Skip to main content
NCBI home page
The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2023 Mar 9;228(5):591–603. doi: 10.1093/infdis/jiad058

Potential Anti-Mpox Virus Activity of Atovaquone, Mefloquine, and Molnupiravir, and Their Potential Use as Treatments

Daisuke Akazawa 1,#, Hirofumi Ohashi 2,#, Takayuki Hishiki 3,#, Takeshi Morita 4,#, Shoya Iwanami 5,#, Kwang Su Kim 6,7,8,#, Yong Dam Jeong 9,10,#, Eun-Sil Park 11, Michiyo Kataoka 12, Kaho Shionoya 13,14, Junki Mifune 15, Kana Tsuchimoto 16, Shinjiro Ojima 17, Aa Haeruman Azam 18, Shogo Nakajima 19, Hyeongki Park 20, Tomoki Yoshikawa 21, Masayuki Shimojima 22, Kotaro Kiga 23, Shingo Iwami 24,25,26,27,28,29,30, Ken Maeda 31, Tadaki Suzuki 32, Hideki Ebihara 33, Yoshimasa Takahashi 34, Koichi Watashi 35,36,37,38,39,✉,5
PMCID: PMC10469127  PMID: 36892247

Abstract

Background

Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling.

Methods

We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment.

Results

Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51–5.2 μM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations.

Conclusions

These data suggest that atovaquone would be a potential candidate for treating mpox.

Keywords: mpox, antiviral, atovaquone, dihydroorotate dehydrogenase, mefloquine, molnupiravir, pan-orthopoxvirus

In a drug repurposing approach, we screened 132 approved drugs using an mpox virus infection cell system and identified potential drug candidates. Atovaquone was predicted to have clinical impact by mathematical modeling.

Mpox virus (MPXV) is a zoonotic virus classified in the Orthopoxvirus genus of the Poxviridae family that causes smallpox-like diseases including rash, scab, fever, and head and body aches in humans [1–3]. After the first cases of human infection were reported in Congo in 1970, mpox cases have been primarily reported in Central and West African countries, with rare reports in other countries that have been linked with importation and travel from endemic African regions. In May 2022, an outbreak of human mpox was reported and involved more than 80 000 cases in over 100 countries, mainly in Europe and North America at the time of writing of this article [4]. The World Health Organization declared this mpox outbreak a global health emergency on 23 July 2022.

Tecovirimat and brincidofovir are approved by the US Food and Drug Administration (FDA) for the treatment of smallpox [5]. Tecovirimat targets the viral-encoded VP37 protein to inhibit the envelopment of intracellular mature virions, whereas brincidofovir is a lipid conjugate prodrug of the nucleotide analogue cidofovir, which inhibits viral DNA replication [6–8]. Although these 2 drugs have been approved for smallpox treatment through the FDA animal efficacy rule, their effectiveness against MPXV infection in humans has not been well documented. A recent clinical study did not show any convincing clinical benefit of brincidofovir in mpox patients but instead showed that the drug caused liver damage resulting in treatment cessation [9]. Due to concerns over the international spread of mpox, there is increasing demand for effective and safe clinical treatments for mpox.

This study employed a drug repurposing approach to identify already-approved drugs that exhibit anti-MPXV activity in a virological infection assay. We also quantitatively investigated the antiviral dynamics under clinical drug concentrations and predicted the impact on patient viral load to identify clinically relevant drug candidates.

METHODS

Cell Culture

Detailed culture conditions of VeroE6, A549, and Huh-7 cells are described in Supplementary Methods.

Infection Assay

MPXV was handled in a biosafety level 3 facility. MPXV stocks of the Zr-599 and Liberia strains were prepared by propagating viruses in VeroE6 cells; virus infectious titers were determined by plaque assay [10]. Conditions of the virus infection are described in Supplementary Methods in detail.

Drug Library and Screening

The drug library used for screening included 65 antiviral, 33 antifungal, and 34 antiprotozoal/antiparasitic drugs (Selleck) that are approved in countries as shown in Supplementary Table 1. The screening methods and the criteria are described in Supplementary Methods in detail.

Immunofluorescence Analysis

Viral proteins were detected by indirect immunofluorescence analysis. The methods are described in Supplementary Methods in detail.

Real-Time PCR

MPXV DNA and human dihydroorotate dehydrogenase (DHODH) gene expression was analyzed using real-time polymerase chain reaction (PCR), which is described in Supplementary Methods in detail.

Cell Viability Assay

Cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) or the WST assay (Cell Counting Kit-8; Dojindo) according to the manufacturer's protocol.

Time-of-Addition Assay

The assay was performed in VeroE6 cells which were inoculated with MPXV at an multiplicity of infection (MOI) of 0.1 under the same condition as Figure 1. Detailed conditions are described in Supplementary Methods.

Figure 1.

Figure 1.

