RGS (regulator of G-protein signaling) proteins attenuate GPCR signals and provide an intriguing alternative to direct receptor targeting for modulating GPCR pathways. A recent effort led by Richard Neubig at Michigan State University (Blazer, et al ACS Chem Neurosci. 2015 Apr 20) provides a big boost to validation of RGS proteins as drug targets. Their development of a selective RGS4 inhibitor that ameliorates movement disorders in a mouse model of Parkinson’s disease (PD) is the first demonstration of in vivo efficacy for an RGS inhibitor, a result which is likely to generate interest in therapeutic targeting of RGS4 as well as other members of the RGS family.
RGS proteins attenuate GPCR signaling immediately downstream of receptor interaction with heterotrimeric G proteins by acting as GAPs (GTPase accelerating proteins) for Gα proteins and converting them to the inactive, GDP-bound state. There are 20 known RGS proteins in humans, and their potential for pharmaceutical targeting has been explored since the discovery of their function in the 90’s (Siderovski, Druey). Given their tissue-specific expression and selectivity for Gα proteins, targeting an RGS protein could potentially provide a way to fine tune the effects of GPCR ligands; e.g. potentiating or prolonging agonist signals. This approach could be used as a monotherapy to modulate endogenous ligand effects, or to limit the side effects and increase the efficacy of drugs that target GPCRs. Despite their tantalizing therapeutic potential, development of inhibitors has been slow for a number of reasons, including a dearth of HTS-compatible assays and the challenge of disrupting the Gα-RGS interaction with a small molecule.
Neubig and colleagues used an innovative flow cytometry-based protein-protein interaction assay, in which RGS4 and Gα proteins were immobilized on luminescent beads (Luminex), to perform a small molecule screen. They identified a thiazolidine compound that binds to an active site cysteine of RGS4, blocking its binding to Gα with low nanomolar potency and inhibiting the GAP activity (Blazer, et al 2011). The original inhibitor was subsequently optimized to reduce off target activity and increase solubility (Turner, et al 2012) resulting in compound, CCG-203769, which was investigated in more detail in the recent study by Blazer, et al.
CCG-203769 binds RGS4 covalently, with an IC50 of 17 nM in the bead-based protein interaction assay, and it shows selectivity between 8- and >6,000-fold for four other RGS proteins tested using the same assay. They also tested the effect of CCG-203769 on glycogen synthase kinase 3β, because related thiazolidine inhibitors of that enzyme are in clinical trials; the IC50 was more than 300-fold higher than for RGS4. Because CCG-203769 is a thiol reactive covalent inhibitor, Neubig’s team also performed broad activity profiling with biochemical and cellular assays. They found some off target activity with GPCRs in the biochemical (ligand binding) assays, but none using secondary cellular assays (cAMP) for the same receptors. Cellular activity of CCG-203769 was demonstrated by its ability to inhibit the translocation of GFP-tagged RGS4 from the cytoplasm to the membrane in HEK-293 cells, consistent with its mechanism of blocking the interaction with Gα.
Most notably, in the in vivo studies, CCG-203769 showed pharmacological activity consistent with an emerging model for RGS4 as a therapeutic target in PD. RGS4 was recently shown to regulate excitatory synapse plasticity by mediating convergent signaling through adenosine and dopamine receptors in neurons, and RGS4 knockout mice had far fewer movement deficits than wild type mice in a PD model based on dopamine depletion (Lerner, et al 2012). When Neubig’s team administered CCG-203769 to mice treated with a dopamine antagonist, there was a rapid reversal of slow movement behaviors used to model PD. This result provides pharmacological evidence that inhibiting RGS4 might provide, for the first time, an alternative to levodopa therapy for treating PD. Moreover, a recent RGS4 siRNA knockdown study suggested that RGS4 might also be a target for reducing the dyskinesia associated with long term levodopa treatment, which can become more debilitating than the PD symptoms (Ko, et al 2014).
The development of a potent, specific RGS inhibitor by Neubig and his colleagues has taken several years, and the effort has paid off in the support it has provided for RGS4 as a new molecular target for PD. Their work also provides overall support for pharmacological targeting of other RGS proteins, which have been linked to diverse diseases, including cancer, cardiovascular disorders in addition to other neurodegenerative diseases.
Look at GPCR drug discovery from a whole new angle: use our RGSscreen Assay Service.