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# Molecular mechanism of the antiviral effect of Tranislast against vaccinia and Mpox viruses

Jacques Fantini, Henri Chahinian, Didier Raoult

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Jacques Fantini, Henri Chahinian, Didier Raoult. Molecular mechanism of the antiviral effect of Tranislast against vaccinia and Mpox viruses. 2025. hal-04815321v2

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# Molecular mechanism of the antiviral effect of Tranislast against vaccinia and Mpox viruses

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Jacques Fantini<fnref n="1" />*, Henri Chahinian<fnref n="1" /> and Didier Raoult<fnref n="2" />

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**Abstract:** We show that vaccinia and Mpox viruses share a common site of interaction for the glycosaminoglycan chondroitin sulfate, a cell surface receptor for many animal and human viruses. This site, located on the homologous D8L (vaccinia) and E8L (Mpox) proteins, comprises the remarkable triad of amino acids, R44, Y104 and E105 that delimit a crevice in which chondroitin sulfate spreads, forming a high-affinity molecular complex. The same site is recognized by Tranilast, an approved drug showing antiviral effects against vaccinia virus in vitro. Remarkably, Tranilast and chondroitin sulfate exhibit a globally electronegative surface electrostatic potential, explaining why both molecules interact with an electropositive site on viral proteins. Physicochemical studies with reconstituted lipid monolayers at the air water interface gave an experimental validation of these findings. In the presence of Tranilast, the interaction of recombinant E8L with membrane lipids was abrogated for electronegative gangliosides. Based on this dual structural-physicochemical analysis, we propose a common inhibition mechanism of Tranilast against infection by vaccinia and Mpox viruses. Since Tranilast is a widely used drug in Japan, safe and with a very low cost, it would be interesting to test it as a potential inhibitor of the Mpox virus.

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**Keywords:** Tranilast; Mpox virus; vaccinia virus; glycosaminoglycans; chondroitin sulfate; gangliosides; lipid rafts; antiviral

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## 1. Introduction

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Given the growing risk of pandemics due to virus outbreaks [1-3], there is an urgent need for an arsenal of antiviral molecules that can neutralize any circulating or emerging virus [4]. In this context, the successive alerts linked to the Mpox virus over the last two years have challenged the scientific community and health authorities [5-7]. Recently, a systematic study carried out on a panel of 767 approved drugs highlighted the antiviral properties of Tranilast (2-[[[(E)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]amino]benzoic acid) on the vaccinia virus [8]. However, the antiviral mechanisms of action of this molecule initially used in the treatment of bronchial asthma [9] remained undetermined. Since vaccinia and Mpox viruses display very strong similarities in genome and protein sequences [10], it seemed important to us to continue our research efforts in this area. To this end, we decided to focus our attention on the earliest stages of the infection cycle by investigating possible effects of Tranilast at this level. Vaccinia virus, like a wide variety of microbial pathogens [11] including other enveloped viruses [12-14], begins its replication cycle by interacting with the proteoglycans of the host cell plasma membrane [15]. More specifically, it is the glycosaminoglycans of these surface molecules that attract and bind the envelope proteins [16], combining electrostatic, van der Waals), aromatic-aromatic ($\pi$-$\pi$) interactions and hydrogen bonds [17,18]. In addition, the release of solvent molecules has been characterized as an important parameter that controls glycosaminoglycan-protein interactions [19]. In the present work, we used molecular modeling approaches that have been previously validated by our group with several viruses such as endemic coronaviruses [20], SARS-CoV-2 [21], HIV-1 [22], and Mpox [23].

