G protein-coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from the sensation of light to the contractility of the heart. GPCR kinases (GRKs) phosphorylate active GPCRs at sites in their cytoplasmic loops and C-terminal tails, thereby promoting uncoupling of these receptors from heterotrimeric G proteins and ultimately their internalization. Although GRKs allow cells to adapt to changes in their environment and protect against damage incurred by sustained signaling, aberrant GRK activity is strongly associated with diseases such as heart failure and cardiac hypertrophy. Furthermore, inhibition of GRK activity is expected to enhance the action of many drugs that promote GPCR signaling. During and since the last funding cycle, our lab has made substantial advances in the identification and development of GRK selective small molecule inhibitors. We determined the crystal structure of GRK2 in complex with a selective RNA aptamer, and then used this macromolecule as a tool to identify the FDA-approved drug paroxetine as a selective inhibitor of GRK2 activity in vitro and in vivo that improves outcome in myocardial infarcted mice. Furthermore, we conducted several ligand-induced thermal stability screens that identified additional chemical scaffolds that potently and selectivity inhibits GRK2 and GRK5. Subsequent rational design based on crystal structures of these leads in complex with various GRKs led to the development of more potent compounds, one of which assisted us in determining the atomic structure of GRK5. In the first aim, we will further develop and characterize hybrid inhibitors of GRK2 based on the two most promising scaffolds.
The second aim i s devoted to testing how these compounds affect GRK2 recruitment to membranes and receptors and how they perform in cell-based and whole animal models relevant to human disease. In the third aim, we will use our inhibitors to help investigate how GRK5 interacts with membranes and Ca2+*CaM, which together regulate the entry of GRK5 into the nucleus where it promotes the expression of genes that cause cardiac hypertrophy. Collectively, these studies are designed to create a chemical tool box that can be used to help decipher the function of specific GRKs in living cells and disease states, to take a significant step closer towards development of new therapeutic agents for the treatment of heart disease, and to achieve a better understanding of how GRKs interact with their cellular targets.
G protein-coupled receptor (GPCR) kinases (GRKs) regulate the activity of most of the ~800 GPCRs in the human genome and play an adaptive role in cells. Some GRKs are also overexpressed in pathophysiological conditions. For example, GRK2 and GRK5 are strongly implicated in the progression of heart failure and cardiac hypertrophy, respectively. This proposal seeks to develop chemical probes that selectively inhibit GRKs in cells. These probes could also serve as therapeutic leads for the treatment of heart disease and as useful tools to further investigate how GRKs interact with their cellular targets.
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