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 Iα cAMP-dependent Protein kinase (PKA-Iα), 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-Iα 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.