A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen
Tóm tắt
Regulators 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. Peptide 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. Though 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.
Tài liệu tham khảo
Pierce KL, Premont RT, Lefkowitz RJ: Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 2002, 3 (9): 639-650. 10.1038/nrm908.
Hepler JR: Emerging roles for RGS proteins in cell signalling. Trends Pharmacol Sci. 1999, 20 (9): 376-382. 10.1016/S0165-6147(99)01369-3.
Tesmer JJ, Berman DM, Gilman AG, Sprang SR: Structure of RGS4 bound to AlF4--activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell. 1997, 89 (2): 251-261. 10.1016/S0092-8674(00)80204-4.
Berman DM, Kozasa T, Gilman AG: The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J Biol Chem. 1996, 271 (44): 27209-27212. 10.1074/jbc.271.44.27209.
Roof RA, Sobczyk-Kojiro K, Turbiak AJ, Roman DL, Pogozheva ID, Blazer LL, Neubig RR, Mosberg HI: Novel Peptide Ligands of RGS4 from a One-Bead, One-Compound Library. Chem Biol Drug Des. 2008, 72: 111-119. 10.1111/j.1747-0285.2008.00687.x.
Roof RA, Jin Y, Roman DL, Sunahara RK, Ishii M, Mosberg HI, Neubig RR: Mechanism of action and structural requirements of constrained peptide inhibitors of RGS proteins. Chem Biol Drug Des. 2006, 67 (4): 266-274. 10.1111/j.1747-0285.2006.00373.x.
Roman DL, Talbot JN, Roof RA, Sunahara RK, Traynor JR, Neubig RR: Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay. Mol Pharmacol. 2007, 71 (1): 169-175. 10.1124/mol.106.028670.
Kimple AJ, Willard FS, Giguere PM, Johnston CA, Mocanu V, Siderovski DP: The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face. Biochim Biophys Acta. 2007, 1774 (9): 1213-1220.
Tetko IV, Gasteiger J, Todeschini R, Mauri A, Livingstone D, Ertl P, Palyulin VA, Radchenko EV, Zefirov NS, Makarenko AS, Tanchuk VY, Prokopenko VV: Virtual computational chemistry laboratory – design and description. J Comput Aided Mol Des. 2005, 19 (6): 453-463. 10.1007/s10822-005-8694-y.
Thompson SJ, Hattotuwagama CK, Holliday JD, Flower DR: On the hydrophobicity of peptides: Comparing empirical predictions of peptide log P values. Bioinformation. 2006, 1 (7): 237-241.
Lucast LJ, Batey RT, Doudna JA: Large-scale purification of a stable form of recombinant tobacco etch virus protease. BioTechniques. 2001, 30 (3): 544-6.