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First, we studied the vaccinia virus D8L envelope protein that interacts with cell surface glycosaminoglycans at the earliest step of virus infection [15]. After identifying the chondroitin sulfate binding site on the D8L protein, we show that Tranilast also recognizes this site, suggesting a competitive inhibition mechanism

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<footnote n="1">¹INSERM UMR UA 16, Aix-Marseille Université, Marseille, France</footnote>

<footnote n="2">²IHU Méditerranée Infection, 19-21 boulevard Jean Moulin, 13005 Marseille, France; 3 Aix-Marseille Univ., Microbes Evolution Phylogeny and Infections (MEPHI), 27 boulevard Jean Moulin, 13005 Marseille, France.</footnote>

<footnote>*Correspondence: jacques.fantini@univ-amu.fr</footnote>
blocking virus infection. Finally, we show that this site is structurally conserved on the Mpox virus E8L protein and that it is also recognized by Tranilast. Incidentally, this site was previously identified as an annular ganglioside binding domain recognized by lipid rafts, which also display an electronegative surface potential [23]. We propose a general inhibition mechanism of Tranilast against infection by vaccinia viruses and Mpox virus.

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## 2. Materials and Methods

**2.1. Molecular modeling simulations.** Ab initio structure predictions Mpox virus E8L (Uniprot entry Q8V4Y0) were performed by submitting the amino acid sequences to the Robetta server (https://robetta.bakerlab.org) as previously described [24]. The 3D structure of vaccinia virus D8L (Uniprot entry P20508) was performed by homology modeling and energy minimization of the E8L structure. Chondroitin sulfate (pdb 1c4s) and Tranilast (retrieved from pdb 2vd0) were submitted to energy minimization with the Polak-Ribière algorithm of the Hyperchem software using root-mean-square (RMS) gradient of 0.01 kcal. Å⁻¹.mol⁻¹ as the convergence condition. Molecular docking studies were performed using a protocol allowing complete conformational freedom of proteins and ligands (flexible docking) using Hyperchem software. The starting point for docking was done by manual positioning on each ligand on the viral proteins with full consideration of the surface electrostatic potential of both partners. In addition, a series of 20 randomly positioned ligand molecules covering the whole surface of the protein were analyzed. Energy minimization of each system was then performed with the Polak-Ribière conjugate gradient algorithm, with CHARMM force field in Hyperchem, using a maximum of 3 × 10⁵ steps, and a root-mean-square (RMS) gradient of 0.01 kcal. Å⁻¹.mol⁻¹ as the convergence condition as described previously [25]. The energy of interaction of each molecular complex was estimated with the Ligand Energy Inspector function of the Molegro Molecular Viewer software [23].

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**2.2. Amino acid sequence alignments.** The amino acid sequences of D8L and E8L proteins were aligned with Blast [26,27].

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**2.3. Langmuir microtensiometry measurements.** The recombinant E8L protein from Mpox virus (strain Zaire-96-I-16) was purchased from Proteogenix (Schiltigheim France). The lyophilized protein (100 µg in phosphate buffered saline, pH 7.5) was reconstituted in 200 µl of pure water, stored at 4°C and used in the days following solubilization according to the recommendation of the manufacturer. Phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Gangliosides (ref. 1525) were purchased from Matreya (State College, PA, USA). All lipids were dissolved in chloroform:methanol (1:1, vol:vol) at a concentration of 1 mg/ml and stored at -20°C. Tranislast (2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic Acid N-(3,4-Dimethoxycinnamoyl)anthranilic Acid) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lipid monolayers of the indicated composition were prepared at the air-water interface by spotting small droplets of the lipid solution with a 10 µl Hamilton syringe on the surface of a pure water or Tranilast solution subphase (800 µl). The surface pressure of the monolayer was measured in real-time with a Kibron µThrough apparatus. Since Tranilast is barely soluble in water [28], we prepared an oversaturated solution of the drug at a concentration of 100 µM, which slightly exceeds its limit of solubility. Under these conditions, stable monolayers of either POPC or gangliosides could be prepared and tested for E8L protein interaction.

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The final concentration of E8L was 125 nM. The data are expressed as variations of the surface pressure ($\Delta\pi$ in mN/m⁻¹) as a function of time which allowed to determine the initial velocity of the interaction.