Open in a new tab

Anti-MPXV activity of atovaquone, mefloquine, and molnupiravir. A, Schematic representation of the MPXV infection assay. VeroE6 cells were treated with MPXV at a multiplicity of infection of 0.1 and with or without drugs. In the screening, survived cell numbers at 72 hours were measured with a high-content imaging analyzer (Supplementary Figure 1A). B, Morphology of MPXV-infected cells following drug treatment was observed at 72 hours postinoculation by microscopy. Uninfected cells were also observed as a negative control (image a). Scale bar, 200 μm. C, MPXV protein production was detected at 24 hours postinoculation by immunofluorescence in uninfected (image a) or infected VeroE6 cells upon treatment with drugs. Scale bars, 50 μm. Red, MPXV proteins; blue, nuclei. B and C, Images b–f drug treatments: 0.1% DMSO, 5 μM tecovirimat, 5 μM atovaquone, 5 μM mefloquine, 5 μM molnupiravir. Abbreviations: CPE, cytopathic effect; DMSO, dimethyl sulfoxide; MPXV, mpox virus.

Knockdown Assay

siRNA against the human DHODH (siDHODH) and the control siRNA (siControl) were purchased from Dharmacon. The sequences and detailed analytical methods are described in Supplementary Methods.

Western Blotting

The detailed methods are described in Supplementary Methods.

Rescue Assay

A549 cells were inoculated with MPXV for 1 hour and then incubated with medium supplemented with or without 4 μM atovaquone with either 1 mM orotate or dimethyl sulfoxide (DMSO) for 23 hours. Intracellular DNA was extracted and the MPXV DNA was quantified by real-time PCR as previously described.

Electron Microscopy

Cells were treated with or without MPXV at an MOI of 0.1 and with each drug for 24 hours. The cells were analyzed as described in Supplementary Methods.

Mathematical Analysis

Quantification of dose-response relationships of drugs, determination of synergism between atovaquone and tecovirimat, prediction of the anti-MPXV effect of the drugs, and drug impact on MPXV infection are shown in detail in Supplementary Material Note 2.

Drug Cotreatment

VeroE6 cells were incubated with the drugs singly or in combination at different concentrations for 1 hour during virus inoculation and 29 hours after inoculation. The detailed methods are described in Supplementary Methods.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism 9 software. The statistical method is described in each figure legend.

RESULTS

Identification of Approved Drugs Exhibiting Anti-MPXV Activity

Using a cell-based MPXV infection screening approach, we focused on libraries of clinically approved drugs consisting of 132 antiviral, antifungal, and antiparasitic/antiprotozoal agents (Supplementary Table 1), as these classes include drugs reported to have a wide range of antiviral activity, such as remdesivir, itraconazole, and ivermectin [11–13]. For the initial screening, we treated VeroE6 cells with MPXV at a MOI of 0.1 together with the tested compounds, and then cytopathic effects were assessed at 72 hours postinfection by microscopic observation and quantification of cell viability by high-content imaging analyzer (Figure 1A). The assay system was validated by observations that treatment with MPXV resulted in robust cytopathology that reduced cell viability to <1% (Figure 1B image b and Supplementary Figure 1A) and observations that cells were protected by treatment with a known MPXV inhibitor, tecovirimat, as a positive control [10] (Figure 1B image c and Supplementary Figure 1A). In this assay, an increase in cell survival would suggest that the tested drug inhibits virus infection/replication without cytotoxicity. In a screening at a concentration of 10 μM, 21 drugs showed > 20-fold higher cell viability than DMSO-treated controls (Supplementary Figure 1A, above the red line). As candidates, we focused on atovaquone (anti-Pneumocystis jiroveci), mefloquine (antimalarial), and molnupiravir (anti-severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) as orally applicable drugs that are currently available clinically and exhibited remarkable anti-MPXV activity at 3.3 μM in the second screening (Supplementary Figure 1B; see Supplementary Material Note 1 for details). These drugs protected cells from MPXV-induced cytopathic effects at 72 hours postinfection (Figure 1B images d, e, and f).

To validate the anti-MPXV effect of these drugs, we treated VeroE6 cells with MPXV using the same protocol and detected intracellular MPXV proteins at 24 hours postinfection (before sign of cytopathology emerged). As shown in Figure 1C, these drugs dramatically reduced the production of MPXV proteins in the infection assay (Figure 1C images d, e, and f). These results confirmed the anti-MPXV activity of atovaquone, mefloquine, and molnupiravir.

Anti-MPXV Activity Dose-Response Curves for Atovaquone, Mefloquine, and Molnupiravir

The anti-MPXV activity of atovaquone, mefloquine, and molnupiravir (Figure 2A) was assessed by quantifying viral DNA in infected cells following 30 hours of treatment (before onset of MPXV-induced cytopathic effects) with the drugs at varying concentrations (Figure 2B). We also examined the reported MPXV inhibitors tecovirimat and cidofovir [14–16] as positive controls. Cell viability was simultaneously quantified at different drug concentrations to examine any possible cytotoxic effects of the drugs (Figure 2C). As shown in Figure 2B and 2C, atovaquone, mefloquine, and molnupiravir reduced intracellular MPXV DNA levels in a dose-dependent manner without inducing cytotoxic effects. The 50% and 90% maximal virus inhibitory concentrations (IC50 and IC90, summarized in Supplementary Table 2) and 50% maximal cytotoxic concentrations (CC50) are shown in Figure 2B and 2C. These 3 drugs exhibited greater anti-MPXV potency (lower IC50 and IC90 values) than cidofovir. In particular, the calculated IC50 values of atovaquone and molnupiravir were lower than the reported maximum drug concentration (Cmax) in treated patients (Supplementary Table 3), suggesting the possibility that the antiviral potency of these drugs is clinically relevant.

Figure 2.

Figure 2.

