A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen.
BACKGROUND
Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Galpha 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.


RESULTS
Peptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Galphao interaction with an IC50 of 28 microM. 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.


CONCLUSIONS
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.
Roof
Rebecca A
RA
Department of Pharmacology, University of Michigan,1301 MSRB III SPC 5632, 1150 W, Medical Center Dr, Ann Arbor, MI 48109, USA. roofra@ninds.nih.gov
Roman
David L
DL
Clements
Samuel T
ST
Sobczyk-Kojiro
Katarzyna
K
Blazer
Levi L
LL
Ota
Shodai
S
Mosberg
Henry I
HI
Neubig
Richard R
RR
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D020710:RGS Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019151:Peptide Library; D010456:Peptides, Cyclic; D020710:RGS Proteins; C098799:RGS4 protein; D003545:Cysteine