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### 3. Results

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**3.1. In silico studies.** As a preamble to our molecular docking study, we analyzed the surface electrostatic potential of chondroitin sulfate and Tranilast. As can be seen in **Figure 1A**, these two molecules have a very electronegative potential (colored in red). This suggests that the initial interaction of these molecules with viral envelope proteins is based on electrostatic interactions [29]. It is therefore necessary to look for cationic zones in viral envelope proteins. Regarding the vaccinia virus, the D8L protein was identified as the protein allowing the initial attachment of the virus to the glycosaminoglycans of the cell surface, in particular chondroitin sulfate [15]. Thus, we performed a series of docking studies of chondroitin sulfate on this protein. Several initial conditions were tested but only one high affinity complex was obtained. In this complex, chondroitin sulfate is located in a specific cationic crevice in a globally electropositive surface of the D8L protein (**Figure 1B, left panel**). The calculated binding energy for this complex is -111.7 kJ/mol⁻¹ (**Table 1**). The interaction is based on a characteristic triad of amino acids comprising R44, Y104 and E105 (**Figure 1B, right panel**). The side chains of these amino acids form a clamp around the ligand, which stabilizes it in the crevice. Complementary interactions involving the other amino acids of the binding site (e.g. N46, S65, K108) then reinforce the complex through a network of hydrogen bonds.

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a.

<figure><img src="image_3.png" /></figure>

**Figure 1.** Chondroitin sulfate and Tranilast bind to the same cationic area on the surface of the viral envelope protein D8L. **A.** Electrostatic surface potential of chondroitin sulfate and Tranilast. The scale indicates that electronegative surfaces are colored in red, electropositive in blue and neutral in white. **B.** Docking of chondroitin sulfate on vaccinia virus D8L envelope protein. The electrostatic surface protein is visible in the left panel. A dissection of the binding site is shown in the right panel. In both cases chondroitin sulfate is represented in yellow atomic spheres. **C.** Docking of Tranilast (yellow spheres) on D8L (same representations as in **B**).
The complete analysis of the amino acids contributing to the interaction is given in **Table 1**. This analysis shows the expected presence of cationic amino acids (R44, K108, K175) for only one anionic amino acid (E105).

In the second step, we performed a similar analysis with Tranilast (Figure 1C). The results showed that Tranilast occupies the same interaction site as chondroitin sulfate at the cationic region of the vaccinia virus D8L protein. In this case, the estimated energy of interaction, although slightly lower, remains significant (-77.0 kJ.mol⁻¹). The triad of amino acids involved in the recognition of chondroitin sulfate is also involved in the complex with Tranilast (Table 1). The molecular complex between Tranilast and the vaccinia virus D8L protein involved a combination of electrostatic interactions (R44 with the carboxylate group of the drug), a π-π interaction (Y104 with the aromatic ring bearing the carboxylate group of the drug), and two weak Hydrogen bonds (between the drug and amino acids N46 and S64). As the size of Tranilast is smaller than chondroitin sulfate (Figure 1A), it is quite logical that its binding energy is also lower. However, it represents 69% of the energy of interaction of the D8L-chondroitin sulfate complex.

<table><caption>**Table 1.** Binding energy of GAG (chondroitin sulfate) and Tranilast on D8L (vaccinia virus) or E8L (Mpox virus).</caption><thead><tr><th>Amino acid</th><th>Vaccinia D8L GAG</th><th>Vaccinia D8L Tranilast</th><th>Mpox E8L GAG</th><th>Mpox E8L Tranilast</th></tr></thead><tbody><tr><td>T39</td><td>-</td><td>-6.2</td><td>1.3</td><td>-8.3</td></tr><tr><td>R44</td><td>-21.9</td><td>-19.3</td><td>-23.4</td><td>-21.3</td></tr><tr><td>N46</td><td>-13.7</td><td>-3.4</td><td>-11.4</td><td>-3.1</td></tr><tr><td>S64</td><td>-12.5</td><td>-3.5</td><td>-6.2</td><td>-4.0</td></tr><tr><td>S/T65</td><td>-7.7</td><td>-1.5</td><td>-3.4</td><td>-3.0</td></tr><tr><td>Y104</td><td>-18.5</td><td>-14.1</td><td>-17.4</td><td>-11.0</td></tr><tr><td>E105</td><td>-9.7</td><td>-5.5</td><td>-12.1</td><td>-5.5</td></tr><tr><td>K108</td><td>-10.2</td><td>-5.1</td><td>-12.1</td><td>-4.4</td></tr><tr><td>K/N175</td><td>-3.5</td><td>-7.3</td><td>-</td><td>-2.2</td></tr><tr><td>Total</td><td>-111.7</td><td>-77.0</td><td>-98.6</td><td>-74.6</td></tr></tbody></table>

Calculated from molecular models of each ligand bound to the indicated virus protein in Figures 1 and 2 and expressed as ΔG in kJ.mol⁻¹. For the sake of clarity only key amino acids are mentioned. Note that S65 and K175 in D8L are changed in T6 and N175 in E8L.