Open in a new tab

Dose-response curves for anti-mpox virus (MPXV) activity and cytotoxicity of the drugs. A, Chemical structures of the drugs. B, VeroE6 cells were infected with MPXV at a multiplicity of infection of 0.03 for 1 hour and were washed out, followed by incubation with varying concentrations of each drug for a further 29 hours. Anti-MPXV activity was examined by quantifying MPXV DNA in cells. Relative MPXV DNA levels (y-axis, log scale), determined by setting dimethyl sulfoxide (DMSO)-treated control cells as 100%, are shown against the drug concentration (x-axis, log scale). The 50% and 90% maximal inhibitory concentrations (IC50 and IC90) are indicated above the graphs. C, Cell viability was measured following treatment with drugs at the indicated concentrations using the CellTiter-Glo Luminescent Cell Viability Assay, with DMSO-treated control cells set as 100%. The calculated 50% maximal cytotoxic concentrations (CC50) are shown above the graphs.

Viral Life Cycle Step Targeted by the Identified Drugs

In the viral life cycle, MPXV attaches to target cells and enters cells to deliver the viral core into the cytoplasm (entry phase, Figure 3A). After early transcription, protein synthesis, and core uncoating, the viral DNA replicates as well as drives intermediate and late transcriptions, and is then assembled into virions in the cytoplasmic viral factories, followed by a stepwise virion maturation process resulting in the production of progeny infectious virions (postentry phase; Figure 3A) [17]. To determine which phase in the viral life cycle is inhibited by the identified drugs, we performed time-of-addition assay in which cells were treated with the drugs at varying time points (Figure 3B, left) to distinguish the entry and postentry phases [18,19]. Drugs were administered either (1) throughout the assay (whole life cycle), (2) for the initial 2 hours (entry phase), or (3) for the last 22 hours after viral infection (postentry and reinfection phase) (Figure 3B, left). The positive control tecovirimat, which inhibits virion maturation, exhibited significant antiviral activity under conditions (1) and (3) but not (2), whereas heparin, which reportedly inhibits viral entry [20], showed significant anti-MPXV activity in condition (2) (Figure 3B, right), thereby validating the time-of-addition assay. In this assay, mefloquine showed significant anti-MPXV activity under condition (2), in addition to conditions (1) and (3) (Figure 3B, right), consistent with the reported inhibition of the entry of multiple viruses, including coronaviruses and Ebola virus [19, 21], although there are no reports of its effect on poxviruses. In contrast, atovaquone and molnupiravir predominantly reduced MPXV DNA levels in cells treated under condition (3) but not condition (2) (Figure 3B, right), suggesting that atovaquone and molnupiravir target the postentry phase.

Figure 3.

Figure 3.

Open in a new tab

Viral life cycle step targeted by drugs. A, Schematic illustration of the MPXV life cycle. Target steps of the positive control compounds, heparin and tecovirimat, are also shown. B, Time-of-addition assay. Left, experimental schedules of the assays in which cells were treated with drugs at 3 times: (1) whole, throughout the assay for 24 hours; (2) entry, for the initial 2 hours (1 hour with the MPXV inoculum and the following 1 hour after inoculation); or (3) postentry, for the last 22 hours after inoculation. Right, MPXV DNA levels in infected VeroE6 cells treated with the drugs under conditions (1), (2), or (3) were determined and are shown relative to that of the DMSO-treated control. Data were expressed as mean ± standard deviation (SD), and the statistical analysis was performed with 1-way analysis of variance followed by Dunnett multiple comparison test vs each DMSO-treated control. ns P ≥ .05, * P < .05, ** P < .005, *** P < .001, **** P < .0001. Abbreviations: DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; EV, extracellular virion; IV, immature virion; MPXV, mpox virus; MV, mature virion; ns, not significant; WV, wrapped virion.

Critical Role of Dihydroorotate Dehydrogenase in MPXV Replication

Molnupiravir is a nucleoside analogue that targets polymerization of the genome of different virus classes [22–25], consistent with the observed inhibition of MPXV replication process by molnupiravir in Figure 3B. Atovaquone targets the cytochrome bc1 complex to inhibit mitochondrial electron transport [26] and also inhibits parasite DHODH in the pyrimidine biosynthesis pathway [27], which regulates the replication of a wide range of viruses, including influenza viruses, coronaviruses, and flaviviruses [28]. To validate the contribution of DHODH to MPXV replication, we knocked down the endogenous DHODH gene and examined the intracellular MPXV DNA levels after viral infection. By transfection with a specific siRNA against DHODH that reduced both RNA and protein levels of DHODH (Figure 4A), the intracellular MPXV DNA level was decreased by over 60% in viral-infected cells (Figure 4B). Supplementation with orotate, the product of DHODH (Figure 4C), significantly rescued the reduced MPXV replication caused by atovaquone (Figure 4D). Moreover, other DHODH inhibitors, leflunomide and teriflunomide [29], reduced intracellular MPXV DNA levels in a dose-dependent manner (Figure 4E). These data suggest that the DHODH-mediated pyrimidine biosynthesis pathway is important for the efficient replication of MPXV, which is likely to be a target for the anti-MPXV activity of atovaquone.

Figure 4.

Figure 4.

