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. 2026 Jan 20;49(5):100949. doi: 10.1016/j.bj.2026.100949

Discovery of BPR2-D2 compound in inhibiting Chikungunya viral RNA synthesis by replicon systems

Yi-Ju Hsu a,1, Tram-Anh Thi Phan b,1, Hui-Chung Lin c, Der-Jiang Chiao c, Szu-Cheng Kuo c, Syh-Jae Lin d, Rei-Lin Kuo a,b,d,e,f,⁎
PMCID: PMC13634103  PMID: 41571103

Abstract

Chikungunya virus (CHIKV), an alphavirus in the Togaviridae family, is transmitted to humans through mosquito bites and can lead to severe complications in newborns and elders. Its RNA genome functions as mRNA to produce viral nonstructural proteins, which assemble into replication complex for synthesizing new viral genomic and subgenomic RNA within the cytoplasm. Previous studies have established a CHIKV replicon system, that can express CHIKV replication complex from viral genomic RNA and an eGFP reporter from subgenomic RNA, to study viral RNA replication and screen antivirals against the CHIKV RNA replication. Additionally, researchers have discovered that BPR2-D2, which is classified to furanocoumarin, exhibits potential as a broad-spectrum antiviral drug. In this research, we applied the replicon system to evaluate the antiviral activity of BPR2-D2 against CHIKV RNA replication. The effective concentration was 10.47 ± 0.02297 nM for inhibiting 50 % of CHIKV genomic RNA replication with high selective index. Moreover, we validated the antiviral effect of BPR2-D2 with the authentic infection of Sindbis virus, which is also an arthritogenic alphavirus. The molecular docking analysis was applied to suggest possible targets of BPR2-D2 among CHIKV nonstructural proteins. Taken together, this research demonstrated that BPR2-D2 can be a promising antiviral against CHIKV.

Graphical abstract

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Highlights

  • •

    BPR2-D2, a coumarin derivative, is able to inhibit Chikungunya virus (CHIKV) RNA replication with a high selective index.

  • •

    BPR2-D2 can inhibit replication of Sindbis virus, which is an arthritogenic alphavirus as CHIKV.

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    The CHIKV replicon systems can be surrogates for studying CHIKV RNA replication and antiviral screening.

1. Introduction

Chikungunya virus (CHIKV), which is an old-world arbovirus and belongs to the genus of Alphavirus in the family of Togaviridae, is transmitted by Aedes aegypti and Aedes albopictus. Infection of the virus may cause fever, inflammatory arthralgia and arthritis and may also be life-threatening in newborn infants and elders [1,2]. Due to the increasing global traveling and transportation, spreading of the virus has become one of the latest concerns in public health.

The CHIKV particle consists of a single positive-stranded RNA genome, featuring a 5′ cap and 3’ poly(A) tail, which serves as viral mRNA for the translation of a viral nonstructural polyproteins called P123 and P1234. The polyprotein can be further processed to nsP1-4 by the viral encoded protease [3]. The nsPs form replication complex to replicate viral genomic RNA and synthesize viral 26S subgenomic RNA, encoding viral structural proteins: the capsid protein (C), the major envelop proteins E1-2, and the accessory proteins E3, TK and TF [4]. In the replication complex, the nsP1 possesses methyltransferase (MTase) and guanylyltransferase (Gtase) activities for transferring methyl group from S-adenosylmethione to GTP to create m7G-capped viral mRNA. Additionally, the nsP1 anchors the replication complex to the plasma membrane [[5], [6], [7], [8]]. The nsP2 contains RNA helicase region at the N-terminal region [9,10], while its C-terminal papain-like cysteine protease domain is responsible for cleaving the precursor nonstructural polyprotein [11]. The nsP3 is essential for viral replication and pathogenesis. The N-terminal macrodomain of nsP3 has ADP-ribosyl-binding and hydrolase activities, which are critical for effective viral RNA replication [12,13]. The C-terminal region of nsP3 modulates viral replication through interactions with host factors [14]. Finally, the nsP4, the RNA-dependent RNA polymerase (RdRP), serves as a core component of the replication complex, responsible for synthesizing viral RNA [15,16].

Since CHIKV infection could cause severe complications, developing effective antivirals against CHIKV is crucial. The nsPs represent promising drug targets, as the nsPs are essential for viral RNA synthesis and replication. Specifically, antivirals targeting on the protease and RdRP activities of nsP2 and nsP4, respectively, have been extensively studied [[17], [18], [19]]. Furthermore, the MTase and GTase functions of the nsP1 are potential targets [20]. Residues of Arg70, Arg92, and Asp152 in the nsP1 are critical for binding of GTP and SAM [21,22], hence small molecules bound to the sites may disrupt the MTase activity to block viral mRNA production [23]. Additionally, despite its incompletely understood mechanism of CHIKV nsP3 in regulating viral RNA replication, the nsP3 marcodomain is a recognized antiviral target. Residues Asp10, Gly32, Gly112 and Tyr114 within this domain, key positions for either ADP-ribosyl-binding or hydrolase activity, are crucial for viral replication [12,13]. Compounds targeting the positions could inhibit CHIKV replication [24,25].

Due to the pathogenicity and limited treatment options, CHIKV is classified to a risk group 3 pathogen, requiring biosafety level 3 containments for research. Consequently, CHIKV replicon and virus-like replicon particle (VRP) systems, designed for use in lower biosafety level facilities have been established by previous research. CHIKV replicon systems that express the replication complex allow for the study of viral RNA synthesis and antiviral screening [[26], [27], [28], [29], [30], [31]]. In addition, CHIKV VRPs are applicable to mimic single-cycle viral infection without producing progeny CHIKV viral particles [[32], [33], [34]]. By presenting the CHIKV structural proteins required for viral receptor binding, the VRPs can be used to test the effectiveness of neutralizing antibodies and antiviral compounds [[35], [36], [37]].