Since the degrees of freedom of chondroitin sulfate at the host cell surface are much more limited than those of Tranilast in solution, it is reasonable to conclude that Tranilast acts by competitive inhibition, interfering with the initial attachment of vaccinia virus to cell surface glycosaminoglycans.

Next, we studied the E8L protein of Mpox virus which shares 97% of amino acid sequence homology with vaccinia virus D8L protein (Figure 2).
<table><caption>**Figure 2.** Amino acid sequence alignment of D8L (vaccinia virus) and E8L (Mpox virus). Key amino acid residues involved in chondroitin sulfate and Tranilast binding are in bold and red. Differences between vaccinia D8L (amino acids highlighted in yellow) and Mpox E8L (amino acids highlighted in cyan) are indicated. Uniprot accession numbers are P20508 for D8L and Q8V4Y0 for E8.</caption><tr><td>D8</td><td>MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGKLVRINFKGGYISGGFLPNE</td><td>60</td></tr><tr><td>E8L</td><td>MPQQLSPINIETKKAISDTRLKTLDIHYNESKPTTIQNTGKLVRINFKGGYISGGFLPNE</td><td>60</td></tr><tr><td>D8L</td><td>YVLSSLHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEAKKHDDGLIIISIF</td><td>120</td></tr><tr><td>E8L</td><td>YVLSTIHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEAKKHDDGIIIAIF</td><td>120</td></tr><tr><td>D8L</td><td>LQVSDHKNVYFQKIVNQLDSIRSANTSAPFDSVFYLDNLLPSTLDYFTYLGTTIKHSADA</td><td>180</td></tr><tr><td>E8L</td><td>LQVSDHKNVYFQKIVNQLDSIRSANMSAPFDSVFYLDNLLPSTLDYFTYLGTTINHSADA</td><td>180</td></tr><tr><td>D8L</td><td>VWIIFPTPINIHSDQLSKFRTLLSSSNHDEGKPYYITENYRNPYKLNDDTQVYYSGEIIRA</td><td>240</td></tr><tr><td>E8L</td><td>AWIIFPTPINIHSDQLSKFRTLLSSSNHEGKPHYITENYRNPYKLNDDTQVYYSGEIIRA</td><td>240</td></tr><tr><td>D8L</td><td>ATTSPARENYFMRWLSDLRETCSYYQKYIEGNKTFAIIAIVFVFILTAILFLMSRRYSR</td><td>300</td></tr><tr><td>E8L</td><td>ATTSPVRENYFMKWLSDLREACFSYYQKYIEGNKTFAIIAIVFVFILTAILFLMSQRYSR</td><td>300</td></tr><tr><td>D8L</td><td>EKQN 304</td><td></td></tr><tr><td>E8L</td><td>EKQN 304</td><td></td></tr></table>

As shown in **Figure 3A**, the structure of the E8L-chondroitin sulfate complex obtained by docking is very similar to that of the complex involving the D8L protein. The glycosaminoglycan recognizes the same crevice on the surface of the E8L protein. Despite two amino acid changes at the binding site (at positions 65 and 175), the interaction energy remains high (-98.6 kJ·mol⁻¹) (**Table 1**). The main amino acids defining the binding site are the same as for the D8L protein, notably the characteristic triad R44-Y104-E105. The presence of asparagine instead of lysine at position 175 of the E8L protein of the Mpox virus has a slight impact on the interaction, since this amino acid no longer contributes to the interaction with chondroitin sulfate. However, N175 is involved in the recognition of Tranilast by the E8L protein (**Figure 3B**). In this case, the amide group of the side chain of N175 forms a Hydrogen bond with one of the two O-methyl group of the drug (**Figure 4A**). It even seems that the interaction with the drug is slightly improved compared to the D8L protein of the vaccinia virus. Indeed, it can thus be estimated that in the case of Mpox virus, Tranilast reaches 76% of the binding energy of the glycosaminoglycan compared with 69% for the vaccinia virus (**Table 1**).

