An NMR-Based Antagonist Induced Dissociation Assay for Targeting the Ligand-Protein and Protein-Protein Interactions in Competition Binding Experiments
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- 作者：Marcin Krajewski ; Ulli Rothweiler ; Loyola D'Silva ; Sudipta Majumdar ; Christian Klein ; Tad A. Holak
- 刊名：Journal of Medicinal Chemistry
- 出版年：2007
- 出版时间：September 6, 2007
- 年：2007
- 卷：50
- 期：18
- 页码：4382 - 4387
- 全文大小：187K
- 年卷期：v.50,no.18(September 6, 2007)
- ISSN：1520-4804
文摘
We present an NMR-based antagonist induced dissociation assay (AIDA) for validation of inhibitor actionon protein-protein interactions. As opposed to many standard NMR methods, AIDA directly validates theinhibitor 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 smallfragment of a protein fused to glutathione S-transferase (GST) can effectively substitute the large proteincomponent. We successfully used a GST-tagged N-terminal 73-residue p53 domain for binding studieswith 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 associatedwith tumorigenesis, and are thus the subject of anticancer therapy strategies.