BPR2-D2, 8-benzoyl-4-methyl-9-phenyl-furo[2,3-h]chromen-2-one, is classified to furanocoumarin, which is one of coumarin derivatives and a potential antiviral against different types of viruses [38]. Previous research discovered that BPR2-D2 inhibits replication of influenza virus, enteroviruses, and SARS-CoV-2 [39,40]. In the present study, we evaluated the antiviral activity of BPR2-D2 against CHIKV RNA replication by using a CHIKV replicon system and validated the anti-alphavirus function by an infection model of Sindbis virus (SINV), which is also an arthritogenic alphavirus as CHIKV. Furthermore, the molecular docking analysis was applied for suggesting possible targets of BPR2-D2 among CHIKV nsPs. Collectively, this research demonstrated that BPR2-D2 can be a promising compound against CHIKV and other alphaviruses.

2. Materials and Methods

2.1. Cell culture and cytotoxicity assay

U-2 OS cells, a human osteosarcoma cell line, and BHK-21 cells, a cell line generated from hamster kidney fibroblasts, were cultivated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % Fetal Bovine Serum (FBS) and maintained in an incubator at 37 °C with 5 % CO2. To examine the cytotoxicity of BPR2-D2, kindly provided by Jim-Tong Horng at Chang Gung University, U-2 OS cells were seeded in 96-well plates for 24 h and then treated with various concentrations of BPR2-D2 for 48 h. Cells were subsequently fixed with 3.7 % paraformaldehyde and stained with 0.1 % crystal violet solution. After removing the staining solution and washing the plates with water, the absorbance of the crystal violet-stained cells that attached on the plates was measured using a microtiter plate reader at a wavelength of 570 nm. The cell survival percentage was calculated by comparing the absorbance to that of control cells treated with vehicle alone.

2.2. Recombinant baculovirus expressing CHIKV replicon

The recombinant baculovirus expressing CHIKV wild type and RdRP mutant (DDAA) replicons with eGFP reporter were constructed as previously described [30]. Briefly, a synthetic CHIKV replicon DNA containing nonstructural protein ORF and structural gene 26S promoter fused with eGFP open reading frame (ORF) was constructed to a modified baculovirus expressing vector pFastBac1 with the glycoprotein ORF of vesicular stomatitis virus (VSVG) insertion, denoted as pFastBac1-VSVG-CHIKV replicon-eGFP. The Bac-to-Bac expression system (Invitrogen) was applied with the transfer vector pFastBac1-VSVG-CHIKV replicon-GFP to generate a recombinant baculovirus expressing CHIKV replicon-GFP following the manufacturer's protocol. The recombinant baculovirus was titrated by BacPAK Baculovirus Rapid Titer kit (Takara Bio).

2.3. Transduction of recombinant baculoviruses following BPR2-D2 treatment

U-2 OS cells were seeded in 6-well plates with 5 × 105 cells/well and maintained in DMEM containing 10 % FBS at 37 °C for 24 h. After washing with PBS, cells were incubated with the recombinant baculovirus expressing CHIKV replicon and eGFP reporter in PBS at the multiplicity of infection (MOI) of 0.1. After 1 h incubation at 37 °C, cells were washed with PBS and then added DMEM with 10 % FBS containing indicated concentrations of BPR2-D2. At 24 h post drug treatment, eGFP expression of transduced cells was observed under a fluorescent microscope. The baculovirus expressing GFP-fused Rab5a (CellLight Early Endosomes-GFP, Invitrogen) was applied as a control group. The procedures of transduction and treatment were performed as described above.

2.4. Infection of SINV with BPR2-D2 treatment and plaque formation assay

U-2 OS cells were seeded in 6-well plates with 3 × 105 cells/well and cultivated with DMEM containing 10 % FBS at 37 °C for 24 h. Cells were then washed with PBS and then incubated with SINV (Ar-339 strain, ATCC) in serum-free DMEM at an MOI of 0.1 for 1 h. After removing the inoculates, cells were incubated with DMEM containing 10 % FBS and the indicated concentrations of BPR2-D2. At 24 h after treatment, supernatants of the treated U-2 OS cells were collected for plaque formation assay with BHK-21 cells. BHK-21 cells were seeded in 6-well plates with 8 × 105 cells/well for 24 h. Supernatants collected from SINV-infected U-2 OS cells were serially diluted with DMEM, as indicated 10-fold dilutions, and added onto monolayers of BHK-21 cells for 1 h. After removing the inoculates, cells were overlaid with a mixture of DMEM containing 2 % FBS and 1 % agarose and incubated at 37 °C for 48 h. Subsequently, cells were fixed with 3.7 % paraformaldehyde and stained with 0.1 % crystal violet solution.