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<figure><img src="image_4.png" /><figcaption>**Figure 3.** Docking of chondroitin sulfate (A) and Tranilast (B) and Mpox virus envelope protein E8L (same representations as in Figure 1B and C). Note that K175 in D8L is replaced by N165 in E8L.</figcaption></figure>

## 3.2. Physicochemical studies

Physicochemical analysis of E8L-Tranilast interactions were then performed with a specifically designed protocol that, to the best of our knowledge, has never been used for assessing the binding of an antiviral drug to a viral protein (Figure 4). This new protocol allowed us to evaluate in the same experiment the interaction of Tranilast with E8L and the inhibitory effect of the drug. In the first series of experiments, E8L was added underneath a monolayer of gangliosides. We observed a progressive increase of the surface pressure which indicates that the protein readily interacts with these membrane lipids (Figure 4A). In a second experiment, we substituted the water subphase with a Tranilast solution (Figure 4A). Under these conditions, the interaction between E8L and gangliosides was totally abrogated. Finally, E8L was injected underneath a monolayer of phosphatidylcholine, a zwitterionic membrane lipid (Figure 4B). In this case, the surface pressure increase induced by the protein was only slightly affected by Tranilast, which further suggests a specific effect of the drug on E8L-ganglioside interactions. The reproducibility of these data is shown in the histograms of Figure 5. The effect of Tranilast on the E8L-ganglioside interactions is statistically significant, whereas the effect of the drug on E8L-phosphatidylcholine appears more erratic.

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<figure><img src="image_5.png" /></figure>

**Figure 4. Kinetics of Interaction of recombinant Mpox E8L protein with reconstituted membrane systems.** For each condition, the data show a representative kinetics of interaction of recombinant Mpox E8L (125 nM) with a monolayer of mixed gangliosides (A) or phosphatidylcholine (POPC) (B) prepared at initial surface pressure of 17.5 mN.m⁻¹ in absence or presence of Tranilast (Tran) at 100 µM as indicated in the graphs. In the absence of the drug, the subphase is pure water. The data show the variations of the surface pressure (Δπ) expressed in mN.m⁻¹ as a function of time following the injection of E8L in the indicated subphase.

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<figure><img src="image_6.png" /></figure>

**Figure 5. Effect of Tranilast on E8L-membrane interactions.** (A) Initial velocity of the interaction. (B) Variations of the surface pressure 20 minutes after the injection of E8L (125 nM) in the subphase. In absence of the drug, the subphase is pure water. When indicated the subphase is an oversaturated solution of Tranilast (Tran, 100 µM). All data are expressed as mean ± SD (n=3). The inhibitory effect of Tranilast on the E8L-ganglioside interaction is statistically significant (p<0.005 in T-test). GG, gangliosides; POPC, phosphatidylcholine.

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## 4. Discussion

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Glycosaminoglycans such as heparan sulfate and chondroitin sulfate play a crucial role in Mpox virus infection. These molecules, present on the surface of host cells, serve as primary receptors for Mpox virus attachment [30]. They may also serve as targets for the development of antiviral treatments [31-33]. Molecules structurally similar to glycosaminoglycans could be used to block viral attachment, thereby preventing infection [34].

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However, the approach we used is different since we did not search for structural analogues of chondroitin sulfate. Instead, we analyzed the fingerprint of the chondroitin sulfate binding site in the D8L and DE8L proteins (Figure 6).

