Vidofludimus inhibits porcine reproductive and respiratory syndrome virus infection by targeting dihydroorotate dehydrogenase

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) infection has caused huge economic losses in global swine industry over the last 37 years. PRRSV commercial vaccines are not effective against all epidemic PRRSV strains. In this study we performed a high-throughput screening (HTS) of an FDA-approved drug library, which contained 2339 compounds, and found vidofludimus (Vi) could significantly inhibits PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages (PAMs). Compounds target prediction, molecular docking analysis, and target protein interference assay showed that Vi interacts with dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in the de novo pyrimidine synthesis pathway. Furthermore, PRRSV infection was restored in the presence of excess uridine and cytidine which promote pyrimidine salvage, or excess orotate which is the product of DHODH in the de novo pyrimidine biosynthesis pathway, thus confirming that the antiviral effect of Vi against PRRSV relies on the inhibition of DHODH. In addition, Vi also has antiviral activity against Seneca virus A (SVA), encephalomyocarditis virus (EMCV), porcine epidemic diarrhea virus (PEDV), and pseudorabies virus (PRV) in vitro. These findings should be helpful for developing a novel prophylactic and therapeutic strategy against PRRSV and other swine viral infections.

Keywords: PRRSV infection; Vidofludimus; antiviral; broad-spectrum; dihydroorotate dehydrogenase.

Figure 1

High-throughput screening (HTS) for inhibitors of PRRSV infection from an FDA-approved drug library. A HTS assay timeline. B HTS assay flowchart. C Each dot represents the percent inhibition of PRRSV (0.01 MOI) achieved with by compound (5 μM). D IFA of infected Marc-145 cells treated with one of the four designated compounds. PRRSV N protein is colored green, and brightfield-imaged cells show CPE. Scale bars, 500 μm. E CC₅₀ and EC₅₀ of Ta (i), Vi (ii), Be (iii) and Co (iv). Insets show the structure of each compound.

Figure 2

Vidofludimus anti‑PRRSV activity in Marc‑145 cells. A Viability of Marc-145 cells treated with the indicated concentrations of Vi for 48 h. B Western blot of N-protein in cells infected with PRRSV and treated with the indicated concentrations of Vi. C Relative PRRSV ORF7 mRNA levels determined by qRT-PCR. GAPDH was used as reference control. D Virus titration by TCID₅₀ calculation. E Light microscopy and IFA detection of Marc-145 cells (PRRSV-infected and Vi-treated), at 48 hpi. Green, PRRSV N-protein; blue, nucleus. Scale bars, 500 μm. F and G Western blot of N-protein and TCID₅₀ detection in cells infected with different PRRSV genotypes Vi or DMSO treatment. H EC₅₀ of Vi on PRRSV NADC30-like strain FJ1402. I EC₅₀ of Vi on PRRSV classical strain S1. Error bars represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 3

Vidofludimus anti‑PRRSV activity in PAMs. A Viability of PAMs treated with the indicated concentrations of Vi for 48 h. B Western blot of N-protein in cells infected with PRRSV and treated with the indicated concentrations of Vi. C Relative PRRSV ORF7 mRNA levels determined by qRT-PCR. GAPDH was used as reference control. D Virus titration by TCID₅₀ calculation. Error bars represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns: not significant.

Figure 4

Time-of-addition analysis of vidofludimus anti-PRRSV activity. A Schematic illustration of the time-of-addition experiment. DMSO was used as the solvent of Vi. And an equal volume of DMSO was used for treating viruses as black solvent control. B TCID₅₀ detection for virucidal activity assay. C PRRSV ORF7 mRNA levels determination by qRT-PCR in virus binding assay. GAPDH was used as reference control. D PRRSV ORF7 mRNA levels determination by qRT-PCR in virus internalization assay. GAPDH was used as reference control. E PRRSV ORF7 mRNA levels determination by qRT-PCR in virus replication assay. GAPDH was used as reference control. F Virus titration by TCID₅₀ calculation with cell supernatant in virus release assay. G PRRSV ORF7 mRNA levels determination by qRT-PCR in virus binding assay in PAMs. β-actin was used as reference control. H PRRSV ORF7 mRNA levels determination by qRT-PCR in virus replication assay in PAMs. β-actin was used as reference control. Error bars represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns: not significant.

Figure 5

Target analysis of vidofludimus. A Docked conformation of chloDHODH with Vi. The compound and protein are represented as sticks and cartoons, respectively. Vi is colored orange and the protein chloDHODH is colored green. The binding site is shown as cavity structure. The binding energy of the Vi-chloDHODH complex is marked with an asterisk. B RMSD values of chloDHODH (black), Vi (red), and complex (Vi & chloDHODH, blue) over the 100 ns simulation time. C Number of hydrogen bonds involved in the interaction between chloDHODH and Vi during the MD simulation.

Figure 6

Vidofludimus anti-PRRSV activity is mediated by DHODH. A Effect of DHODH overexpression on PRRSV replication in Marc-145 cells. B Viability of Marc-145 cells treated with the indicated concentrations of siRNAs for 48 h. C Effect of DHODH interference on PRRSV replication in Marc-145 cells. D Effect of DHODH interference on Vi anti-PRRSV activity in Marc-145 cells. E and F Image J analysis of DHODH and PRRSV N protein quantification in (D). G Effect of Vi treatment on DHODH proviral activity in Marc-145 cells. H Image J analysis of PRRSV N protein quantification in (G). I Viability of Marc-145 cells treated with the indicated concentrations of 6-AU for 48 h. J Western blot of N-protein in PRRSV-infected cells with indicated concentrations of 6-AU or DMSO.

Figure 7

Effect of DHO, ORO, uridine, and cytidine on vidofludimus anti-PRRSV activity. Marc-145 cells were infected with PRRSV (0.1 MOI) with treatment of Vi and indicated concentrations of DHO, ORO, uridine, or cytidine for 48 h. DMSO served as the treatment control. A, C, E, and G Viability of Marc-145 cells treated with the indicated concentrations of DHO, ORO, uridine, or cytidine. B, D, F, and H Western blot of N-protein in cells infected with PRRSV and treated with the indicated compounds or DMSO.

Figure 8

Broad-spectrum antiviral activity of vidofludimus against SVA, EMCV, PEDV, and PRV. A Viability of BHK-21, Vero, and PK-15 cells pretreated with the indicated concentrations of Vi for 18 h, 16 h, and 20 h, respectively. Western blot and TCID₅₀ were used to examine the inhibition activity of Vi against SVA (B and C), EMCV (D and E), PEDV F and G and PRV (H and I). Error bars represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns: not significant.

Figure 9

The schematic diagram of PRRSV inhibition by vidofludimus. Dihydroorotate dehydrogenase (DHODH), the key rate-limiting enzyme of the fourth step reaction in the de novo synthesis of pyrimidine (blue arrow), is important in generating UMP required for viral replication. By targeting and inhibiting DHODH activity, vidofludimus (Vi) blocks the production of orotate (ORO) and finally inhibits the synthesis of viral RNA. In addition, the effect of DHODH inhibitors can be disturbed by the salvage pathway (green arrow). On the other hand, Vi can also inhibit PRRSV adsorption on target cells, and its mechanism may be related to PRRSV adsorption-related protein receptors.