Open in a new tab

Atovaquone inhibited MPXV replication through inhibition of DHODH. A, Human DHODH gene knockdown by transfection with specific siRNA into human lung-derived A549 cells. At 48 hours posttransfection of 10 nM of control (siControl) or human DHODH-specific siRNA (siDHODH), A549 cells were lysed to extract total RNA and protein for detection of human DHODH RNA by real-time PCR (left) and proteins DHODH and β-actin by western blotting (right). Data were expressed as mean ± SD, and the statistical analysis was performed by unpaired 2-tailed Student t test. B, At 48 hours posttransfection with siRNA, A549 cells were inoculated with MPXV for 1 hour and cultured for a further 23 hours, and MPXV DNA levels were quantified. Data were expressed as mean ± SD, and the statistical analysis was performed with the unpaired 2-tailed Student t test. C, Metabolic cascade of DHODH-mediated pyrimidine de novo synthesis. D, Rescue of MPXV replication by complementation with exogenous orotate. A549 cells inoculated with MPXV were treated with or without 4 μM atovaquone in the presence or absence of 1 mM orotate for 23 hours, and intracellular MPXV DNA levels were evaluated. Data were expressed as mean ± SD, and the statistical analysis was performed with 1-way analysis of variance followed by Holm-Sidak multiple comparison test. E, Dose-dependent anti-MPXV activity of DHODH inhibitors, leflunomide and teriflunomide. Anti-MPXV activities were examined as in Figure 2B at the indicated concentrations. ns, P > .05, * P < .05, ** P < .005, *** P < .001. Abbreviations: CAD, carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase; DHODH, dihydroorotate dehydrogenase; MPXV, mpox virus; PCR, polymerase chain reaction; PRPP, 5-phosphoribosyl-1-pyrophosphate; UDP, uridine diphosphate; UMP, uridine monophosphate; UMPS, UMP synthase; UTP, uridine triphosphate.

Electron Microscopic Analysis of Drug-Treated Infected Cells

Infection of cells with MPXV induces the formation of cytoplasmic viral factories to initiate the assembly of virions that sequentially forms crescents, immature virions, and mature virions, which further follow the maturation process to wrapped virions and extracellular virions [30]. To examine the effect of the identified drugs, which inhibit the postentry step, on virion maturation and the formation of cytoplasmic viral factories, we used transmission electron microscopy to examine the intracellular morphology of infected drug-treated cells, including cytoplasmic viral factories and the virions produced. As shown in Figure 5, the cytoplasm of infected cells exhibited regions with high electron density at perinuclear areas, indicative of cytoplasmic viral factories (Figure 5C) [30]. In contrast, no such regions were observed in uninfected cells (Figure 5A). These factories contained crescents, circular immature virions, and dense mature virions (Figure 5D). Wrapped virions were also observed outside of the factories (Figure 5B). Upon tecovirimat treatment, a significant number of cells exhibited accumulation of crescents, immature virions, and mature virions at the cytoplasmic viral factories (Figure 5E and 5F), confirming that this drug inhibits the maturation to wrapped virions but not viral replication before virion production. In contrast, treatment with atovaquone and molnupiravir resulted in a dramatic reduction in the number of infected cells, and few virions were observed in these cells, even though they possessed cytoplasmic viral factories (Figure 5G–5J). In contrast to tecovirimat, these observations suggest that atovaquone and molnupiravir inhibit MPXV replication before virion maturation.

Figure 5.

Figure 5.

Open in a new tab

Atovaquone and molnupiravir reduced virion production in MPXV-infected cells. VeroE6 cells infected with (B–J) or without (A) MPXV at a multiplicity of infection of 0.1 were incubated following drug treatment for 24 hours: (B–D) 0.1% DMSO; (E and F) 5 μM tecovirimat; (G and H) 5 μM atovaquone; (I and J) 5 μM molnupiravir. The cells were trypsinized and observed by transmission electron microscopy. A total of 150 cells were observed for each sample, and the figure shows representative images of infected cell morphology. The images in D, F, H, and J show the boxed areas in C, E, G, and I, respectively, at higher magnification. B, Higher-magnification image of the inset in D with 90° rotation. A and C–J, Scale bars, 2 μm. B, Scale bar 200 nm. Abbreviations: *, cytoplasmic viral factories; C, crescent; DMSO, dimethyl sulfoxide; IV, immature virion; MPXV, mpox virus; MV, mature virion; N, nucleus; WV, wrapped virion.

Impact of Antiviral Drugs on MPXV Infection in Clinical Settings

Combining the pharmacokinetics (PK) information (summarized in Supplementary Table 3) of these approved drugs when administered in patients with the observed dose-dependent antiviral activity information (pharmacodynamics [PD] information: summarized in Supplementary Table 2), we calculated the antiviral effect for clinical drug concentrations using the PK/PD model (Eq. 11 in Supplementary Material Note 2; Figure 6A). We assumed a simple 1-compartment model [18] with the reported maximum drug concentrations in plasma (Cmax) and half-life values of tecovirimat [31], cidofovir [32], atovaquone [33], mefloquine [34], and molnupiravir [35] by administration at approval doses (Supplementary Table 3). We found that cidofovir and molnupiravir show antiviral effects after administration but these declined rapidly, reflected by their short half-lives, whereas the antiviral effects of tecovirimat and atovaquone are maintained at high levels during drug treatment (Figure 6A). That of mefloquine was estimated to gradually decline after administration (Figure 6A).

Figure 6.

Figure 6.

