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.

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.

Objective To examine and quantify the sexual dimorphism in pathologic features manifested in the musculoskeletal and cardiopulmonary systems and incidence of mortality in the tumor necrosis factor–transgenic (TNF‐Tg; Tg3647 strain) mouse model of inflammatory erosive arthritis. Methods Kaplan‐Meier survival estimates were determined in male and female Tg3647 mice and sex‐matched wild‐type (WT) littermate mice. Longitudinal and cross‐sectional pathologic outcomes in the musculoskeletal and cardiopulmonary systems were assessed via ultrasound, micro–computed tomography, grip strength measurements, histologic and serologic analyses, flow cytometry, and skeletal muscle physiologic measures. Results Compared to male Tg3647 mice (n = 30), female Tg3647 mice (n = 34) had significantly shorter lifespans (P < 0.001) and exhibited the following pathologic features (n = 4–6 per group; P < 0.05 versus male Tg3647 littermates): gross deficits in body mass and muscle weight, early‐onset inflammatory arthritis with severity of end‐stage arthritis that was as severe as that seen in male transgenic mice, and early onset and increased severity of inflammatory interstitial lung disease (ILD). Histologically, the ILD observed in Tg3647 mice was characterized by inflammatory cell accumulation and pulmonary arteriole thickening, which was concomitant with the presence of right ventricular hypertrophy, a feature that was also more severe in the female compared to male Tg3647 mice (P < 0.05). No sexual dimorphisms in TNF‐induced deficient grip strength, axial skeletal growth, or bone loss were found. Globally, the extent of the pathologic changes observed in female Tg3647 mice was greater than that observed in male Tg3647 mice when each group was compared to their sex‐matched WT littermates. Conclusion These findings indicate that TNF selectively drives the early onset of arthritis and progression of pathologic changes in the cardiopulmonary system in female Tg3647 mice. These results in the Tg3647 mouse identify it as a suitable model to better understand the mechanisms underlying sexual dimorphism and cardiopulmonary disease in the setting of inflammatory arthritis and other connective tissue diseases.

G protein-coupled receptor kinases (GRKs) have been implicated in human diseases ranging from heart failure to diabetes. Previous studies have identified several compounds that selectively inhibit GRK2, such as paroxetine and balanol. Far fewer selective inhibitors have been reported for GRK5, a target for the treatment of cardiac hypertrophy, and the mechanism of action of reported compounds is unknown. To identify novel scaffolds that selectively inhibit GRK5, a differential scanning fluorometry screen was used to probe a library of 4480 compounds. The best hit was amlexanox, an FDA-approved anti-inflammatory, anti-allergic immunomodulator. The crystal structure of amlexanox in complex with GRK1 demonstrates that its tricyclic aromatic ring system forms ATP-like interactions with the hinge of the kinase domain, which is likely similar to how this drug binds to IκB kinase ε (IKKε), another kinase known to be inhibited by this compound. Amlexanox was also able to inhibit myocyte enhancer factor 2 transcriptional activity in neonatal rat ventricular myocytes in a manner consistent with GRK5 inhibition. The GRK1 amlexanox structure thus serves as a springboard for the rational design of inhibitors with improved potency and selectivity for GRK5 and IKKε.

IntroductionA pivotal effect of lymphatic vessel (LV) function in joint homeostasis was identified in the tumor necrosis factor-transgenic (TNF-Tg) mouse model of rheumatoid arthritis (RA). Specifically, loss of LV contractions is associated with progressive synovitis and erosions. Furthermore, draining lymph node expansion is a biomarker of arthritic progression, and both macrophages and lymphatic endothelial cells express inducible nitric oxide synthase (iNOS), which disrupts LV contraction and transport of immune cells to the draining lymph nodes. Therefore, to directly assess these relationships, we tested the hypothesis that TNF-Tg mice with global genetic ablation of iNOS (iNOS−/−) will show delayed draining lymph node expansion, maintained LV contractions, and decreased synovitis and erosions.MethodiNOS−/−× TNF-Tg female and male mice, and control littermates (iNOS−/−, TNF-Tg, and WT), were examined with (1) ultrasound to determine popliteal lymph node (PLN) volume and (2) near-infrared imaging (NIR) to assess popliteal LV contraction frequency, and differences between genotypes were assessed at 3, 4, 5, and 6 months of age. Knees and PLN were harvested at 4 months in females and 6 months in males, to assess synovitis, bone erosions, and cellular accumulation in PLN sinuses via histology.ResultsInitially, an increase in PLN volume was observed for both female and male iNOS−/−× TNF-Tg and TNF-Tg compared to their WT and iNOS−/− counterparts at 2 and 3 months, respectively. Subsequently, TNF-Tg PLNs continue to increase in volume, while iNOS−/−× TNF-Tg did not increase in volume from the initial timepoints. WT and iNOS−/− PLN volume was unchanged throughout the experiment. LV contraction frequency was increased at 4 months in females and 5 months in males, in the iNOS−/−× TNF-Tg mice compared to the TNF-Tg. Synovitis and erosions were moderately reduced in iNOS−/−× TNF-Tg versus TNF-Tg knees in females, while no differences in knee pathology were observed in males.ConclusionsGenetic iNOS ablation maintains draining lymph node volume and LV function during TNF-induced inflammatory arthritis and is associated with moderately decreased joint inflammation and damage.