FIG. 1.

(A) PF-429242 inhibits LCMV and LASV GPC cleavage by S1P. 293T cells (2.5 × 10⁵/M6 well) were transfected with 0.1 μg of pC-LCMV-GPC, pC-LASV-GPC, or pC-LCMV-GPCf using Lipofectamine 2000 (10 μl/μg DNA), and at 5 h posttransfection, the transfection mixture was removed, the cells were washed once, and fresh medium containing PF-429242 (1, 10, or 30 μM) or the vehicle (DMSO) was added. At 36 h posttransfection, GPC and GP2 expression levels in cell lysates were determined by WB using a polyclonal serum to Flag. As a control, we also determined the level of actin in each sample (bottom). The cleavage sequences within GPC recognized by S1P or furin are indicated (arrows). (B) In vitro activity of PF-429242. Soluble S1P was preincubated with the indicated concentrations of PF-429242 for 20 min prior to the addition of the substrate Succ-YISRRLL-MCA (20 mM). The reaction was carried out at room temperature with 25 mM Tris-HCl-25 mM MES (pH 7.5)-1 mM CaCl₂, and enzymatic activity measurements were performed by detecting the liberated AMC in a fluorescence plate reader. Plots represent initial rates at the indicated inhibitor concentration with 50% inhibition at circa 200 nM PF-429242 (asterisk).