Open in a new tab

Mathematical prediction of the impact of the identified drugs on viral load dynamics in clinical settings. A, Time-dependent antiviral effects predicted by PK/PD modeling of tecovirimat (600 mg, oral twice a day for 14 days), cidofovir (5 mg/kg, intravenous once a week for 2 weeks), atovaquone (750 mg, oral twice a day for 21 days), mefloquine (25 mg/kg, single oral dose), and molnupiravir (800 mg, oral twice a day for 5 days). B, Viral infection dynamics in the presence or absence of tecovirimat, cidofovir, atovaquone, mefloquine, and molnupiravir by PK/PD/VD models are shown. Gray lines show the predicted viral load in patients in the absence of treatments. Colored lines show the expected viral load in the presence of treatments. Dashed lines indicate the detection limit of MPXV DNA. C and D, Cumulative viral load (area under the curve in B) and the duration of virus shedding (time until the viral load is below the detection limit in B) were calculated for untreated (gray bars) and treated (colored bars) patients. Abbreviations: MPXV, mpox virus; PD, pharmacodynamics; PK, pharmacokinetics; VD, viral dynamics.

To evaluate the impact of drug treatment on MPXV-infected patients after onset of rash in patients (ie, day 0 is the date of onset of rash), we developed a mathematical model (PK/PD/viral dynamics model, Eqs 7–11 in Supplementary Material Note 2) integrating the PK/PD information with viral dynamics information (summarized in Supplementary Table 4) (Supplementary Figure 2). As shown in Figure 6B, in the absence of antiviral treatment, the mathematical model (corresponding to Eqs 3 and 4 in Supplementary Material Note 2) predicted that MPXV viral load would exponentially increases for the first 0.71 days and then peak, which would be followed by a gradual decline (Figure 6B, gray lines, and Supplementary Figure 3). Based on the expected viral load, we calculated the cumulative viral RNA burden (ie, area under the curve of viral load) (Figure 6C and Supplementary Figure 2) and the duration of viral shedding (Figure 6D and Supplementary Figure 2). Based on the time-dependent antiviral effect of the drugs, we predicted the impact of the antiviral effects on the dynamics of MPXV infection when the drugs are administrated on day −1, which corresponds to the date for the first viral load sampling, after the onset of rash (Figure 6B, colored lines). Interestingly, our quantitative simulations predicted that atovaquone would be the most effective in the clinical setting, reducing the cumulative viral load by 91.6% (Figure 6C) and reducing the duration of virus positivity in serum by 7.16 days compared with untreated control subjects (Figure 6D). As we explored in a recent study [36], it is required for reductions of viral shedding that antiviral treatments start before the viral load hits its peak.

We here used the previously reported mean values of the viral load (genomes/mL) in the blood [37] for parameter estimations for our quantitative simulation (Supplementary Figure 3 and Supplementary Material Note 2). It should be noted that the peak PCR viral load in blood occurs near the first day of rash appearance, meaning viral load may have already peaked. A time-course individual-level clinical viral load from different specimens covering whole MPXV infection is required to accurately evaluate the effect of antivirals; however, clinical viral load data are currently quite limited.

Combined Treatment With Atovaquone and Tecovirimat

The clinical outcome of an antiviral treatment regimen can be improved by combining drugs, as is employed in the treatment of human immunodeficiency virus and hepatitis C virus infections [38, 39]. The mathematical prediction of antiviral effects in clinical doses in Figure 6 estimated atovaquone as the first priority for a drug candidate. Additionally, as there are possible differences in the mode of action of atovaquone and tecovirimat, we examined the antiviral activity of the combination of these drugs using the MPXV infection assay. Cells were treated with combinations of the drugs at various concentrations for 30 hours, after which intracellular viral DNA and cell viability were evaluated. Compared with the dose-dependent reduction in viral DNA levels observed with single treatment with atovaquone or tecovirimat, the combination of these drugs further reduced viral DNA levels (Figure 7A). We did not observe any significant cytotoxicity at any of the drug concentrations tested (Figure 7B). We then compared the observed experimental antiviral activity with theoretical predictions calculated using a classical Bliss independence model that assumes the drugs act independently (Supplementary Material Note 2) [18, 40, 41]. The difference between the observed values and theoretical predictions suggests that atovaquone and tecovirimat exhibit a synergistic activity over a broad range of concentrations, especially at lower concentration ranges (Figure 7C red, synergistic effect). The higher concentrations of each drug alone showed substantial activity, and therefore the calculated synergistic effects appear to be low. Thus, combination treatment with atovaquone enhances the antiviral activity of tecovirimat.

Figure 7.

Figure 7.

Open in a new tab

Cotreatment with tecovirimat and atovaquone. A. Viral DNA in VeroE6 cells cotreated with tecovirimat and atovaquone at varying concentrations (tecovirimat, 0, 0.94, 1.88, 3.75, and 7.5 nM [× 2 dilution]; atovaquone, 0, 0.38, 0.51, 0.68, and 0.90 μM [× 1.3 dilution]) for 30 hours was quantified and results are shown relative to the dimethyl sulfoxide (DMSO)-treated control. B, Cell viability measured at 30 hours posttreatment. C, Heatmap of synergy scores for tecovirimat and atovaquone is shown based on a Bliss independence model. Red, white, and blue indicate the synergistic, additive, and antagonistic interactions between the 2 drugs, respectively.

