Suppression of poxvirus replication by SC144

Abstract

Poxviruses are a significant threat to the health of human and economically important animals. Although smallpox, one of the most devastating infectious diseases, is eradicated, the rapid rise of mpox and other poxviral diseases call for the development of antivirals. Here, we show that SC144, a reported small molecule inhibitor of cellular gp130 pathway, suppresses the replication of monkeypox, cowpox, and vaccinia viruses in cultured cells. Interestingly, SC144 likely suppresses vaccinia virus replication independent of gp130. We further show that this suppression of poxvirus replication is achieved by inhibiting viral DNA replication. With EC₅₀ in sub-micromolar range and CC₅₀ of over 250 μM, SC144 is potent and selective, making it a promising candidate for further development as an antiviral against poxviruses.

Keywords: Antiviral; DNA replication; Monkeypox(mpox) virus; Poxviruses; SC144; Vaccinia virus.

Fig. 1.. SC144 suppresses the replication of poxviruses in both primary and transformed cells.

(A) Chemical structure of SC144. (B) HFFs were infected with VACV at MOI = 2 for 24 h in the presence of vehicle (DMSO) or 10 μM SC144. Viral titers were measured by a plaque assay. (C) HFFs were infected with VACV at MOI = 0.01 for 48 h in the presence of vehicle or 10 μM SC144 and the virus titers were measured by a plaque assay using BS-C-1 cells. (D) SC144 treatment does not alter HFF viability. HFFs were grown in the presence of vehicle or 10 μM SC144 for 48 h. Cell viability was determined by trypan blue exclusion assay using a hemocytometer. (E) HeLa cells were infected with VACV in the presence of vehicle or 10 μM SC144. Viral titers were measured by a plaque assay at 24 hpi (MOI = 2) and 48 hpi (MOI = 0.01). (F) A549 cells were infected with VACV in the presence of vehicle or 10 μM SC144. Virus titers were measured by a plaque assay at 24 hpi (MOI = 2) and 48 hpi (MOI = 0.01). (G) HFFs were infected with MOI =0.01 of vLGluc in the presence of vehicle or a series of concentration of SC144 or tecovirimat. Gaussia luciferase activities were measured at 24 hpi to determine the EC₅₀ value using GraphPad Prism (version 10.2.3) by applying the “log(inhibitor) vs. normalized response – variable slope” model. The horizontal dashed line indicates 50% inhibition of virus replication. (H) HFFs were treated with vehicle or a series of concentration of SC144 or tecovirimat. CCK8 assay was performed at 24 h post treatment to calculate the CC₅₀ value using GraphPad Prism (version 10.2.3) by applying the “log(inhibitor) vs. normalized response – variable slope” model. The horizontal dashed line indicates 50% reduction in cell viability. Error bars represent standard deviation of at least three biological repeats. Whenever indicated, cells were treated with 40 μg/mL AraC, which was used as a positive control. Error bars represent standard deviation of at least three biological repeats. For statistical analysis (GraphPad Prism 10.2.3) the mean of all groups was compared to that of the vehicle using one-way ANOVA comparison and the Dunnett method for determination of significance. For P-values, ns = P > 0.05; **** = P ≤ 0.0001.

Fig. 2.. SC144 suppresses VACV DNA synthesis and subsequently post-replicative gene expression.

(A) Cascade expression of VACV genes and the recombinant reporter VACVs that encode Gaussia luciferase under viral early promoter (C11R; vEGluc), viral intermediate promoter (G8R; vIGluc), or viral late promoter (F17R; vLGluc). (B) SC144 does not affect VACV early gene expression. HFFs were infected with vEGluc at MOI = 2 for 4 h in the presence or absence of 10 μM SC144. Gaussia luciferase activities was measured using a Pierce Gaussia Luciferase Flash Assay Kit (Thermo Scientific) using a GloMax Luminometer (Promega) according to the manufacturer’s instructions. (C-D) SC144 suppresses VACV intermediate and late gene expression. HFFs were infected with vIGluc (C) or vLGluc (D) at MOI = 2 for 8 h in the presence or absence of 10 μM SC144. Gaussia luciferase activities was measured as described above. (E) HFFs were infected with VACV at MOI =2 in the presence of vehicle or 10 μM SC144 for 8 h. Relative amounts of viral DNA were determined by real-time quantitative PCR using VACV-specific primers. Cells treated with 40 μg/mL AraC was used as a positive control. Error bars represent standard deviation of at least three biological repeats. For statistical analysis (GraphPad Prism 10.2.3) the mean of all groups was compared to that of the vehicle using one-way ANOVA comparison and the Dunnett method for determination of significance. For P-values, ns = P > 0.05; **** = P ≤ 0.0001.

