Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target

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, e...

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Bibliographic Details
Published in:Analytical biochemistry 2016-03, Vol.497, p.8-17
Main Authors: Chaudhry, Charu, Davis, Jonathan, Zhang, Yong, Posy, Shana, Lei, Ming, Shen, Henry, Yan, Chunhong, Devaux, Brigitte, Zhang, Litao, Blat, Yuval, Metzler, William, Borzilleri, Robert M., Talbott, Randy L.
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Assay
Caspase 3 - metabolism
Cell Line
Fluorescence Resonance Energy Transfer - methods
HTRF
Humans
Mice, Inbred BALB C
Off-rate
Peptidomimetics - chemistry
Peptidomimetics - pharmacology
Protein Binding
Protein Interaction Domains and Motifs
Smac
X-Linked Inhibitor of Apoptosis Protein - antagonists & inhibitors
X-Linked Inhibitor of Apoptosis Protein - chemistry
X-Linked Inhibitor of Apoptosis Protein - metabolism
XIAP
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2015.11.026