herpes simplex virus, Zika virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and dengue virus (19)(20)(21)(22)(23). In addition, topical trifluridine was used as a treatment for orthopoxvirus infection (24,25). We confirmed the anti-MPXV activity of the three compounds by detecting viral protein production in MPXV-infected cells by immunofluorescence analysis. VeroE6 cells infected with MPXV at an MOI of 0.1 for 1 h were incubated with each compound for another 23 h and then fixed to detect anti-MPXV protein together with 49,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The cells did not show cytopathology under these conditions at 24 h after virus inoculation (Fig. 1B, blue). As shown in Fig. 1B, MPXV protein expression was drastically reduced upon treatment with gemcitabine, trifluridine, and MPA ( Fig. 1Be to Bg), which was similar to results obtained for tecovirimat and brincidofovir ( Fig. 1Bc and Bd). These antiviral effects were also observed in the human-derived cell line Huh7 cells (Fig. 1C), suggesting that the anti-MPXV activity of these compounds was not dependent on the cell type.
To examine the activity of these compounds against multiple orthopoxviruses, we analyzed their effect on infection assays using the MPXV Liberia strain (West African strain, clade IIa), MPXV clinical isolate in the 2022 outbreak, SPL-mpx2A7 strain (clade IIb), vaccinia virus, and cowpox virus. As shown in Fig. 2, gemcitabine, trifluridine, and MPA clearly reduced the expression of viral proteins in cells inoculated with all viruses (Fig. 2). These results suggest that gemcitabine, trifluridine, and MPA possess antiviral activities against a wide range of orthopoxviruses.
Dose response curve of anti-MPXV activity for gemcitabine, trifluridine, and MPA. To quantify the antiviral effect of drugs, we compared the results from the two methods, the plaque assay (26) and the quantification of intracellular viral DNA (27), frequently used in the previous studies. For plaque assay, VeroE6 cells were infected with MPXV and cocultured with each compound in a 24-well plate. After 72 h of infection, cells were fixed and were then stained to count plaque number (see Fig. S2 in the supplemental material). For viral DNA quantification assay, VeroE6 cells were infected with MPXV and cocultured with each compound in a 96-well plate. After 24 h of infection, intracellular viral DNA levels were analyzed by real-time PCR. As a comparison of the 50% maximal inhibitory concentrations (IC 50 ) calculated by the two methods showed no significant difference (see Table S2 in the supplemental material), we employed the viral DNA  IMPDH as a Target for Anti-MPXV Agents Microbiology Spectrum cytotoxicity (Fig. 3C). Cytotoxicity was further assessed using Huh7, primary human hepatocytes (PHH), and primary human peripheral blood mononuclear cells (PBMC) (see Fig.  S3 in the supplemental material). The 50% and 90% maximal inhibitory concentrations (IC 50 and IC 90 ) as well as the 50% maximal cytotoxic concentrations (CC 50 ) of each compound are shown in Fig. 3B and C and in Fig. S3. These results indicated that the three compounds exhibited dose-dependent anti-MPXV activity without cytotoxicity and presented a lower IC 90 than brincidofovir. Gemcitabine, trifluridine, and MPA target the postentry phase in MPXV life cycle. A schematic diagram of the MPXV life cycle is shown in Fig. 4A. MPXV attaches to the surface of a target cell to enter intracellularly, where the viral core is delivered into the cytoplasm (entry phase) (Fig. 4A). Through early transcription, protein synthesis, and uncoating of the core, viral DNA replicates and drives intermediate and late transcription, which is followed by viral assembly in a specific compartment called the cytoplasmic viral factory, and further stepwise virion maturation produces infectious virions (postentry phase) (Fig. 4A) (29). To clarify the phase of the MPXV life cycle that is inhibited by the compounds, we performed a time-of-drug-addition assay, in which the entry and postentry phases are distinguished by changing the treatment time of the compound (  (Fig. 4B, left). We assessed the antiviral activity by detecting intracellular viral DNA using real-time PCR for each condition. Brincidofovir, a positive control that inhibits viral genome replication, showed significant antiviral activity in conditions a and c but not in condition b (Fig. 4B, right) (28). In contrast, heparin, which is reported to inhibit viral entry, showed significant antiviral activity under condition b (Fig. 4B, left). These results indicate that our time-of-drug-addition assay could successfully distinguish entry inhibitors from those that inhibit viral replication. As shown in Fig. 4B, gemcitabine, trifluridine, and MPA were significantly reduced under conditions a and c but not under condition b, suggesting that these compounds inhibit the postentry phase in the MPXV life cycle.