2.5. Reverse transcription and quantitative PCR (RT-qPCR) and primers

Total RNA of recombinant baculovirus-transduced cells was extracted by TRIzol reagent (Invitrogen) following the manufacturer's instruction. Total RNA was subjected to RT reaction by SuperScript III First-Strand Synthesis System (Invitrogen) with oligo-dT primer, for detecting CHIKV genomic/messenger RNA and subgenomic RNA. The cDNA obtained from RT reaction was applied to qPCR by KAPA SYBR FAST qPCR Mix Kit (KAPA Biosystems) with the nsP1-targeting primer set: 5′-5′-TGC,GTA,CCC,CAT,GTT,TGA,GG-3′ and 5′-GTC,CGA,CAT,CATCCT,CCT,TGC-3′, and the GFP-targeting primer set: 5′-CCA,CAT,GAA,GCA,GCA,CGA,CTT-3′ and 5′-GGT,GCG,CTC,CTG,GAC,GTA-3’. The expression level of β-actin mRNA was used as a control for normalization. The qPCR primers for β-actin were 5′-GCT,CGT,CGT,CGA,CAA,CGG,CTC-3′ and 5′-CAA,ACA,TGA,TCT,GGG,TCA,TCT,TCT,C-3’.

2.6. Immunoblotting and antibodies

Cell extracts were heated in a sample buffer and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously [41]. The separated proteins on gels were transferred to PVDF membranes and reacted with anti-CHIKV nsP2 (GTX636962, GeneTex), anti-GFP (GTX113617, GeneTex), and anti-β-tubulin (MA5-16308, Invitrogen) antibodies, respectively. After incubating with the species-specific secondary antibodies conjugated with horseradish peroxidase (HRP), anti-mouse IgG-HRP (18-8817-30, Rockland) and anti-rabbit IgG-HRP (18-8816-33, Rockland), the membranes were reacted with chemiluminescence HRP substrate (Immobilon Western, Millipore) and visualized by X-ray film or ChemiDoc Imaging System (BioRad). The intensity of the bands on immunoblots was quantified by TotalLab 1D software.

2.7. Molecular docking analysis

The 3D crystal structures of CHIKV nonstructural proteins were obtained from the Protein Data Bank (PDB), including nsP1 (PDB ID: 7FGH), nsP2 helicase domain (PDB ID: 6JIM), nsP2 protease domain (PDB ID: 3TRK), nsP3 macrodomain (PDB ID: 3GPG), and nsP4 in the replicatin complex (PDB ID: 7Y38). The structure of ligand, BPR2-D2 (8-benzoyl-4-methyl-9-phenyl-furo[2,3-h]chromen-2-one), was retrieved from PubChem in SDF file and then converted to PDB file. The UCSF Chimera software version 1.17.3 (University of California, San Francisco) incorporating with AutoDock Vina 1.1.2 (The Scripps Research Institute) was applied for molecular docking [42] to predict the potential binding sites and docking scores of CHIKV nsPs and BPR2-D2.

2.8. Statistical analysis

The One-Way ANOVA was used to test the significance of the differences among groups (by the GraphPad Prism software). The error bars represented the standard deviations. P value of less than 0.05 between two groups was considered as a statistically significant difference.

3. Results

3.1. BPR2-D2 effectively inhibits replication of CHIKV RNA

Previous studies showed that BPR2-D exhibits the anti-influenza virus activity by targeting viral RNPs with a high selective index, and that BPR2-D2 may possess broad-spectrum antiviral properties against other RNA viruses, such as enterovirus 71, coxsackievirus B3 and human rhinovirus-2 [39]. To investigate inhibitory effect of BPR2-D2 on replication of CHIKV, we utilized a baculovirus-based CHIKV replicon system in U-2 OS cells [30]. Initially, cytotoxicity assays using crystal violet staining, as described in the section of the Materials and Methods, revealed that the cytotoxic concentration of BPR2D2 at which 50 % inhibition of U-2 OS cell viability (CC50) exceeded 80 μM after 48 h of treatment, while the solvent for 80 μM of BPR2-D2, 0.8 % of DMSO, did not significantly affect cell viability [Fig. 1]. U-2 OS cells were transduced with the recombinant baculovirus, which expresses CHIKV genomic RNA encoding nsP genes and an eGFP ORF under the control of CHIKV 26S subgenomic RNA promoter. Subsequently, cells transduced with the replicon were treated with varying concentrations of BPR2-D2 for 24 h. The results showed a reduction of eGFP reporter expression at 12.5 nM [Fig. 2A]. The reduction was quantified by immunoblotting with anti-CHIKV nsP2 and anti-GFP antibodies, respectively [Fig. 2B]. The results showed decreasing trends of nsP2 and eGFP protein levels under BPR2-D2 treatment at 12.5 nM, suggesting that BPR2-D2 might inhibit both of genomic and subgenomic RNA synthesis of CHIKV. To verify the specificity of BPR2-D2 in inhibiting the step of CHIKV RNA replication, we determined whether BPR2-D2 could affect the mRNA or protein production from a recombinant baculovirus, CellLight Early Endosomes-GFP (Invitrogen), expressing GFP-fused Rab5a under a CMV promoter. U-2 OS cells were transduced with this control GFP-baculovirus and treated with BPR2-D2 at concentrations of 2.5 and 12.5 nM. Unlike the results obtained with the CHIKV replicon, BPR2-D2 at these concentrations did not inhibit GFP expression, as observed by fluorescence microscopy and immunoblotting [Fig. 2C and D].

Fig. 1.

Fig. 1

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Determination of the cytotoxicity for BPR2-D2 in U-2 OS cells. U-2 OS cells were cultivated in 96-well plates and then treated with the indicated concentrations of either BPR2-D2 or DMSO for 48 h. Each condition was repeated with 8 wells of the cells. The cells were then stained with crystal violet solution (A). The absorbance of the stained cells that attached on the plates was measured at a wavelength of 570 nm. The cell survival percentage was calculated by comparing the absorbance to that of control cells without treatment (B).

Fig. 2.

