FIGURE 3.
Estimation of IC50 values for low molecular weight antagonists of the uPA-uPAR interaction. Panel A shows the linear relationship of the association rate of uPAR (vobs; 10−18 mol/s per mm2) to a high density of immobilized pro-uPAS356A (5800 RU ∼ 0.13 × 10−12 mol/mm2) as a function of the analyzed uPAR concentration (0.5–0.0008 nm). The SDS-PAGE analysis to the left shows the quality of the purified proteins after reduction and alkylation, whereas the raw sensorgrams recorded by surface plasmon resonance for the interaction of a serial 2-fold dilution of uPAR starting at 1 nm are shown to the right. The reproducibility of this data set is illustrated by the repeat analysis of 0.5 nm uPAR at the end of the experiment (shown in blue). vobs was calculated as ΔRU/s from 280 to 415 s and was converted to mol/s assuming that 1 RU ∼ 1 pg/mm2. Panel B shows the competition profile for 0.5 nm uPAR binding to immobilized pro-uPAS356A by a serial 3-fold dilution of the linear peptide antagonist AE120 (300–0.005 nm). The efficacy of the obtained inhibition is visualized by the recorded sensorgrams, and the quality of the data is demonstrated by the repeat analysis of 0.5 nm uPAR without competitor (blue curve). The green curves in A and B are buffer runs. Panel C, the residual levels of unoccupied uPAR in the presence of various concentrations of peptide antagonists were subsequently calculated from recorded vobs (as exemplified for AE120 in B) and the corresponding standard curves (A). The resulting inhibition profiles are shown along with their four-parameter logistic fits for GFD, a cyclic decapeptide derived from the β-hairpin of GFD (AE234) and its scrambled control (AE235), a linear nonapeptide antagonist (AE105), as well as a pseudosymmetrical analog (AE120) and its scrambled control (AE151). The derived IC50 values are shown in Table 1. suPAR, soluble uPAR.