Development of peptide inhibitors of RGS4 involves the following:

• Design of peptide inhibitors based on the structure of RGS4 bound to Galphao.
• Synthesis and purification of individual peptides.
• Evaluation of peptide activity in a single turnover GTPase assay.
• Expression and purification of proteins in E. coli.
• Analysis of structure-activity relationships.
• Design and synthesis of a 2.5 million member focused one-bead, one-compound peptide library using a mix and split scheme.
• Screening the bead based library for binding to a fluorescently labeled target.
• Verification of hit peptide-beads for binding RGS4 using flow cytometry.
• Characterization of hit peptides in multiple assays for binding to and inhibiting the function of RGS4 using a fluorometric assay, a flow cytometry protein-protein interaction assay, and a functional radiochemical assay.

HONORS

• ASPET Young Investigator Best Paper first place award winner, 2007
• International Union of Pharmacology first place young investigator award winner, 2006
• ASPET travel award winner, 2007 (for Experimental Biology), 2006 (for the International Union of Pharmacology Meeting) and 2005 (for Experimental Biology)
• Rackham Travel award recipient, 2006 and 2005
• Member of the Chemistry-Biology Interface Program Student Committee 2006 and 2005
• American Peptide Society Travel award recipient, 2005
• Chemistry-Biology Interface Program Fellowship January 2004-August 2005

RGS inhibitors

Jameson EE, Roof RA, Whorton MR, Mosberg HI, Sunahara RK, Neubig RR Kennedy RT
Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues.
J Biol Chem., 280: 7712-7719 (2005)

Barron DM, Chatterjee SK, Ravindra R, Roof R, Baloglu E, Kingston DG, Bane S
A fluorescence-based high-throughput assay for antimicrotubule drugs
Anal. Biochem., 315: 49-56 (2003)

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., 67: 266-274 (2006)

Roman DL, Talbot JN, Roof RA, Sunahara RK, Traynor JR, Neubig RR
Identification of small molecule inhibitors of Regulator of G-protein Signaling 4 (RGS4) using a high throughput flow cytometry protein interaction assay (FCPIA)
Mol. Pharmacol, 71: 169-175 (2007)

Kent UM, Pascual L, Roof RA, Ballou DP, Hollenberg PF
Mechanistic studies with N-benzyl-1-aminobenzotriazole-inactivated CYP2B1: differential effects on the metabolism of 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, and benzphetamine
Arch Biochem. Biophys., 423: 277-287 (2004)