DISCUSSION

In this study, we screened a library of approved drugs for anti-MPXV activity using a cell culture infection assay and identified atovaquone, mefloquine, and molnupiravir as candidate drugs. The IC50 of atovaquone was 0.516 μM, which is within the concentration range for clinical use, with 31.3 μM as the plasma Cmax and 67.0 hours as the half-life [33]. In the mathematical modeling to translate the in vitro antiviral activity to the clinical efficacy [18, 19, 36, 42], atovaquone was predicted to exhibit sustained anti-MPXV activity following administration at approved doses and induce rapid viral decay in infected patients, reducing the cumulative viral load and shortening the time until virus elimination. Another potential application for atovaquone is combination use with approved anti-orthopoxvirus agents such as tecovirimat. Interestingly, addition of atovaquone to tecovirimat in cell culture experiments resulted in a further reduction in MPXV DNA levels without cytotoxic effects. Given the good tolerability profile of atovaquone in clinical settings [43], our present data provide an attractive idea for supplementing atovaquone to improve the current approved anti-orthopoxvirus treatment.

Mechanistically, molnupiravir is a nucleoside analogue that targets the polymerization of the genome of a variety of different viruses, including hepatitis C virus, norovirus, chikungunya virus, and coronaviruses [22–25]. It is speculated that molnupiravir also targets polymerization of the MPXV genome, and this should be investigated in future studies. In this study, molnupiravir was predicted to show only a mild anti-MPXV effect in clinical doses by our mathematical analysis, in contrast to its anti–SARS-CoV-2 activity in clinical settings [44]. This can be explained, at least in part, by different strength of antiviral activities between the 2 viruses: IC50, IC90, and IC99 of molnupiravir for MPXV were 1.35, 3.25, and around 10 μM, while those for SARS-CoV-2 were 0.22, 0.74, and around 1 μM, respectively [44]. Molnupiravir is estimated to require 4- to 10-fold higher concentration to achieve anti-MPXV activity compared with anti–SARS-CoV-2 activity. Mefloquine reportedly inhibits the cell entry of multiple viruses, including coronaviruses and Ebola virus, although the mode of action remains unclear [19, 21]. Our analysis of atovaquone also suggests that DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, is important for efficient replication of MPXV. In addition to the previous report that the DHODH inhibitor brequinar inhibited the replication of other poxviruses, Cantagolo virus, vaccinia virus, and cowpox virus [45], we showed the essential role of the DHODH-mediated pyrimidine biosynthesis pathway in the anti-MPXV activity of DHODH inhibitors. Following administration of leflunomide (a rheumatoid arthritis agent) at 5–20 mg daily, clinical Cmax of 107–192 μM and half-life of 2 weeks for teriflunomide, an active metabolite of leflunomide, have been reported [46], suggesting another candidate for an anti-MPXV agent in clinical use. This study primarily focused on the evaluation of approved drugs for drug repurposing, but further development focusing on the ubiquinone structure, including atovaquone analogs, would be a relevant approach for developing new anti-MPXV drugs. Also, searches for new DHODH inhibitors should enable the development of more potent anti-MPXV agents in the future.

The antiviral activities of atovaquone were observed not only against the Zaire strain of MPXV, but also the Liberia strain of MPXV, vaccinia virus, and cowpox virus. Atovaquone's antiviral activities were also reproduced in multiple cell lines, including monkey and human kidney-, lung-, and liver-derived cell lines (Supplementary Figures 4 and 5). However, a limitation of our study was that it used only a cell culture infection assay without animal model experiments and patient studies. In vivo animal infection models for MPXV have been reported, including wild rodents (eg, prairie dog, squirrel, dormouse) and laboratory animals (eg, cynomolgus and rhesus macaques, inbred mice) [47], although there is no one model that can recapitulate all aspects of MPXV infection in humans. To evaluate the drug effect on pathogenesis as well as viral load, nonhuman primates would be a relevant model candidate to be used in the future. Considering the limited amount of research regarding this virus to date and the current outbreak and spread of MPXV across multiple continents, we believe rapid analyses using cell culture infection assays will be of benefit in providing scientific evidence to propose alternative treatment options for this infectious disease and to minimize its international spread. It should also be highlighted that these drugs exhibit antiviral activity against multiple orthopoxviruses, making them candidates for controlling a wide range of orthopoxviruses, potential zoonotic, and bioterrorism agents. Further studies on animals and human subjects in the future should lead to the development of alternative and/or better treatments.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Supplementary Material

jiad058_Supplementary_Data
Click here for additional data file. (4MB, pdf)

Contributor Information

Daisuke Akazawa, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Hirofumi Ohashi, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Takayuki Hishiki, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Takeshi Morita, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Shoya Iwanami, Interdisciplinary Biology Laboratory, Graduate School of Science, Nagoya University, Nagoya, Japan.

Kwang Su Kim, Interdisciplinary Biology Laboratory, Graduate School of Science, Nagoya University, Nagoya, Japan; Department of Science System Simulation, Pukyong National University, Busan, South Korea; Department of Mathematics, Pusan National University, Busan, South Korea.

Yong Dam Jeong, Interdisciplinary Biology Laboratory, Graduate School of Science, Nagoya University, Nagoya, Japan; Department of Mathematics, Pusan National University, Busan, South Korea.

Eun-Sil Park, Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.

Michiyo Kataoka, Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.

