Figure 1. Identification of paroxetine as an inhibitor of GRK2.

(a) Schematic of the GRK2-aptamer interaction used in the flow cytometry bead binding assay. Biotinylated GRK2 (bGRK2) was immobilized to streptavidin coated beads and bound by a fluorescein labeled aptamer (C13.28-FAM). (b) Representative binding and control isotherms for C13.28-FAM and bGRK2, wherein C13.28-FAM exhibited a dissociation constant (K_d) of 1.5 ± 0.9 nM (n=11) for bGRK2. (c) Competitive inhibition of C13.28-FAM binding by a panel of known GRK2 inhibitors. Data shown are representative mean values ± SEM of three or more experiments, performed in duplicate (see Table 1). (d) Primary screen identifying two small molecule inhibitors of the GRK2-aptamer interaction. Typical screening Z′ factors were ≥ 0.90 (0.62 for data shown) with 10 μM C13.28 as the positive control and DMSO as the negative control. Hits (boxed data points) were defined by their ability to decrease the fluorescence intensity below 3 σ from the negative (i.e. uninhibited) controls. (e) Structures of primary screening hits from the Prestwick Chemical Library. (f) Confirmation dose-response titrations of P-851 and P-835 against 2.0 nM C13.28-FAM as measured by the flow cytometry bead binding assay. (g) Changes in melting temperature (T_m) induced by incubation of 200 μM inhibitor or Mg²⁺·ATP with GRK2. Data shown are representative of three or more experiments performed in duplicate (f) or triplicate (g).