Fig. 2

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Specifically inhibitory effect of BPR2-D2 on CHIKV RNA synthesis evaluating with a recombinant baculovirus-based CHIKV replicon and a control GFP-expressing baculovirus. U-2 OS cells were transduced with either a recombinant baculovirus-expressing CHIKV replicon containing eGFP ORF driven by 26S subgenomic RNA promoter (A and B) or a recombinant baculovirus expressing GFP-fused Rab5a driven by a CMV promoter (CellLight Early Endosomes-GFP, Invitrogen) (C and D) at an MOI of 0.1, and subsequently treated with BPR2-D2 at the indicated concentrations, as diagrammed in the upper panel. GFP expressed from CHIKV replicon and the control baculovirus was observed under a fluorescent microscope (A and C). The expression of CHIKV nsP2 and GFP was examined by immunoblotting with anti-CHIKV nsP2 and anti-GFP antibodies, respectively (B and D). Relative protein expression levels from three repeats of the expression were quantified by TotalLab 1D gel image analysis software (lower panels of B and D).

We validated CHIKV RNA synthesis by RT-qPCR with primers targeting the nsP1 region. As shown in [Fig. 3A], a dose-dependent decrease in CHIKV genomic RNA was observed in BPR2-D2-treated cells. As eGFP is encoded by the subgenomic RNA of the replicon, RT-qPCR targeting the eGFP region confirmed a reduction in combined CHIKV genomic and subgenomic RNA levels upon BPR2-D2 treatment [Fig. 3B], while GFP-Rab5a mRNA levels from the control GFP-baculovirus-transduced cells remained unchanged under these treatments [Fig. 3C]. The results demonstrated that BPR2-D2 specifically inhibits CHIKV RNA replication. We further determined that the effective concentration at which 50 % inhibition of CHIKV genomic RNA synthesis (EC50) was 10.47 ± 0.02297 nM [Fig. 4A], and that the EC50 for inhibiting genomic/subgenomic RNA was 9.864 ± 0.03637 nM [Fig. 4B]. Collectively, these results conclude that BPR2-D2 effectively inhibits CHIKV RNA synthesis with a high selective index (CC50/EC50).

Fig. 3.

Fig. 3

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Examination of CHIKV RNA synthesis upon BPR2-D2 treatment. Total RNA was extracted from U-2 OS cells that transduced with either a CHIKV replicon or a control baculovirus and treated with BPR2-D2. RT-qPCR was performed with oligo-dT RT primer and nsP1 and GFP-specific PCR primer sets, respectively, for detecting positive-stranded CHIKV genomic RNA (A), combination of genomic and subgenomic RNA (B), and GFP mRNA from the control baculovirus-transduced cells (C). The experiments were triplicated. Compared to no BPR2-D2 treatment group, ** indicates p < 0.01, and *** indicates p < 0.001.

Fig. 4.

Fig. 4

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Determination of the EC50 of BPR2-D2 in inhibiting CHIKV RNA synthesis. Total RNA was extracted from U-2 OS cells that transduced with CHIKV replicon and treated with BPR2-D2 at concentrations of 0, 5, 7.5, 10, 15, 20, and 25 nM, respectively. RT-qPCR was performed with oligo-dT RT primer and nsP1 and GFP-specific PCR primer sets, respectively, for detecting positive-stranded CHIKV genomic RNA (A) and combination of genomic and subgenomic RNA (B). The experiments were triplicated. EC50 was calculated by the GraphPad Prism software.

Although we detected the decreasing levels of CHIKV RNAs, we did not rule out the possibility that BPR2-D2 might suppress CHIKV mRNA translation and reduce the nsP2 and eGFP levels. To examine the possibility, we transduced U-2 OS cells with a CHIKV replicon that is mutated in nsP4 region [30] and then examined eGFP and viral protein synthesis. We did not detect eGFP, which is translated from subgenomic RNA, by immunoblotting [Supplementary Fig. S1], verifying that the mutant lost the RdRP activity, as shown in previous research [30]. Additionally, the result showed that the protein level of CHIKV nsP2, which is translated from viral genomic RNA, was not changed under BPR2-D2 treatment [Supplementary Fig. S1], indicating that BPR2-D2 does not inhibit CHIKV polyprotein translated from viral genomic RNA.

3.2. Evaluation of the antiviral effect of BPR2-D2 during alphavirus infection

Previous results showed that BPR2-D2 can inhibit CHIKV RNA synthesis by using a CHIKV replicon system. We further determined whether BPR2-D2 has antiviral activity during authentic alphavirus infection. SINV, which is also an arthritogenic alphavirus as CHIKV, was applied as an infection model. U-2 OS cells were infected with SINV and then treated with BPR2-D2 at concentrations of 2.5, 12.5, and 62.6 nM. We found that SINV-induced cytopathic effect on U-2 OS cells was reduced with treatments of BPR2-D2 at 12.5 and 62.6 nM [Fig. 5A]. Supernatants of the infected cells were collected for titrating the infectious virus with indicated 10-fold dilutions by plaque formation assay [Fig. 5B]. The results showed that SINV titers were reduced more than 20 folds and 40 folds with BPR2-D2 treatment at 12.5 and 62.6 nM, respectively [Fig. 5C], indicating that BPR2-D2 contains antiviral activity against alphavirus infection.

Fig. 5.

