Figure 4

Quenching of the intrinsic tryptophan fluorescence of CCL17 and CCL22 by hit molecules.

CCL17 and CCL22 were titrated with increasing concentrations (0–25 μM) of GPN279 (a, ○) or GPN136 (b,◻), respectively and the change in tryptophan fluorescence was monitored as a function of the added ligand. As a control, the CCR4 antagonist, C-021, was tested on CCL17 (c,•) and CCL22 (d,◼). The fluorescence emission spectrum (300–400 nm) of each sample was measured at an excitation wavelength of 285 nm. The fluorescence emission spectra were corrected by subtracting the emission scan of the compound alone. DMSO had no effect on the fluorescence measurements. Titration data were normalized by dividing the measured fluorescence (F) by the initial fluorescence in the absence of the compound (F0) at 340 nm. Solid lines show the fit of the data to a second order equation: (RL)² + (RL) × (-Ro-Lo-K_D) + Ro × Lo = 0 where (RL) = ((Ro + Lo + K_D) ± ((-Ro-Lo-K_D)²-4 × Ro × Lo)^1/2)/2 and where Ro, the concentration of CCL17 or CCL22 was set at 1.5 μM; Lo is the initial concentration of the neutraligand; K_Dis the dissociation constant of the neutraligand for the chemokine; and RL is the fractional concentration of chemokine and ligand complex. Data are means (dots) and bars are SD values of three independent experiments.