Observation of intracellular structures by electron microscopic analysis. Poxvirus infection induces the formation of an intracellular structure called the cytoplasmic factory, which represents a hallmark of infected cells and is involved in virion assembly (31)(32)(33). We then observed the intracellular morphological features of the compound-treated cells by transmission electron microscopy. Tecovirimat, a particle maturation inhibitor, was used as the positive control (34). Crescents, immature, mature, and wrapped particles were observed in DMSO-treated MPXV-infected cells ( Fig. 5Ab and Ac), while crescents, immature, and mature virions but not wrapped virions were observed in tecovirimattreated cells (Fig. 5Bd and Bh), which is consistent with the mode of action of tecovirimat. In contrast, few virions were observed in gemcitabine-, trifluridine-, and MPA-treated cells (Fig. 5Be to Bg and Bi to Bk). These observations are consistent with the results obtained for gemcitabine, trifluridine, and MPA, which suppress the phase before virion assembly.
Mycophenolic acid suppresses MPXV replication through inhibition of IMPDH. Among the three identified compounds, gemcitabine and trifluridine are nucleoside analogs that are likely to target viral polymerase similar to brincidofovir (20)(21)(22)28). Therefore, we analyzed the mechanism of action of MPA against MPXV. MPA inhibits IMP dehydrogenase (IMPDH), which is the rate-limiting enzyme of GTP de novo synthesis (19,23,35) (Fig. 6A). IMPDH is composed of the following two isoforms: IMPDH1 and IMPDH2. To examine the role of IMPDH in MPXV replication, we transfected Huh7 cells with or without small interfering RNA (siRNA) targeting IMPDH1/2 or randomized control siRNA. At 48 h posttransfection, we confirmed the knockdown of both endogenous IMPDH1 and IMPDH2 at both the mRNA and protein levels ( Fig. 6Bi and Bii; see also Fig. S4 in the supplemental material). At 48 h posttransfection with siRNA, we infected the cells with MPXV for 24 h and quantified the intracellular MPXV DNA by real-time PCR to examine the MPXV replication levels. As shown in Fig. 6Biii and Fig.  S4B, the MPXV DNA levels were significantly reduced in IMPDH1/2-depleted cells.
To further examine the relevance of the guanosine nucleotide synthetic pathway in the anti-MPXV activity of MPA, we performed a rescue experiment by complementing the MPA treatment with guanosine, a downstream product of the IMPDH-catalyzing step. MPXV-infected Huh7 cells were treated with MPA in the presence or absence of varying concentrations of guanosine and examined to detect viral DNA in cells at 24 h posttreatment. As shown in Fig. 6C, supplementation with guanosine clearly recovered the MPA-mediated reduction of viral DNA levels in a dose-dependent manner, suggesting that IMPDH and its guanosine synthesis pathway are targets for the observed anti-MPXV activity of MPA. Thus, IMPDH may be crucial for the efficient replication of MPXV.
IMPDH inhibitors reduce MPXV propagation. The above results indicate that IMPDH is a potential target for the development of anti-MPXV agents. Therefore, the effects of known IMPDH inhibitors mycophenolate mofetil, AVN-944, merimepodib, and ribavirin were investigated. MPXV-infected Huh7 cells were incubated with these IMPDH inhibitors for 24 h to assess MPXV replication by detecting intracellular MPXV DNA levels by real-time PCR and cytotoxicity by the WST assay. All of these IMPDH inhibitors clearly reduced MPXV DNA levels in a dose-dependent manner without showing cytotoxic effects ( Fig. 7A and B), thus supporting the essential role of IMPDH

DISCUSSION
In this study, we screened a compound library using an MPXV infection cell culture assay and identified 74 compounds as first hits. Among the hit compounds, we focused on the three compounds gemcitabine, trifluridine, and MPA and showed that they inhibited multiple strains of MPXV, vaccinia virus, and cowpox virus. Gemcitabine and trifluridine present anti-MPXV activities that are equivalent to or more potent than that of brincidofovir, and these nucleoside analogs are likely to show similar targeting of viral polymerization as brincidofovir and become incorporated into the viral genome or interfere with viral polymerase, resulting in the suppression of viral genome replication (22,36,37). Gemcitabine targets poliovirus RNA polymerase to inhibit viral replication (38). The ability of gemcitabine and trifluridine to target MPXV polymerization was supported by the fact that these compounds inhibited the postentry phase and reduced intracellular virion accumulation.