Fig. 3.. The suppression of gp130 does not affect VACV replication in HFFs.

(A) HFFs were transfected with either a negative control siRNA or three different siRNAs that target the IL6ST gene (gp130) using the following siRNAs obtained from Qiagen for 48 h. Hs_IL6ST_1 FlexiTube siRNA, GeneGlobe ID: SI00033719; Hs_IL6ST_5 FlexiTube siRNA, GeneGlobe ID - SI03050544; Hs_IL6ST_3 FlexiTube siRNA, GeneGlobe ID - SI00033733. RNA was extracted from the HFFs using TRIzol reagent (Ambion) and purified with the Invitrogen PureLink RNA mini kit (Thermo Fisher Scientific). A total of 500 ng of RNA was used to create cDNA with random hexamer primers and the SuperScript III first-strand synthesis kit (Invitrogen). The CFX96 Real-Time PCR Detection System (Bio-Rad) was used to measure the relative levels of specific mRNAs with All-in-One 2X qPCR mix (GeneCopoeia) and gene-specific primers. The PCR settings were: an initial denaturation at 95°C for 3 minutes, followed by 39 cycles of denaturation at 95°C for 10 seconds, annealing and fluorescence reading at 64°C for 30 seconds, and extension at 72°C for 30 seconds. 18S RNA was used as an internal control for normalization. (B) HFFs were transfected with the indicated siRNAs. After 24 h, the cells were infected with vLGLuc at MOI = 0.01. One-hour post infection the media was changed, and the cells were transfected again with the respective siRNAs. Gaussia luciferase activities were measured at 24hpi to determine the levels of viral replication. (C-D) Chemical inhibitor of gp130 does not affect VACV replication. HFFs were infected with vLGluc at MOI = 2 (C) or 0.01 (D). At 1hpi the media was changed to add 40 μg/mL AraC, 10 μM SC144 or indicated concentrations of bazedoxifene. Gaussia luciferase activities was measured at 8hpi (C) or 24hpi (D) using a Pierce Gaussia Luciferase Flash Assay Kit (Thermo Scientific) using a GloMax Luminometer (Promega) according to the manufacturer’s instructions. (E) Bazedoxifene treatment does not alter HFF viability. HFFs were grown in the presence of vehicle or 20 μM bazedoxifene for 48 h. Cell viability was determined by trypan blue exclusion assay using a hemocytometer. Error bars represent standard deviation of at least four biological repeats in Fig 3A and 3B and at least three biological repeats for the remaining figures. For statistical analysis (GraphPad Prism 10.2.3) the mean of all groups was compared to that of the vehicle using one-way ANOVA comparison and the Dunnett method for determination of significance. For P-values, ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Fig. 4.. SC144 effectively suppresses the replication of MPXV and CPXV in HFFs.

HFFs were infected with MPXV-MA001 2022 isolate at (A) MOI = 0.01 for 48 h or (B) MOI = 1 for 24 h in the presence of vehicle (DMSO) or 10 μM SC144. Virus titers were measured by a plaque assay in BS-C-1 cells. (C) HFFs were infected with Brighton Red strain of CPXV at MOI of 0.01 and treated with vehicle or 10 μM SC144 for 48 h. Viral yields were titrated using a plaque assay using BS-C-1 cells. HFFs treated with 40 μg/mL AraC were used as a positive control. Error bars represent standard deviation of at least three biological repeats. For statistical analysis (GraphPad Prism 10.2.3) the mean of all groups was compared to that of the vehicle using one-way ANOVA comparison and the Dunnett method for determination of significance. For P-values, **** = P ≤ 0.0001.