Work

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

Year: 2006

Type: article

Abstract: Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named ligand-regulated competition (LiReC), in an effort to find non-ATP competitive small-molecule regulators for type Ialpha cAMP-dependent Protein kinase (PKA-Ialpha), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Ialpha complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high-throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small-molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP-binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.

Source: Analytical Chemistry

DOI: 10.1021/ac061104g

Authors: S. Adrian Saldanha, Gregory Kaler, Howard B. Cottam, Ruben Abagyan, Susan S. Taylor

Institutions: Scripps Research Institute, Howard Hughes Medical Institute, University of California San Diego, University of California San Diego Medical Center, Moores Cancer Center

Language: English

Topic: Protein Kinase Regulation and GTPase Signaling

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: Cancer Research Institute, California Breast Cancer Research Program, National Institutes of Health, National Institute of General Medical Sciences

Awards: G361034356, PROTEIN KINASE: PRIMARY STRUCTURE AND CAMP INTERACTION, G4507138344, Protein Kinase - Primary Structure and cAMP Interaction, Assembly and regulation of dynamic PKA macromolecular signaling systems