title: Assay principle for modulators of protein-protein interactions and its application to non-ATP-competitive ligands targeting protein kinase A.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, La Jolla, California 92093, USA.
authors.forename: S Adrian
authors.initials: SA
authors.lastname: Saldanha
authors.forename: Gregory
authors.initials: G
authors.lastname: Kaler
authors.forename: Howard B
authors.initials: HB
authors.lastname: Cottam
authors.forename: Ruben
authors.initials: R
authors.lastname: Abagyan
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases