Development of Small Molecule RGS Inhibitors as a Mechanism to Modulate G-Protein Signaling.

Regulator of G-protein Signaling (RGS) proteins are important regulatory molecules in the transduction of G-Protein Coupled Receptor (GPCR) signaling. They function by directly binding to G alpha subunits and accelerating GTP hydrolysis, thus potently inhibiting GPCR signaling. We and others have proposed that small molecule inhibitors of RGS proteins may provide a novel mechanism for therapeutic intervention in diseases stemming from deficiencies in GPCR signaling. This thesis details the identification and characterization of two novel classes of small molecule RGS inhibitors with unique properties. These compounds were identified from a series of high throughput screens performed by myself and others in our laboratory. The CCG-63802 class of molecules includes the first examples of reversible inhibitors of RGS4. These compounds can inhibit the in vitro binding and activity of several RGS proteins with IC50 values in the 3-100 micromolar range. They function by binding to RGS4 near a site thought to be important for allosteric regulation by endogenous acidic phospholipids. The second class of molecules, typified by CCG-50014, includes the most potent RGS4 inhibitors identified to date. This compound irreversibly inhibits RGS4 with nanomolar potency (IC50 30±6 nM) by covalently interacting with at least one cysteine on the RGS. In spite of the thiol dependence of these compounds, several members of this class can inhibit RGS binding and activity on G protein alpha subunits in living cells. Future work with these compounds is focused upon testing their activity in a variety of isolated organ and whole-animal studies. It is hoped that these compounds will provide a foundation for the development of new, more active RGS inhibitors with potential clinical and/or research utility.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78783/1/llblazer_1.pd

Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound Library

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65605/1/j.1747-0285.2008.00687.x.pd

Selectivity and anti-Parkinson's potential of thiadiazolidinone RGS4 inhibitors

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein−protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson’s disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3β, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 μM. CCG-203769 enhances Gαq-dependent cellular Ca2+ signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson’s disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson’s disease and other neural disorders

Use of Flow Cytometric Methods to Quantify Protein‐Protein Interactions

A method is described for the quantitative analysis of protein‐protein interactions using the flow cytometry protein interaction assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead‐associated fluorescence in a flow cytometer. This method can be used to calculate protein‐protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the regulator of G‐protein signaling protein, RGS19, in either a saturation or a competition format. An adaptation of this method that is compatible for high‐throughput screening is also provided. Curr. Protoc. Cytom. 51:13.11.1‐13.11.15. © 2010 by John Wiley & Sons, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152948/1/cpcy1311.pd

Allosteric Inhibition of the Regulator of G Protein Signaling–Gα Protein–Protein Interaction by CCG-4986

Regulator of G protein signaling (RGS) proteins act to temporally modulate the activity of G protein subunits after G protein-coupled receptor activation. RGS proteins exert their effect by directly binding to the activated Gα subunit of the G protein, catalyzing the accelerated hydrolysis of GTP and returning the G protein to its inactive, heterotrimeric form. In previous studies, we have sought to inhibit this GTPase-accelerating protein activity of the RGS protein by using small molecules. In this study, we investigated the mechanism of CCG-4986 [methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitro-benzenesulfinimidoate], a previously reported small-molecule RGS inhibitor. Here, we find that CCG-4986 inhibits RGS4 function through the covalent modification of two spatially distinct cysteine residues on RGS4. We confirm that modification of Cys132, located near the RGS/Gα interaction surface, modestly inhibits Gα binding and GTPase acceleration. In addition, we report that modification of Cys148, a residue located on the opposite face of RGS4, can disrupt RGS/Gα interaction through an allosteric mechanism that almost completely inhibits the Gα–RGS protein–protein interaction. These findings demonstrate three important points: 1) the modification of the Cys148 allosteric site results in significant changes to the RGS interaction surface with Gα; 2) this identifies a “hot spot” on RGS4 for binding of small molecules and triggering an allosteric change that may be significantly more effective than targeting the actual protein-protein interaction surface; and 3) because of the modification of a positional equivalent of Cys148 in RGS8 by CCG-4986, lack of inhibition indicates that RGS proteins exhibit fundamental differences in their responses to small-molecule ligands

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling ProteinsS⃞

Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((2E)-2-(1,3-benzothiazol-2-yl)-3-[9-methyl-2-(3-methylphenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]prop-2-enenitrile)], inhibit the interaction between RGS4 and Gαo with an IC50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 > 19 = 16 > 8 ≫ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to Gαo. On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology

A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties

Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells