An NMR-Based Antagonist Induced Dissociation Assay for Targeting the Ligand−Protein and Protein−Protein Interactions in Competition Binding Experiments

• Published in: Journal of medicinal chemistry Vol. 50; no. 18; pp. 4382 - 4387
• Main Authors: Krajewski, Marcin, Rothweiler, Ulli, D'Silva, Loyola, Majumdar, Sudipta, Klein, Christian, Holak, Tad A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 06.09.2007
• Subjects: Binding, Competitive; Biological and medical sciences; Cyclin A - chemistry; Cyclin-Dependent Kinase 2 - chemistry; Cyclin-Dependent Kinase Inhibitor p27 - chemistry; Fundamental and applied biological sciences. Psychology; Glutathione Transferase - chemistry; Glutathione Transferase - genetics; Humans; Interactions. Associations; Intermolecular phenomena; Ligands; Magnetic Resonance Spectroscopy; Medical sciences; Miscellaneous; Molecular biophysics; Nitrogen Isotopes; Pharmacology. Drug treatments; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins c-mdm2 - chemistry; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Retinoblastoma Protein - chemistry; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - genetics; Ligand binding; Biological fixation; Targeting; Molecular interaction; NMR spectrometry; Antagonist; Competitive inhibition; Protein
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm070365v

We present an NMR-based antagonist induced dissociation assay (AIDA) for validation of inhibitor action on protein−protein interactions. As opposed to many standard NMR methods, AIDA directly validates the inhibitor potency in an in vitro NMR competition binding experiment. AIDA requires a large protein fragment (larger than 30 kDa) to bind to a small reporter protein (less than 20 kDa). We show here that a small fragment of a protein fused to glutathione S-transferase (GST) can effectively substitute the large protein component. We successfully used a GST-tagged N-terminal 73-residue p53 domain for binding studies with the human MDM2 protein. Other interactions we studied involved complexes of CDK2, cyclin A, p27, and the retinoblastoma protein. All these proteins play a key role in the cell division cycle, are associated with tumorigenesis, and are thus the subject of anticancer therapy strategies.