Kaho Shionoya, Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.

Junki Mifune, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Kana Tsuchimoto, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Shinjiro Ojima, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Aa Haeruman Azam, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Shogo Nakajima, Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Hyeongki Park, Interdisciplinary Biology Laboratory, Graduate School of Science, Nagoya University, Nagoya, Japan.

Tomoki Yoshikawa, Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.

Masayuki Shimojima, Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.

Kotaro Kiga, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Shingo Iwami, Interdisciplinary Biology Laboratory, Graduate School of Science, Nagoya University, Nagoya, Japan; Institute of Mathematics for Industry, Kyushu University, Fukuoka, Japan; Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto, Japan; Interdisciplinary Theoretical and Mathematical Sciences Program, RIKEN, Saitama, Japan; NEXT-Ganken Program, Japanese Foundation for Cancer Research, Tokyo, Japan; Science Groove, Inc, Fukuoka, Japan; MIRAI, Japan Science and Technology Agency, Saitama, Japan.

Ken Maeda, Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.

Tadaki Suzuki, Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.

Hideki Ebihara, Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.

Yoshimasa Takahashi, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

Koichi Watashi, Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan; Interdisciplinary Biology Laboratory, Graduate School of Science, Nagoya University, Nagoya, Japan; Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Department of Applied Biological Science, Tokyo University of Science, Noda, Japan; MIRAI, Japan Science and Technology Agency, Saitama, Japan.

Notes

Acknowledgments . Huh-7 and A549 cell lines were kindly provided by Dr Francis V. Chisari at the Scripps Research Institute and Dr Masayoshi Fukasawa at the Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, respectively.

Financial support. This work was supported by the Agency for Medical Research and Development (grant numbers JP21fk0108427, JP21fk0108589, JP22fk0310504, JP22jm0210068, and JP20wm0325007); the Japan Society for the Promotion of Science (grant number JP20H03499); the Japan Science and Technology Agency (grant numbers JPMJMI22G1 MIRAI program, and JPMJMS2021 and JPMJMS2025 Moonshot R&D program); and the Takeda Science Foundation.

