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Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

  1. Author:
    Nikolovska-Coleska, Z.
    Wang, R. X.
    Fang, X. L.
    Pan, H. G.
    Tomita, Y.
    Li, P.
    Roller, P. P.
    Krajewski, K.
    Saito, N. G.
    Stuckey, J. A.
    Wang, S. M.

  2. Author Address

    Univ Michigan, Dept Internal Med, Ctr Comprehens Canc, Ann Arbor, MI 48109 USA. Univ Michigan, Dept Med Chem, Ctr Comprehens Canc, Ann Arbor, MI 48109 USA. Univ Michigan, Dept Radiat Oncol, Ann Arbor, MI 48109 USA. Georgetown Univ, Med Ctr, Vincent T Lombardi Canc Res Ctr, Washington, DC 20057 USA. NCI, Frederick Canc Res & Dev Ctr, Med Chem Lab, Frederick, MD 21702 USA. Univ Michigan, Inst Life Sci, Ann Arbor, MI 48109 USA Wang, SM, Univ Michigan, Dept Internal Med, Ctr Comprehens Canc, 1500 E Med Ctr Dr, Ann Arbor, MI 48109 USA
    1. Year: 2004
    2. Date: SEP 15
  2. Journal: Analytical Biochemistry
    1. 332
    2. 2
    3. Pages: 261-273
  4. Type of Article: Article
  1. Abstract:

    The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a initiated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH2) showed the highest affinity (K-d = 17.92 nM) and a large dynamic range (DeltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (K-i values) for inhibitors in the FP-based competitive binding assay conditions.. and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain. (C) 2004 Elsevier Inc. All rights reserved

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External Sources

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  2. DOI: 10.1016/j.ab.2004.05.055
  3. View record in Web of Science®
  4. WOS: 000223604100008

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