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Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target.
- Source :
-
Analytical biochemistry [Anal Biochem] 2016 Mar 15; Vol. 497, pp. 8-17. Date of Electronic Publication: 2015 Dec 30. - Publication Year :
- 2016
-
Abstract
- XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.<br /> (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Caspase 3 metabolism
Cell Line
Humans
Mice, Inbred BALB C
Peptidomimetics chemistry
Protein Binding
Protein Interaction Domains and Motifs
X-Linked Inhibitor of Apoptosis Protein chemistry
Fluorescence Resonance Energy Transfer methods
Peptidomimetics pharmacology
X-Linked Inhibitor of Apoptosis Protein antagonists & inhibitors
X-Linked Inhibitor of Apoptosis Protein metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0309
- Volume :
- 497
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 26743718
- Full Text :
- https://doi.org/10.1016/j.ab.2015.11.026