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

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

Marcin Krajewski, Ulli Rothweiler, Loyola D'Silva, Sudipta Majumdar, Christian Klein, Tad A Holak

J Med Chem. 2007 Sep 6;50(18):4382-7.

PMID: 17696513

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411981	CDK2, GST tagged human			Price

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