Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2–Gβ₁γ₂ Complex

Interactions between proteins and cell membranes are critical for biological processes such as transmembrane signaling, and specific components of the membrane may play roles in helping to organize or mandate particular conformations of both integral and peripheral membrane proteins. One example of a signaling enzyme whose function is dependent on membrane binding and whose activity is affected by specific lipid components is G protein-coupled receptor (GPCR) kinase 2 (GRK2). Efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by heterotrimeric Gβγ subunits but also on the presence of highly negatively charged lipids, in particular phosphatidylinositol 4′,5′-bisphosphate (PIP₂). We hypothesized that PIP₂ may favor a distinct orientation of the GRK2–Gβγ complex on the membrane that is more optimal for function. In this study, we compared the possible orientations of the GRK2–Gβγ complex and Gβγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation vibrational and attenuated total reflectance Fourier transform infrared spectroscopic methods. Our results indicate that PIP₂ affects the membrane orientation of the GRK2–Gβ₁γ₂ complex but not that of complexes formed with anionic phospholipid binding deficient mutations in the GRK2 pleckstrin homology (PH) domain. Gβ₁γ₂ exhibits a similar orientation on the lipid bilayer regardless of its lipid composition. The PIP₂-induced orientation of the GRK2–Gβ₁γ₂ complex is therefore most likely caused by specific interactions between PIP₂ and the GRK2 PH domain. Thus, PIP₂ not only helps recruit GRK2 to the membrane but also “fine tunes” the orientation of the GRK2–Gβγ complex so that it is better positioned to phosphorylate activated GPCRs

Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine

In heart failure, the β-adrenergic receptors (βARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, <b>14as</b>, with an IC₅₀ of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, <b>14as</b> showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC₅₀ for over 7 h. Three of these inhibitors, including <b>14as</b>, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of these inhibitors

Structure-Based Design, Synthesis, and Biological Evaluation of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors

G protein-coupled receptors (GPCRs) are central to many physiological processes. Regulation of this superfamily of receptors is controlled by GPCR kinases (GRKs), some of which have been implicated in heart failure. GSK180736A, developed as a Rho-associated coiled-coil kinase 1 (ROCK1) inhibitor, was identified as an inhibitor of GRK2 and co-crystallized in the active site. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, Takeda103A, a library of hybrid inhibitors was developed. This campaign produced several compounds possessing high potency and selectivity for GRK2 over other GRK subfamilies, PKA, and ROCK1. The most selective compound, <b>12n</b> (CCG-224406), had an IC₅₀ for GRK2 of 130 nM, >700-fold selectivity over other GRK subfamilies, and no detectable inhibition of ROCK1. Four of the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors