Highlights on the mode of action of ST-246, an anti-orthopoxviral compound

Abstract number: P576

Duraffour S., Vigne S., Vermeire K., Crance J.M., Garin D., Jordan R., Hruby D.E., Yang G., Andrei G., Snoeck R.

Objectives: ST-246 is a highly potent and orally bioavailable anti-orthopoxviral molecule that targets the F13L protein of vaccinia virus (VACV). We investigated part of its mechanism of action by using small interfering RNAs, i.e., siF13Ls, designed to specifically knockdown the F13L gene and by characterising ST-246 resistant strains of VACV and camelpox virus (CMLV).

Methods: The VACV strains Copenhagen, Western Reserve (WR) expressing GFP as a C-terminal fusion protein with F13L or an F13L deleted virus expressing GFP from the F13L locus, and the Iran CMLV strain were used. Cultures of human embryonic lung fibroblasts (HEL) were transfected with the siF13L of interest, and infected 24h later at a MOI of 0.01. The effects of siRNA were determined by real-time RT-PCR, viral titrations and flow cytometry. For the selection of resistant viruses, VACV and CMLV were propagated in HEL cells under increasing concentrations of ST-246 for approximately 17 passages. Strains exhibiting at least 100-fold increase of their wild type 50% inhibitory concentration (IC50) were defined as resistant.

Results: Real-time RT-PCR and viral titrations data indicated that siF13Ls (100 nM) knocked down the VACV and CMLV F13L gene transcripts by more than 85% and reduced their replication by 90%. At lower siF13L concentrations, VACV and CMLV replication was inhibited in a different way, which is in agreement with our previously published data on the anti-VACV and -CMLV activity of ST-246. Flow cytometry analysis showed a reduction of VACV-WR replication expressing a F13L-GFP, but not of a F13L deleted VACV-WR, by siF13Ls and ST-246 and validated a common target, i.e., F13L. Furthermore, the IC50 values of two ST-246 resistant CMLV mutants were >20 mg/ml compared to 0.005 mg/ml for the wild type. In the case of VACV, a 120-fold increase of the IC50 value indicated a ST-246 resistant phenotype. Sequencing of the F13L genes, for both resistant and wild type VACV and CMLV strains, is currently ongoing.

Conclusion: We have shown the inhibitory potency of siF13L inducing a specific silencing of the F13L gene which resulted in the inhibition of VACV and CMLV yields. We demonstrated the mode of action of ST-246 at the protein level and not on mRNA. A single target for ST-246 has been validated in VACV and CMLV. We also isolated ST-246 resistant clones of both viruses and the determination of mutations induced by ST-246 in the F13L gene is currently investigated.