MPA not only has anti-pox virus activity but has also been used to create recombinant vaccinia viruses (26,(39)(40)(41). MPA inhibits the enzymatic activity of IMPDH, the rate-limiting enzyme for the de novo synthesis of guanine nucleotides. In this study, we demonstrated that the anti-MPXV activity of MPA is mediated by the inhibition of IMPDH and the guanine synthetic pathway. Inhibition of IMPDH decreases the guanine nucleotide pool, which likely results in the decreased efficiency of MPXV DNA and/or RNA synthesis. In addition to this mode of action, the inhibition of the nucleic acid synthetic pathway induced the expression of interferon-stimulated genes to inhibit hepatitis C and E virus replication in Huh7 cells (21,42,43). We addressed this possibility by treating Huh7 cells with MPA at anti-MPXV effective concentration ranges; however, we did not observe significant induction of the representative interferon-stimulated genes ISG15 and ISG56 (see Fig. S5 in the supplemental material). Consistent with the essential role of nucleic acid synthesis in the replication of most or all of the virus species, MPA has been reported to inhibit a wide range of viruses, including dengue, Zika, SARS-CoV-2, hepatitis C, Lassa, and Epstein-Barr viruses (19,23,(44)(45)(46). Thus, targeting IMPDH may contribute to the development of pan-antiviral agents beyond anti-orthopoxvirus drugs. Although host cells also require guanine synthesis for survival and function, we observed a significant window for drug concentration ranges showing anti-MPXV activity without cytotoxicity, thus indicating that IMPDH represents a realistic drug target. Actually, IMPDH-targeting agents are in clinical use for the treatment of diseases, including rheumatoid arthritis, psoriasis, and nephrotic syndrome, and are promising targets for the development of new immunosuppressants and anticancer agents. In this study, we identified mycophenolate mofetil, AVN-944, and merimepodib as the most potent compounds against MPXV. Further antiviral analyses under more physiologically relevant conditions, such as primary cells or animal models, would demonstrate the usefulness of IMPDH inhibition in antiviral strategies.
In conclusion, our findings suggest that IMPDH can serve as a potential target for the development of anti-MPXV agents. We found that IMPDH inhibitors exerted antiviral activities against a wide range of orthopoxviruses by inhibiting viral replication. Further studies are ongoing to demonstrate the usefulness of IMPDH-targeting agents in eliminating MPXV, with the goal of improving virus-induced pathogenesis and identifying more potent antiviral agents that target IMPDH.

MATERIALS AND METHODS
Chemical compounds. The anti-vaccinia virus compound library was prepared based on a previous study (17) by selecting compounds from the Inhibitor Library (Selleck; L1100), Anti-infection Compound IMPDH as a Target for Anti-MPXV Agents Microbiology Spectrum Library (Selleck; L3100), and Immunology/Inflammation Compound Library (Selleck; L4100). A list of compounds in the library is presented in Table S1 in the supplemental material. Brincidofovir and AVN-944 were purchased from Cayman Chemical Company and MedChemExpress, respectively. Guanosine and ribavirin were purchased from Sigma-Aldrich. The compounds were dissolved in dimethyl sulfoxide (DMSO). Cell culture. An African green monkey kidney-derived cell line (VeroE6 cells) and a human hepatoma cell line (Huh7 cells) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Fujifilm Wako), which was supplemented with penicillin and streptomycin sulfate (Thermo Fisher Scientific) and 5% fetal bovine serum (FBS) (Nichirei) for VeroE6 or 10% FBS for Huh7. Primary human hepatocytes (PHH) (PhoenixBio) were cultured with DMEM supplemented with 20 mM HEPES, 100 units/mL penicillin, 100 mg/mL streptomycin, 10% FBS, 44 mM NaHCO 3 , 5 ng/mL epidermal growth factor (EGF), and 50 nM dexamethasone, as described previously (47). Primary human peripheral blood mononuclear cells (PBMC) (Lonza) were cultured with X-VIVO-15 (Lonza). The cells were then incubated under 5% CO 2 at 37°C.
Compound screening. VeroE6 cells were seeded at 2 Â 10 4 cells/well in a 96-well plate. At 16 h after seeding, the cells were treated with MPXV Zr-599 (clade I) (48) at an MOI of 0.1 and with 10 mM each compound for 72 h. We confirmed robust cytopathology upon MPXV infection using DMSO as a control (Fig. 1Ab). We screened for compounds that protected cells from MPXV-induced cell death. Cells fixed with 4% paraformaldehyde and then stained with DAPI were counted with an ImageXpress micro confocal high-content imaging analyzer (Molecular Devices), as previously described (18) (see Fig. S1 in the supplemental material). Compounds that increased the number of viable cells by more than 50-fold compared to the DMSO-treated control were selected as the first hits (Fig. S1).
