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

Abstract

Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein-coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the K D for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs.

Paroxetine-Mediated GRK2 Inhibition Reverses Cardiac Dysfunction and Remodeling After Myocardial Infarction

“…Notably, there have been recent reports of small-molecule inhibitors of the Gβγ-mediated membrane translocation of GRK2 (24) being effective in rescuing other models of HF in mice, including having direct sympatholytic activity (26); however, these compounds have issues regarding their suitability for proceeding down the drug development path and do not selectively target GRK2 activity (24). The Food and Drug Administration-approved status of paroxetine increases the potential attractiveness of developing this agent as a GRK2 inhibitor in patients with both HF and depression or using its chemical structure as a platform for further drug development (27).…”

Role of G Protein–Coupled Receptor Kinases 2 and 3 inμ-Opioid Receptor Desensitization and Internalization

“…2). Other approaches previously taken to inhibit GRKs include the broad-spectrum kinase inhibitor staurosporine (Arttamangkul et al, 2012), the PKC/GRK inhibitor 7,8,indol-10-yl]-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione hydrochloride] (Hull et al, 2010), "b-ARK-1 inhibitor" (Iino et al, 2002;Hull et al, 2010), and the antidepressant drug paroxetine (Thal et al, 2012;Homan et al, 2014b). However, none of these approaches combine the cell-permeability, potency, and selectivity profiles of Cmpd101.…”

G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current Trends and Future Perspectives

“…As a consequence, the loss of fluorine is detrimental for kinase inhibition, which is the case for defluoro paroxetine (4d, Table 3). 90,91 In a tubulin phosphorylation assay, 4a showed an 8-fold more potent inhibition of GRK2 phosphorylation with an IC 50 of 2.5 µM. 90 An improved selectivity towards the target kinase was observed over the other GRK isoforms.…”

“…The benzolactam inhibitor is less selective because it more strongly inhibits PKA and PKC (IC 50 values of 2.6 µM and 26 µM, respectively) compared to 4a (IC 50 values of 45 µM for PKA and 220 µM for PKC) 91. …”