References

  • 1. Kozlov M. Monkeypox goes global: why scientists are on alert. Nature 2022; 606:15–6. [DOI] [PubMed] [Google Scholar]
  • 2. Thornhill JP, Barkati S, Walmsley S, et al. Monkeypox virus infection in humans across 16 countries—April-June 2022. N Engl J Med 2022; 387:679–91. [DOI] [PubMed] [Google Scholar]
  • 3. Titanji BK. Neglecting emerging diseases—monkeypox is the latest price of a costly default. Med (N Y) 2022; 3:433–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Centers for Disease Control and Prevention . Mpox Outbreak Global Map. https://www.cdc.gov/poxvirus/mpox/response/2022/index.html. Accessed 24 February 2023.
  • 5. Delaune D, Iseni F. Drug development against smallpox: present and future. Antimicrob Agents Chemother 2020; 64:e01683-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Duraffour S, Lorenzo MM, Zöller G, et al. ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping. J Antimicrob Chemother 2015; 70:1367–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Hutson CL, Kondas AV, Mauldin MR, et al. Pharmacokinetics and efficacy of a potential smallpox therapeutic, brincidofovir, in a lethal monkeypox virus animal model. mSphere 2021; 6:e00927-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Parker S, Touchette E, Oberle C, et al. Efficacy of therapeutic intervention with an oral ether-lipid analogue of cidofovir (CMX001) in a lethal mousepox model. Antiviral Res 2008; 77:39–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Adler H, Gould S, Hine P, et al. Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect Dis 2022; 22:1153–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Saijo M, Ami Y, Suzaki Y, et al. Virulence and pathophysiology of the Congo basin and west African strains of monkeypox virus in non-human primates. J Gen Virol 2009; 90:2266–71. [DOI] [PubMed] [Google Scholar]
  • 11. Bauer L, Ferla S, Head SA, et al. Structure-activity relationship study of itraconazole, a broad-range inhibitor of picornavirus replication that targets oxysterol-binding protein (OSBP). Antiviral Res 2018; 156:55–63. [DOI] [PubMed] [Google Scholar]
  • 12. Jans DA, Wagstaff KM. The broad spectrum host-directed agent ivermectin as an antiviral for SARS-CoV-2? Biochem Biophys Res Commun 2021; 538:163–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Warren TK, Jordan R, Lo MK, et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016; 531:381–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Smee DF, Sidwell RW, Kefauver D, Bray M, Huggins JW. Characterization of wild-type and cidofovir-resistant strains of camelpox, cowpox, monkeypox, and vaccinia viruses. Antimicrob Agents Chemother 2002; 46:1329–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Stittelaar KJ, Neyts J, Naesens L, et al. Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection. Nature 2006; 439:745–8. [DOI] [PubMed] [Google Scholar]
  • 16. Yang G, Pevear DC, Davies MH, et al. An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge. J Virol 2005; 79:13139–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Schramm B, Locker JK. Cytoplasmic organization of poxvirus DNA replication. Traffic 2005; 6:839–46. [DOI] [PubMed] [Google Scholar]
  • 18. Ohashi H, Watashi K, Saso W, et al. Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment. iScience 2021; 24:102367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Shionoya K, Yamasaki M, Iwanami S, et al. Mefloquine, a potent anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) drug as an entry inhibitor. Front Microbiol 2021; 12:651403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Vázquez MI, Esteban M. Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus. J Virol 1999; 73:9098–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Sun W, He S, Martínez-Romero C, et al. Synergistic drug combination effectively blocks Ebola virus infection. Antiviral Res 2017; 137:165–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Costantini VP, Whitaker T, Barclay L, et al. Antiviral activity of nucleoside analogues against norovirus. Antivir Ther 2012; 17:981–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Ehteshami M, Tao S, Zandi K, et al. Characterization of β-d-N4-hydroxycytidine as a novel inhibitor of chikungunya virus. Antimicrob Agents Chemother 2017; 61:e02395-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Kabinger F, Stiller C, Schmitzová J, et al. Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nat Struct Mol Biol 2021; 28:740–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Stuyver LJ, Whitaker T, McBrayer TR, et al. Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture. Antimicrob Agents Chemother 2003; 47:244–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Birth D, Kao WC, Hunte C. Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nat Commun 2014; 5:4029. [DOI] [PubMed] [Google Scholar]
  • 27. Guler JL, White J, Phillips MA, Rathod PK. Atovaquone tolerance in Plasmodium falciparum parasites selected for high-level resistance to a dihydroorotate dehydrogenase inhibitor. Antimicrob Agents Chemother 2015; 59:686–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Zheng Y, Li S, Song K, et al. A broad antiviral strategy: inhibitors of human DHODH pave the way for host-targeting antivirals against emerging and re-emerging viruses. Viruses 2022; 14:928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 1996; 35:1270–3. [DOI] [PubMed] [Google Scholar]
  • 30. Weisberg AS, Maruri-Avidal L, Bisht H, et al. Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants. Proc Natl Acad Sci U S A 2017; 114:E11001–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Grosenbach DW, Honeychurch K, Rose EA, et al. Oral tecovirimat for the treatment of smallpox. N Engl J Med 2018; 379:44–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Cundy KC, Petty BG, Flaherty J, et al. Clinical pharmacokinetics of cidofovir in human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 1995; 39:1247–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. GlaxoSmithKline . Product monograph, PrMEPRON, atovaquone oral suspension, USP 750mg/5mL. GlaxoSmithKline. [Google Scholar]
  • 34. Gutman J, Green M, Durand S, et al. Mefloquine pharmacokinetics and mefloquine-artesunate effectiveness in Peruvian patients with uncomplicated Plasmodium falciparum malaria. Malar J 2009; 8:58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Painter WP, Holman W, Bush JA, et al. Human safety, tolerability, and pharmacokinetics of molnupiravir, a novel broad-spectrum oral antiviral agent with activity against SARS-CoV-2. Antimicrob Agents Chemother 2021; 65:e02428-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Kim KS, Ejima K, Iwanami S, et al. A quantitative model used to compare within-host SARS-CoV-2, MERS-CoV, and SARS-CoV dynamics provides insights into the pathogenesis and treatment of SARS-CoV-2. PLoS Biol 2021; 19:e3001128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Pittman PR, Martin JW, Kingebeni PM, et al. Clinical characterization of human monkeypox infections in the democratic republic of the Congo. medRxiv, 10.1101/2022.05.26.22273379, 29May2022, preprint: not peer reviewed. [DOI] [Google Scholar]
  • 38. Koizumi Y, Ohashi H, Nakajima S, et al. Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection. Proc Natl Acad Sci U S A 2017; 114:1922–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Shen L, Peterson S, Sedaghat AR, et al. Dose-response curve slope sets class-specific limits on inhibitory potential of anti-HIV drugs. Nat Med 2008; 14:762–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Greco WR, Bravo G, Parsons JC. The search for synergy: a critical review from a response surface perspective. Pharmacol Rev 1995; 47:331–85. [PubMed] [Google Scholar]
  • 41. Koizumi Y, Iwami S. Mathematical modeling of multi-drugs therapy: a challenge for determining the optimal combinations of antiviral drugs. Theor Biol Med Model 2014; 11:41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Iwanami S, Ejima K, Kim KS, et al. Detection of significant antiviral drug effects on COVID-19 with reasonable sample sizes in randomized controlled trials: a modeling study. PLoS Med 2021; 18:e1003660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Jacquerioz FA, Croft AM. Drugs for preventing malaria in travellers. Cochrane Database Syst Rev 2009; (4):CD006491. [DOI] [PubMed] [Google Scholar]
  • 44. Ohashi H, Hishiki T, Akazawa D, et al. Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 omicron subvariants, BA.1 and BA.2. Antiviral Res 2022; 205:105372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Schnellrath LC, Damaso CR. Potent antiviral activity of brequinar against the emerging Cantagalo virus in cell culture. Int J Antimicrob Agents 2011; 38:435–41. [DOI] [PubMed] [Google Scholar]
  • 46. Chan V, Charles BG, Tett SE. Population pharmacokinetics and association between A77 1726 plasma concentrations and disease activity measures following administration of leflunomide to people with rheumatoid arthritis. Br J Clin Pharmacol 2005; 60:257–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Domán M, Fehér E, Varga-Kugler R, Jakab F, Bányai K. Animal models used in monkeypox research. Microorganisms 2022; 10:2192. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jiad058_Supplementary_Data
Click here for additional data file. (4MB, pdf)

Articles from The Journal of Infectious Diseases are provided here courtesy of Oxford University Press

RESOURCES