Rebecca A. Roof

Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.

Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

N-(3-fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-yl)butyl)arylcarboxamides as selective dopamine D3 receptor ligands: critical role of the carboxamide linker for D3 receptor selectivity.

N-(3-fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)butyl)arylcarboxamides were prepared and evaluated for binding and function at dopamine D3 receptors (D3Rs) and dopamine D2 receptors (D2Rs). In this series, we discovered some of the most D3R selective compounds reported to date (e.g., 8d and 8j, >1000-fold D3R-selective over D2R). In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized, and while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced binding affinities at D3R by >100-fold (e.g., D3R K(i) for 15b = 393 vs for 8j = 2.6 nM), resulting in compounds with significantly reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine class of compounds and further reveals a point of separation between structure-activity relationships at D3R and D2R.

Mechanism of action and structural requirements of constrained peptide inhibitors of RGS proteins.

Regulators of G‐protein signaling (RGS) accelerate guanine triphosphate hydrolysis by Gα‐subunits and profoundly inhibit signaling by G protein‐coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design of a constrained peptide inhibitor of RGS4 (1: Ac‐Val‐Lys‐[Cys‐Thr‐Gly‐Ile‐Cys]‐Glu‐NH2, S‐S) based on the structure of the Gαi switch 1 region but its mechanism of action was not established. In the present study, we show that 1 inhibits RGS4 by mimicking and competing for binding with the switch 1 region of Gαi and that peptide 1 shows selectivity for RGS4 and RGS8 versus RGS7. Structure–activity relationships of analogs related to 1 are described that illustrate key features for RGS inhibition. Finally, we demonstrate activity of the methylene dithioether‐bridged peptide inhibitor, 2, to modulate muscarinic receptor‐regulated potassium currents in atrial myocytes. These data support the proposed mechanism of action of peptide RGS inhibitors, demonstrate their action in native cells, and provide a starting point for the design of RGS inhibitor drugs.

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

Regulators of G protein signaling accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein‐coupled receptors. The distinct expression patterns and pathophysiologic regulation of regulators of G protein signaling proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation, and structure–activity relationships of a disulfide‐containing cyclic peptide inhibitor of RGS4, YJ34 (Ac‐Val‐Lys‐c[Cys‐Thr‐Gly‐Ile‐Cys]‐Glu‐NH2, S‐S) (Roof et al., Chem Biol Drug Des, 67, 2006, 266). Using a focused one‐bead, one‐compound peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2 (Gly‐Thr‐c[Cys‐Phe‐Gly‐Thr‐Cys]‐Trp‐NH2, S‐S with a free or acetylated N‐terminus) inhibited RGS4‐stimulated Gαo GTPase activity at 25–50 μm. They selectively inhibit RGS4 but not RGS7, RGS16, and RGS19. Their inhibition of RGS4 does not depend on cysteine‐modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.

Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by regulators of G protein signaling proteins 4 or 8 and 7 in permeabilised C6 cells is Gα subtype dependent

J. Neurochem. (2009) 112, 1026–1034.

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

BackgroundRegulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.ResultsPeptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Gαo interaction with an IC50 of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity.ConclusionThough it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.