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<figure><img src="image_7.png" /><figcaption>**Figure 6.** Fingerprint analysis of the glycosaminoglycan binding site of vaccinia D8L and Mpox E8L proteins suggests a competitive inhibition mechanism for Tranilast. The chondroitin sulfate binding site "fingerprint" is colored in dark blue on the surface of D8L (upper panels) and E8L (lower panels) proteins. The protein surface is colored in gray and Tranilast is represented in yellow sticks. Four different views of the complex are shown for each protein. These structural data suggest that Tranilast may act as a competitive inhibitor of glycosaminoglycan binding to D8L and E8L proteins.</figcaption></figure>

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This "surface fingerprint" approach, initiated in 2009 [35], is currently gaining momentum due to the rapid progress in artificial intelligence (AI) [36]. In our case, however, we did not use AI to generate our data, which came in fact from the optimized use of Hyperchem software in a docking configuration [37-39]. We challenged the D8L and E8L chondroitin sulfate fingerprints with Tranilast because this antihistamine molecule has an electrostatic surface potential characteristic that seemed compatible with these viral glycoproteins (Figure 1A). Yet chondroitin sulfate and Tranilast have a distinct geometry. Dynamic docking simulations allowed us to verify the compatibility of both ligands with the virus proteins. It is important to mention that in contrast with rigid docking of glycosaminoglycans onto viral proteins that can be automatically performed with ClusPro [40,41], (e.g. SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2), our method allows the whole molecules to move stochastically and adopt distinct conformations during the simulations. For instance, ClusPro primarily performs rigid body docking, meaning that the bond angles, bond lengths, and torsion angles of the proteins remain fixed during the docking process [40]. Thus, while ClusPro excels at rigid body docking, it does have limitations when dealing with complexes where significant backbone conformational changes occur upon binding.

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Our flexible docking approach recently allowed to highlight the great flexibility of the ivermectin molecule and to explain why this drug interacts with the surface of the spike proteins of successive variants of SARS-CoV-2 [21]. Our work is also based on the elegant study of Hsiao et al. who previously showed that the D8L protein of the vaccinia virus envelope interacts with chondroitin sulfate on the surface of host cells and that this interaction controls the attachment of the virus to these cells [15]. We characterized a chondroitin sulfate interaction site on the D8L protein and showed that this site is also recognized by Tranilast. The strong electronegative surface potential of these two ligands plays a major role in the recognition of the D8L protein, which has a large surface-exposed electropositive zone. The site consists of a crevice delimited by a group of amino acids comprising the triad R44-Y104-E105. Our data are consistent with a previous study that also identified an electropositive crevice in the same region of D8L as a binding site for chondroitin sulfate [42] and a target for neutralizing antibodies [43]. They also indicate that the same crevice and the same amino acid triad are involved in the binding of Tranilast to the D8L protein. Overall, our study suggests a mechanism of competitive inhibition of Tranilast on the initial attachment of vaccinia virus to cell surface glycosaminoglycans and lipid raft gangliosides.

For this experimental part of the study, we developed a new protocol allowing to detect the inhibitory effect of any potential antiviral drug on the physical interaction of a viral protein with membrane lipids. This method is based on real-time microtensiometry measurements of the surface pressure of reconstituted membrane lipid systems. Gangliosides were used preferentially to glycosaminoglycans because they form stable monolayers at the air-water interface, thus allowing surface pressure measurements to detect the binding of the E8L protein. Using this innovative approach, we demonstrated that E8L has a remarkable affinity for membrane gangliosides, which confirmed our previous in silico studies [23]. Indeed, the protein induced a rapid increase of the surface pressure when added in the aqueous subphase underneath ganglioside monolayers. However, in the presence of Tranilast, the protein did no longer interact with gangliosides whereas a slight delay was observed for POPC. Overall, these data confirmed the presence in silico studies which identified a structural Tranilast binding site overlapping the common glycosaminoglycan/ganglioside binding domain of E8L.

The E8L protein of the Mpox virus presents 97% sequence homology with D8L. However, if we consider that the Tranilast binding site is mainly made up of 8 amino acids, two of them are not conserved in the two viruses: serine 65 of D8L is replaced by threonine in E8L, and lysine 175 by asparagine. This results in a clearer difference of 25% between the two binding sites. It was therefore important to check that such a divergence could affect the recognition of glycosaminoglycans and Tranilast. Our docking results showed that this is not the case. In fact, the E8L protein of the Mpox virus interacts perfectly with chondroitin sulfate. Indeed, we observed only a small difference of 7% in the interaction energies of the two complexes (Table 1). Thus, from a functional point of view, the binding sites for chondroitin sulfate and Tranilast are remarkably conserved in vaccinia D8L and Mpox E8L proteins.

Our analysis is based on the identification of the D8L protein of vaccinia virus as a privileged site of interaction between the envelope of this virus and glycosaminoglycans, establishing cell surface chondroitin sulfate as a critical component for vaccinia virus entry [15]. By occupying glycosaminoglycan recognition sites on the D8L (vaccinia virus) and E8L (Mpox virus) envelope proteins, Tranilast is highly likely to act by competitive inhibition, blocking the initial adhesion of these viruses to proteoglycans on the surface of host cells (Figure 6).

Our modeling studies also revealed that Tranilast interacts with D8L and E8L viral glycoprotein proteins through a combination of electrostatic forces, aromatic ring interactions, and hydrogen bonds, which are also operative in glycosaminoglycan protein-interactions. In this respect, it is also interesting to note that Tranilast can inhibit the infection of other viruses such as hepatitis B virus [44] that are also known to interact with glycosaminoglycans [45,46]. Finally, Tranilast may also interfere with late steps of viral cycles, as demonstrated for SARS-CoV-2. In this case, Tranilast has been identified as a potent and selective inhibitor
of the viral protease [47]. That Tranilast may act at different steps of the Mpox virus replication cycle is also suggested by a time-addition experiment [48,49] conducted on vaccinia virus with a luciferase reporter assay [46]. In these experiments, the preincubation of recipient Vero cells with Tranilast had no effect on infection. This clearly indicates that the drug does not bind to the host cell, but rather to one of the virus components, in agreement with our own results. Furthermore, the presence of Tranilast in competition with the virus for 1 hr at 37°C induced a reduction of infection of approximately 35%. This effect is consistent with a mechanism of competitive inhibition during the phase of virus attachment to target cells. Even greater inhibition is obtained if Tran is added 1 hr after incubation of the virus with cells, either at 4°C or 37°C, with the drug remaining present for the next 24 hr. This suggests an effect of the drug on virus-cell fusion or on later phases including post-entry steps that may extend to intracellular biosynthesis. In summary, if we combine our results with those published by Wu et al. [8], we can conclude that Tranilast probably acts at the level of the initial attachment of the virus to target cells and at later stages.

## 5. Conclusions

Our results allow us to propose a molecular mechanism in favor of the repositioning of Tranilast as an antiviral drug in the prevention and treatment of infections by vaccinia and Mpox viruses, and perhaps other viruses. Cell surface glycosaminoglycans such as chondroitin sulfate and lipid raft gangliosides are anionic structures that serve as initial receptors for several human and animal viruses, and such interactions are known targets for antiviral intervention [50,51]. Chondroitin sulfate and Tranilast, which share common electronegative surface properties but have a distinct geometry, have the same binding site on vaccinia and Mpox virus envelope proteins D8L and E8L. Considering the results of the present study, we can anticipate a specific antiviral effect of Tranilast on the early stages of the Mpox virus infection cycle. Since Tranilast is a widely used drug in Japan, safe and with a very low cost, it would thus be interesting to test it as a potential inhibitor of the Mpox virus. Finally, our physicochemical study using Langmuir monolayer technology represents an innovative approach to screen antiviral molecules from their ability to inhibit the interaction of virus adhesins with different lipids used as membrane models.

## Author Contributions

Conceptualization, H.C., J.F. and D.R.; Formal analysis, H.C. and J.F.; Investigation, H.C. and J.F.; Methodology, H.C., J.F. and D.R.; Supervision, J.F.; Writing—original draft, J.F.; Writing—review and editing, H.C., J.F. and D.R.

## Funding

This research received no external funding.

## Institutional Review Board Statement

Not applicable.

## Informed Consent Statement

Not applicable.

## Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

## Conflicts of Interest

The authors declare no conflicts of interest.
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