Identification of Human S100A9 as a Novel Target for Treatment of Autoimmune Disease via Binding to Quinoline-3-Carboxamides
Figure 1
Human S100A9 Is a Target Protein for Quinolines
(A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown.
(B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9.
(C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of HBS-P buffer containing 3 mM EDTA (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca2+ and 10 μM Zn2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM (r2 = 1.00).
(D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude.
(E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈Bmax/2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC50 value of 37 μM was calculated in this experiment using a one-site competition model (r2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data).
(F) Effect of Ca2+ and Zn2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca2+ (1 mM) or Zn2+ (10 μM), with Zn2+ and Ca2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC50 values of 5.5 μM for Zn2+ and 193 μM for Ca2+ were calculated using a sigmoidal dose-response model.