Fig. 5

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BPR2-D2 inhibits replication of SINV. U-2 OS cells were infected with SINV and treated with BPR2-D2 at indicated concentrations, as diagrammed in the upper panel. (A) Cytopathic effect induced by SINV infection was observed under a microscope. (B) Plaque formation asaay with BHK21 cells was applied to determine virus titers in the supernatants of SINV-infected U-2 OS cells. (C) Average virus titers were determined from three repeats of the experiments. Compared to no BPR2-D2 treatment group, ** indicates p < 0.01.

3.3. Molecular docking analysis of the BPR2-D2 targeting on CHIKV nonstructural proteins

Previous results have shown that BPR2-D2 may target CHIKV RNA synthesis. We applied UCSF Chimera software, incorporating with AutoDock Vina, to predict the potential interaction of BPR2-D2 with CHIKV nonstructural proteins. The structure of BPR2-D2 was retrieved from PubChem and converted to PDB format using Dock Prep. The available three-dimensional (3D) crystal structures of CHIKV nsPs were obtained from PDB, including nsP1 (PDB ID: 7FGH); nsP2 helicase domain (PDB ID: 6JIM) and nsP2 protease active site domain (PDB ID: 3TRK), nsP3 N-terminal macrodomain (PDB ID: 3GPG), and nsP4 in the replication complex (PDB ID: 7Y38). The result showed that the putative interaction of BPR2-D2 with nsP1 had the highest binding score, as shown the model in [Fig. 6]A, with a predicted binding energy of −10.8 kcal/mol. The model demonstrated that BPR2-D2 forms a hydrogen bond with Arg92, pi-anion interaction with Asp152, pi-alkyl interactions with Val153 and Ala155, and pi-pi stacking with Tyr248 of nsP1 [Fig. 6B and C]. Residues Arg92 and Asp152 have been found in GTP and SAM-binding sites of nsP1 [[21], [22], [23]]. Additionally, the molecular docking analysis predicted that BPR2-D2 may also bind to functional residues in the N-terminal macrodomain of nsP3 [Fig. 7A], which possesses ADP-ribose binding and hydrolase activities and is critical for the initiation of alphavirus replication [13], with a binding energy of −10.1 kcal/mol. The predicted bindings were a pi-sigma bond with Tyr114, a pi-donor hydrogen bond with Val113, and alkyl or pi-alkyl interactions with Ala22, Val33, and Val113 [Fig. 7B and C]. The Val113 and Tyr114 in the macrodomain of nsP3 are responsible for the phosphate binding, which is important for its mono ADP-ribose hydrolase activity [43,44]. Moreover, the residue Gly32, which may also be involved in ADP-ribose binding and hydrolase functions of nsP3 [13], was predicted to have van der Waals interactions with BPR2-D2. Nevertheless, further experiments and functional assays are required to validate the mechanisms predicted by the molecular docking analysis for BPR2-D2-targeting CHIKV nonstructural proteins.

Fig. 6.

Fig. 6

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Molecular docking analysis for BPR2-D2 with CHIKV nsP1. Interaction of CHIKV nsP1 (PDB ID: 7FGH) and BPR2-D2 was analyzed by the UCSF Chimera software version 1.17.3 incorporating with AutoDock Vina 1.1.2. (A) Molecular binding model for BPR2-D2 (red color) and the ribbon 3D structure of CHIKV nsP1 (blue color). (B) Interactions between specific nsP1 residues and BPR2-D2 were revealed by the docking analysis. (C) The interaction residues of nsP1 with BPR2-D2 include Arg92: conventional hydrogen bond (green color), Asp152: pi-anion interaction (orange color), Tyr248: pi-pi stacking interaction (magenta color), and Val153 and Ala155: pi-alkyl interaction (light purple color).

Fig. 7.

Fig. 7

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Molecular docking analysis for BPR2-D2 and CHIKV nsP3 macrodomain interaction. Interaction of CHIKV nsP3 macrodomain (PDB ID: 3GPG) and BPR2-D2 was analyzed by the UCSF Chimera software incorporating with AutoDock Vina. (A) Molecular binding model for BPR2-D2 (red color) and the ribbon 3D structure of CHIKV nsP3 macrodomain (green color). (B) Interactions of specific residues in nsP3 macrodomain and BPR2-D2 were revealed by the docking analysis. (C) The interactions of nsP3 macrodomain and BPR2-D2 include a pi-donor hydrogen bond with Val113 (light green color), pi-sigma bond with Tyr114 (violet color), pi-pi stacking interaction with Trp148 (magenta color), alkyl or pi-alkyl interactions with Ala22, Val33, and Val113 (light purple color).

4. Discussion

Alphaviruses, such as CHIKV, can cause severe diseases and pose a significant public health threat. As no approved specific antivirals targeting these viruses are available, the development of effective treatment is urgently needed. Previous studies have demonstrated that BPR2-D2 inhibits the replication of several RNA viruses, including influenza A virus, enterovirus A71, coxsackievirus B3, human rhinovirus-2, and SARS-CoV-2 [39,40]. In this study, we further determined whether BPR2-D2 possesses the antiviral activity against CHIKV RNA replication. Initially, we used a recombinant baculovirus-transduced CHIKV replicon system expressing viral nsPs from the genomic RNA and an eGFP reporter from the 26S subgenomic RNA to examine the expression of CHIKV nsPs and eGFP under BPR2-D2 treatment. The results revealed that BPR2-D2 could affect the expression of both viral and reporter proteins from the CHIKV replicon. We further demonstrated that the BPR2-D2 suppresses synthesis of CHIKV genomic RNA by 50 % (EC50) at a concentration of 10.47 nM. In contrast, GFP mRNA expression via transduction with a control GFP baculovirus was not suppressed, indicating that BPR2-D2 specifically interferes with CHIKV RNA synthesis. Furthermore, SINV was applied as a model for authentic alphavirus infection and validated the anti-alphavirus effect of BPR2-D2 upon infection.

