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1.
Figure 4

Figure 4. Paroxetine enhances βAR-mediated cardiomyocyte contractility in vitro. From: Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility.

(a) Representative contraction tracings of single adult ventricular cardiomyocytes showing shortening with a basal twitch and after isoproterenol (Iso) stimulation, and then representative cell shortening basally and after Iso with paroxetine pre-treatment. (b) Quantitation of maximal single myocyte contraction amplitude under corresponding conditions. *, P<0.05 vs. Baseline; **, P<0.05 vs. Iso alone; n=18 myocytes in each condition. (c) Paroxetine treatment significantly increased the percent Iso-mediated change in myocyte contractility. *, P<0.05 paroxetine (plus Iso) vs. control (Iso alone).

David M. Thal, et al. ACS Chem Biol. 2012 November 16;7(11):1830-1839.
2.
Figure 3

Figure 3. Atomic structure of the GRK2·paroxetine-Gβγ complex. From: Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility.

(a) Stereo-view of paroxetine bound in the active site of GRK2. Electron density from an m|Fo| − D|Fc| omit map contoured at 3 σ is shown as a green cage. (b) Schematic of GRK2 interactions with paroxetine. Residues that form hydrogen-bonds (dashed lines) with paroxetine are shown in ball-and-stick representation with the interatomic distances shown in Å. Residues forming van der Waal interactions with paroxetine are shown as labeled arcs with radial spokes that point toward the ligand atoms they interact with. (c and d) Interactions of paroxetine with residues forming the adenine and ribose subsites, respectively. (e) Paroxetine binding stabilizes the AST region of GRK2, which contacts the inhibitor and forms novel interactions with the phosphate-binding loop (P-loop). Carbons for GRK2 and paroxetine are shown in slate and white, respectively. Nitrogens are colored cyan, oxygens red, and sulfur and fluorine yellow.

David M. Thal, et al. ACS Chem Biol. 2012 November 16;7(11):1830-1839.
3.
Figure 2

Figure 2. Paroxetine inhibits GRK2 activity in vitro and in living cells. From: Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility.

Inhibition of GRK1, GRK2 and GRK5 mediated phosphorylation of (a) 500 nM bovine rod outer segments (ROS) or (b) 500 nM tubulin in the presence of 5 μM ATP. (c) Inhibition of GRK2-dependent phosphorylation of the thyrotropin-releasing hormone receptor (TRHR) by paroxetine. HEK293 cells were incubated for 45 min with indicated concentration of inhibitor and then stimulated with TRH for 15 s and agonist-dependent phosphorylation was evaluated using a phospho-site-specific antibody (34). Data points are the representative (a and b) or pooled (c) mean ± SEM values from three or more experiments performed in duplicate or triplicate.

David M. Thal, et al. ACS Chem Biol. 2012 November 16;7(11):1830-1839.
4.
Figure 5

Figure 5. Paroxetine increases βAR-mediated in vivo cardiac contractility but does not affect heart rate. From: Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility.

In vivo cardiac hemodynamic function was determined using Millar catheterization at 1 h after the treatment of phospho-buffered saline (PBS) or paroxetine (10 mg kg−1). (a) Representative original left ventricular (LV) DP/dt data acquired from PBS or paroxetine pre-treated groups in response to increasing doses of isoproterenol (0.1–1.0 ng per mouse) (dotted lines). (b) The mean ± SEM of baseline (B) and isoproterenol dose response (in ng per mouse) of maximal LV +dP/dt (+DP/dt). *, P<0.05 paroxetine vs. PBS (ANOVA), n=14 and 10 mice per group, respectively. (c) Mean ± SEM of baseline (B) and isoproterenol dose response of minimal LV −dP/dt (+DP/dt) as a measure of cardiac relaxation. *, P<0.05 Paroxetine vs. PBS (ANOVA), n=6–9 mice per group. (d) Heart rate (HR) values (mean ± SEM) of mice at baseline (B) and after isoproterenol.

David M. Thal, et al. ACS Chem Biol. 2012 November 16;7(11):1830-1839.
5.
Figure 1

Figure 1. Identification of paroxetine as an inhibitor of GRK2. From: Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility.

(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 (Kd) 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 (Tm) 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).

David M. Thal, et al. ACS Chem Biol. 2012 November 16;7(11):1830-1839.

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