Cytotoxicity assay. The cell viability assay was performed using the Cell Counting Kit-8 (Dojindo). It used tetrazolium salt as the chromogenic reagent, and when reduced, water-soluble formazan with maximum absorption around 460 nm is produced. Cells were cultured in a 96-well plate with each compound. After 24 h (Fig. 7B) or 72 h ( Fig. 3C; see also Fig. S3 in the supplemental material) incubation, 10 mL of reagent was added to each well. The absorbance at 450 nm was measured using a microplate reader after 30 min to 1 h of incubation of the cell with reagent.
Indirect immunofluorescence analysis. The cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.005% digitonin for 15 min at room temperature. Rabbit anti-vaccinia virus antibody (Abcam) and anti-rabbit Alexa Fluor Plus 555 (Thermo Fisher Scientific) were used as the primary and secondary antibodies, respectively. Nuclei were visualized using 4,6-diamidino-2-phenylindole (DAPI), and fluorescence was visualized using a fluorescence microscope (BZ-X710; Keyence). Quantification of the red fluorescence area was performed using a BZ-X analyzer (Keyence).
Real-time PCR/reverse transcriptase PCR analysis. DNA was extracted from the cells using a QIAamp DNA minikit (Qiagen) or MagMAX DNA Ultra 2.0 with cell and tissue extraction buffer (Thermo Fisher Scientific) according to the manufacturer's protocol. Real-time PCR detection of the ATI gene of MPXV was performed using TaqMan gene expression master mix (Thermo Fisher Scientific) following the manufacturer's instructions. The primers and probe used were as follows: forward primer, GAGATTAGCAGACTCCAA; reverse primer, GATTCAATTTCCAGTTTGTAC; and TaqMan probe, FAM-CTAGATTGTAATCTCTGTAGCATTTCCACGGC-TAMRA (48).
RNA was extracted from the cells using an RNeasy minikit (Qiagen) according to the manufacturer's protocol. Real-time reverse transcriptase PCR (RT-PCR) analysis was performed using Fast Virus 1-Step master mix (Thermo Fisher Scientific) following the manufacturer's instructions. The following primer and probe sets were purchased from Thermo Fisher Scientific: IMPDH1, Hs04190080_gH; IMPDH2, Hs00168418_m1; and beta actin, Hs01060665_g1.
Time-of-drug-addition assay. VeroE6 cells were infected with MPXV at an MOI of 5 for 1 h in the presence (a, b) or absence (c) of the compound. After the virus inoculum was removed and washing with PBS was performed, the cells were incubated with medium supplemented with (a, b) or without (c) the compound. 1 h later, the medium in b and c was removed, washing was performed again, and medium without (b) or with (c) the compound was added. After a further 22 h of incubation, the cells were collected to detect viral DNA by real-time PCR.
Electron microscopy analysis. VeroE6 cells were trypsinized and fixed with buffer (2.5% glutaraldehyde, 2% paraformaldehyde [PFA], and 0.1 M phosphate buffer [pH 7.4]) at 4°C, followed by postfixation with 1% osmium tetroxide, staining with 0.5% uranyl acetate, dehydration with a graded series of alcohols, and embedding with epoxy resin (50). Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope. At least 150 cells per sample were observed in ultrathin sections, and representative images are shown in Fig. 5. The viral factory was identified morphologically based on the previous report (33).
Western blot analysis. Cells were lysed with Passive Lysis Buffer (Promega), separated by SDS-PAGE with Bolt Bis-Tris Plus gel (4 to 12%; Thermo Fisher Scientific), and transferred to polyvinylidene difluoride membranes using an iBlot2 instrument (Thermo Fisher Scientific). Anti-IMPDH1 rabbit polyclonal antibody (Invitrogen), anti-IMPDH2 polyclonal antibody (Proteintech), and anti-beta actin monoclonal antibody (Cell Signaling Technology) were used as primary antibodies. SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used to visualize the signals, which were then detected with a ChemiDoc XRS instrument (Bio-Rad).
Exogenous guanosine supplementation analysis. Huh7 cells infected with MPXV for 1 h were treated with or without 12.5, 25, or 50 mM guanosine (Sigma-Aldrich) in the presence or absence of 5 mM MPA. After 24 h of infection, the cells were recovered to detect viral DNA using real-time PCR.
Statistical analysis. Data are presented as the mean 6 standard deviation (SD). All statistical analyses were performed using Student's t test. Values of P , 0.05 (*) and P , 0.01 (**) were considered statistically significant, and N.S. indicates not significant.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.6 MB. We declare no competing interests.