Although increasing trends of CHIKV RNA levels in the replicon-transduced cells were detected with BPR2-D2 treatment at 2.5 nM, the reporter mRNA expression in the control GFP baculovirus-transduced cells was also increased upon BPR2-D2 treatment. Additionally, the phenomenon was not detected during authentic SINV infection. Therefore, the enhancement of reporter expression at 2.5 nM of BPR2-D2 may be due to the transduction rates of recombinant baculoviruses in mammalian cells. Nevertheless, BPR2-D2 could specifically suppress the viral RNA levels produced from the CHIKV replicon system at the concentration of 12.5 nM. Collectively, using the CHIKV replicon model, we conclude that BPR2-D2 inhibits RNA replication of CHIKV.

Several alphaviruses, including CHIKV and Venezuelan equine encephalitis virus, are classified as a risk group 3 pathogens, which are required biosafety level 3 facilities for antiviral research. Consequently, the replicon systems, which can be surrogates for studying CHIKV RNA replication, are applicable and convenient for antiviral screening. The present research adapted a CHIKV replicon system for evaluating antiviral activity of BPR2-D2 against CHIKV. Additionally, these systems may be used for functional studies of CHIKV nsPs and screening anti-CHIKV candidate molecules.

Replication of CHIKV RNA relies on its replication complex formed by the viral nonstructural proteins, nsP1-4, derived from the proteolysis of the precursor viral protein P1234 by the viral protease nsP2 [3]. Among the nsPs, nsP4 serves as the core of the RNA-dependent RNA polymerase, responsible for replicating viral genomic RNA and transcribing the 26S subgenomic RNA [15,16]. Additionally, the nsP1 functions as a capping enzyme for viral mRNA, transferring the methyl group from SAM to GTP and subsequently linking the methylated GTP to viral RNA to form a m7G-capped RNA [7]. Interference with SAM and GTP binding activity in nsP1 can affect the production of the capped viral mRNA and reduce viral protein synthesis. The molecular docking analysis showed that BPR2-D2 might bind to the SAM and GTP binding pocket of nsP1, predicting that the potential mechanism by which BPR2-D2 can strongly inhibit CHIKV mRNA synthesis and block the viral genomic RNA replication. The nsP3 is composed of three domains: the N-terminal macrodomain, the alphavirus unique domain, and the C-terminal hypervariable domain. The macrodomain of nsP3 has ADP-ribose-binding and hydrolysis activities, which are involved in viral replication and virulence [12,13]. The molecular docking analysis also predicted that BPR2-D2 might bind to the residues which are critical for the ADP-ribose hydrolase activity of nsP3 macrodomain, suggesting that BPR2-D2 has potential as anti-CHIKV agent through blocking nsP3 function. Nevertheless, the results from the molecular docking analysis require further validation by applying mutants of the CHIKV replicon that express mutated nsP1 and nsP3 at the predicted binding positions.

The previous study for BPR2-D2 antiviral activity also demonstrated that BPR2-D2 inhibits influenza viral polymerase activity without affecting the expression of the viral polymerase plasmids driven by cellular polymerase II [39]. In addition, a recent study demonstrated that BPR2-D2 could target RdRP of SARS-CoV-2 and suppress the viral replication [40]. Our present study found no suppression of the GFP expression driven by the CMV promoter and cellular polymerase II in the control recombinant baculovirus-transduced cells, suggesting that BPR2-D2 may specifically target the nonstructural proteins of CHIKV to inhibit viral genomic RNA replication and subgenomic RNA production. Nevertheless, although the molecular docking results predict that BPR2-D2 might bind to CHIKV nsP1 and nsP3, further assays examining the activities of these nsPs upon BPR2-D2 treatment are necessary to elucidate the detail mechanism. Moreover, since cellular factors may be required for formation of replication complex during RNA virus replication, more studies are needed for determining whether the broad spectrum of BPR2-D2 antiviral activity is through regulating functions of cellular factors.

5. Conclusion

In this study, we demonstrate that BPR2-D2, a coumarin derivative, is able to inhibit CHIKV RNA replication with a high selective index. Additionally, the inhibitory activity upon authentic alphavirus infection, is validated by SINV infection model. This research provides novel information for the development of antivirals against CHIKV and other alphaviruses.

Funding information

This work was financially supported by the Research Center for Emerging Viral Infections from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. This research was also supported by grants to R.-L. Kuo from the National Science and Technology Council in Taiwan (112-2320-B-182-046-MY3) and the Chang Gung Memorial Hospital (BMRPC09).

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank Drs. Jim-Tong Horng and Chung-Fan Hsieh at Chang Gung University for technical support and helpful discussion. In preparation of this manuscript, we employed the AI tools, ChatGPT (OpenAI) and Gemini (Google), for language editing and grammar correction. The authors affirm their responsibility for scientific content, interpretation, and conclusion of this manuscript.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bj.2026.100949.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

figs1.

