An NMR-based antagonist induced dissociation assay for targeting the ligand-protein and protein-protein interactions in competition binding experiments.
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.
Krajewski
Marcin
M
Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.
Rothweiler
Ulli
U
D'Silva
Loyola
L
Majumdar
Sudipta
S
Klein
Christian
C
Holak
Tad A
TA
D001667:Binding, Competitive; D019925:Cyclin A / Q000737:chemistry*; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D016160:Retinoblastoma Protein / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics
D019925:Cyclin A; D008024:Ligands; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D050760:Cyclin-Dependent Kinase Inhibitor p27; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D005982:Glutathione Transferase; D051357:Cyclin-Dependent Kinase 2