Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

Abstract

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

“…Bovine GRK1 1-535 , bovine and human GRK2 S670A , bovine GRK5, and human palmitoylation deficient GRK6 (pal 2 ) were purified via a common procedure consisting of Ni-NTA affinity, Source15S, and tandem S200 size exclusion chromatography as previously described (Lodowski et al, 2006;Thal et al, 2012). Bovine Gbg-C68S mutant (Gbg), which is not geranylgeranylated and, thus, is not membrane associated, was expressed in High Five cells and purified via Ni·NTA affinity, MonoQ, and tandem S200 size exclusion chromatography as previously reported (Thal et al, 2012).…”

“…For mechanism of inhibition by paroxetine with respect to ATP, the concentration of ATP was varied 1-100 mM, and the concentration of paroxetine was varied 4-1000 mM. Reactions were quenched with SDS loading buffer, separated via SDS-PAGE, dried, and exposed with a phosphorimaging screen prior to quantification via Typhoon imager, as previously reported (Thal et al, 2012). Data were analyzed and inhibition curves were fit via GraphPad Prism.…”

“…Indexing, integration, and scaling were performed with HKL2000 (Otwinowski and Minor, 1997). A molecular replacement solution was achieved with the Phaser module of CCP4 using PDB ID 3V5W as a search model (McCoy et al, 2007;Winn et al, 2011;Thal et al, 2012). Refinement was performed with the Refmac5 module of CCP4 and model building was conducted with Coot (Murshudov et al, 1997;Emsley and Cowtan, 2004;Winn et al, 2011).…”

“…Small molecule inhibitors of GRK2 would also serve as important clinical therapeutics as well as useful biochemical probes to understand GRK2 function in living cells (Shirakawa, 2009). Recently, we identified the selective serotonin reuptake inhibitor (SSRI) paroxetine, a Food and Drug Administration-approved drug, as a relatively selective inhibitor of GRK2 (Thal et al, 2012). Paroxetine was reported to exhibit an IC 50 of 20 mM and up to 50-fold selectivity over other GRKs such as GRK1 and GRK5, which represent the GRK1 and GRK4 subfamilies, respectively .…”

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

“…Available crystal structures of GRK2 lack electron density for nucleotide in the nucleotidebinding pocket, although GRK2-G␤␥ was crystallized in the presence of ATP, and therefore, it is not clear whether the AST is involved in nucleotide coordination. However, the AST is partially ordered in a structure of GRK2 bound to the serotonin reuptake inhibitor paroxetine, mainly due to a few weak van der Waals contacts with the piperidine B ring of paroxetine (69). Thus, a role of AST in other GRKs is not clear.…”