figs1

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References

  • 1.Silva LA, Dermody TS. Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies. J Clin Investig. 2017;127(3):737–749. doi: 10.1172/JCI84417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Torales M, Beeson A, Grau L, Galeano M, Ojeda A, Martinez B, et al. Notes from the field: chikungunya outbreak - Paraguay, 2022-2023. MMWR Morb Mortal Wkly Rep. 2023;72(23):636–638. doi: 10.15585/mmwr.mm7223a5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ten Dam E, Flint M, Ryan MD. Virus-encoded proteinases of the Togaviridae. J Gen Virol. 1999;80(Pt 8):1879–1888. doi: 10.1099/0022-1317-80-8-1879. [DOI] [PubMed] [Google Scholar]
  • 4.Metz SW, Pijlman GP. Springer; Switzerland: 2016. Function of Chikungunya virus structural proteins in Chikungunya virus: advances in biology, pathogenesis, and treatmen; pp. 63–74. [Google Scholar]
  • 5.Frolova EI, Gorchakov R, Pereboeva L, Atasheva S, Frolov I. Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol. 2010;84(22):11679–11695. doi: 10.1128/JVI.01441-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kujala P, Ikaheimonen A, Ehsani N, Vihinen H, Auvinen P, Kaariainen L. Biogenesis of the Semliki Forest virus RNA replication complex. J Virol. 2001;75(8):3873–3884. doi: 10.1128/JVI.75.8.3873-3884.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ahola T, Kaariainen L. Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci U S A. 1995;92(2):507–511. doi: 10.1073/pnas.92.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zhang K, Law YS, Law MCY, Tan YB, Wirawan M, Luo D. Structural insights into viral RNA capping and plasma membrane targeting by Chikungunya virus nonstructural protein 1. Cell Host Microbe. 2021;29(5):757–64 e3. doi: 10.1016/j.chom.2021.02.018. [DOI] [PubMed] [Google Scholar]
  • 9.Gomez de Cedron M, Ehsani N, Mikkola ML, Garcia JA, Kaariainen L. RNA helicase activity of Semliki Forest virus replicase protein NSP2. FEBS Lett. 1999;448(1):19–22. doi: 10.1016/s0014-5793(99)00321-x. [DOI] [PubMed] [Google Scholar]
  • 10.Das PK, Merits A, Lulla A. Functional cross-talk between distant domains of chikungunya virus non-structural protein 2 is decisive for its RNA-modulating activity. J Biol Chem. 2014;289(9):5635–5653. doi: 10.1074/jbc.M113.503433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Hardy WR, Strauss JH. Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans. J Virol. 1989;63(11):4653–4664. doi: 10.1128/jvi.63.11.4653-4664.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.McPherson RL, Abraham R, Sreekumar E, Ong SE, Cheng SJ, Baxter VK, et al. ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence. Proc Natl Acad Sci U S A. 2017;114(7):1666–1671. doi: 10.1073/pnas.1621485114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Abraham R, Hauer D, McPherson RL, Utt A, Kirby IT, Cohen MS, et al. ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication. Proc Natl Acad Sci U S A. 2018;115(44):E10457–66. doi: 10.1073/pnas.1812130115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lark T, Keck F, Narayanan A. Interactions of Alphavirus nsP3 protein with host proteins. Front Microbiol. 2017;8:2652. doi: 10.3389/fmicb.2017.02652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chen MW, Tan YB, Zheng J, Zhao Y, Lim BT, Cornvik T, et al. Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities. Antivir Res. 2017;143:38–47. doi: 10.1016/j.antiviral.2017.04.001. [DOI] [PubMed] [Google Scholar]
  • 16.Tan YB, Lello LS, Liu X, Law YS, Kang C, Lescar J, et al. Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold. Nucleic Acids Res. 2022;50(2):1000–1016. doi: 10.1093/nar/gkab1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Abu Bakar F, Ng LFP. Nonstructural proteins of alphavirus-potential targets for drug development. Viruses. 2018;10(2):71. doi: 10.3390/v10020071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sharma KB, Subramani C, Ganesh K, Sharma A, Basu B, Balyan S, et al. Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection. PLoS Pathog. 2024;20(12) doi: 10.1371/journal.ppat.1012816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Merten EM, Sears JD, Leisner TM, Hardy PB, Ghoshal A, Hossain MA, et al. Identification of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach. Proc Natl Acad Sci U S A. 2024;121(42) doi: 10.1073/pnas.2409166121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kovacikova K, Gorostiola Gonzalez M, Jones R, Reguera J, Gigante A, Perez-Perez MJ, et al. Structural insights into the mechanisms of action of functionally distinct classes of Chikungunya Virus Nonstructural protein 1 inhibitors. Antimicrob Agents Chemother. 2021;65(7) doi: 10.1128/AAC.02566-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jones R, Hons M, Rabah N, Zamarreno N, Arranz R, Reguera J. Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores. Proc Natl Acad Sci U S A. 2023;120(12) doi: 10.1073/pnas.2213934120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Jones R, Bragagnolo G, Arranz R, Reguera J. Capping pores of alphavirus nsP1 gate membranous viral replication factories. Nature. 2021;589(7843):615–619. doi: 10.1038/s41586-020-3036-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Bhutkar M, Saha A, Tomar S. Viral methyltransferase inhibitors: berbamine, venetoclax, and ponatinib as efficacious antivirals against chikungunya virus. Arch Biochem Biophys. 2024;759 doi: 10.1016/j.abb.2024.110111. [DOI] [PubMed] [Google Scholar]
  • 24.Seyedi SS, Shukri M, Hassandarvish P, Oo A, Shankar EM, Abubakar S, et al. Computational approach towards exploring potential anti-chikungunya activity of selected flavonoids. Sci Rep. 2016;6 doi: 10.1038/srep24027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hwu JR, Roy A, Tsay SC, Huang WC, Lin CC, Hwang KC, et al. Computer-Aided Design and Synthesis of (Functionalized quinazoline)-(alpha-substituted coumarin)-arylsulfonate Conjugates against Chikungunya Virus. Int J Mol Sci. 2022;23(14):7646. doi: 10.3390/ijms23147646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Watanabe Y, Suzuki Y, Emi A, Murakawa T, Hishiki T, Kato F, et al. Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon. Biochem Biophys Res Commun. 2022;637:181–188. doi: 10.1016/j.bbrc.2022.11.013. [DOI] [PubMed] [Google Scholar]
  • 27.Varikkodan MM, Chen CC, Wu TY. Recombinant baculovirus: a flexible drug screening platform for Chikungunya virus. Int J Mol Sci. 2021;22(15):7891. doi: 10.3390/ijms22157891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Prosser O, Stonehouse NJ, Tuplin A. Inhibition of Chikungunya virus genome replication by targeting essential RNA structures within the virus genome. Antivir Res. 2023;211 doi: 10.1016/j.antiviral.2023.105523. [DOI] [PubMed] [Google Scholar]
  • 29.Utt A, Das PK, Varjak M, Lulla V, Lulla A, Merits A. Mutations conferring a noncytotoxic phenotype on chikungunya virus replicons compromise enzymatic properties of nonstructural protein 2. J Virol. 2015;89(6):3145–3162. doi: 10.1128/JVI.03213-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lin HC, Chiao DJ, Lin CC, Kuo SC. Facile method for delivering chikungunya viral replicons into mosquitoes and mammalian cells. Sci Rep. 2021;11(1) doi: 10.1038/s41598-021-91830-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lani R, Hassandarvish P, Shu MH, Phoon WH, Chu JJ, Higgs S, et al. Antiviral activity of selected flavonoids against Chikungunya virus. Antivir Res. 2016;133:50–61. doi: 10.1016/j.antiviral.2016.07.009. [DOI] [PubMed] [Google Scholar]
  • 32.Lin HC, Chiao DJ, Shu PY, Lin HT, Hsiung CC, Lin CC, et al. Development of a novel chikungunya virus-like replicon particle for rapid quantification and screening of neutralizing antibodies and antivirals. Microbiol Spectr. 2023;11(2) doi: 10.1128/spectrum.04854-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Glasker S, Lulla A, Lulla V, Couderc T, Drexler JF, Liljestrom P, et al. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout. Virol J. 2013;10:235. doi: 10.1186/1743-422X-10-235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Schmidt C, Perkovic M, Schnierle BS. Development of a sensitive detection method for alphaviruses and its use as a virus neutralization assay. Viruses. 2021;13(7):1191. doi: 10.3390/v13071191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kishishita N, Sasayama M, Takeda N, Sa-Ngasang A, Anuegoonpipat A, Anantapreecha S. Neutralization activity of patient sera collected during the 2008-2009 Chikungunya outbreak in Thailand. J Clin Microbiol. 2015;53(1):184–190. doi: 10.1128/JCM.02744-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Chung WC, Hwang KY, Kang SJ, Kim JO, Song MJ. Development of a neutralization assay based on the pseudotyped chikungunya virus of a Korean isolate. J Microbiol. 2020;58(1):46–53. doi: 10.1007/s12275-020-9384-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lin HC, Chang SF, Su CL, Hu HC, Chiao DJ, Hsu YL, et al. Facile quantitative diagnostic testing for neutralizing antibodies against Chikungunya virus. BMC Infect Dis. 2024;24(1):1076. doi: 10.1186/s12879-024-09973-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Hassan MZ, Osman H, Ali MA, Ahsan MJ. Therapeutic potential of coumarins as antiviral agents. Eur J Med Chem. 2016;123:236–255. doi: 10.1016/j.ejmech.2016.07.056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Shih SR, Horng JT, Poon LL, Chen TC, Yeh JY, Hsieh HP, et al. BPR2-D2 targeting viral ribonucleoprotein complex-associated function inhibits oseltamivir-resistant influenza viruses. J Antimicrob Chemother. 2010;65(1):63–71. doi: 10.1093/jac/dkp393. [DOI] [PubMed] [Google Scholar]
  • 40.Tang WF, Tsai HP, Chin YF, Tsai SK, Lin CC, Ngo ST, et al. Targeting SARS-CoV-2 RNA-dependent RNA polymerase with the coumarin derivative BPR2-D2: evidence from cell-based and enzymatic studies. Biomed Pharmacother. 2025;189 doi: 10.1016/j.biopha.2025.118252. [DOI] [PubMed] [Google Scholar]
  • 41.Kuo RL, Chen CJ, Tam EH, Huang CG, Li LH, Li ZH, et al. Interactome analysis of NS1 protein encoded by influenza A H7N9 virus reveals an inhibitory role of NS1 in host mRNA maturation. J Proteome Res. 2018;17(4):1474–1484. doi: 10.1021/acs.jproteome.7b00815. [DOI] [PubMed] [Google Scholar]
  • 42.Butt SS, Badshah Y, Shabbir M, Rafiq M. Molecular docking using chimera and Autodock Vina software for nonbioinformaticians. JMIR Bioinform Biotechnol. 2020;1(1) doi: 10.2196/14232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhang S, Garzan A, Haese N, Bostwick R, Martinez-Gzegozewska Y, Rasmussen L, et al. Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design. PLoS One. 2021;16(1) doi: 10.1371/journal.pone.0245013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Malet H, Coutard B, Jamal S, Dutartre H, Papageorgiou N, Neuvonen M, et al. The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket. J Virol. 2009;83(13):6534–6545. doi: 10.1128/JVI.00189-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

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