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

Charu Chaudhry; Jonathan Davis; Yong Zhang; Shana Posy; Ming Lei; Henry Shen; Chunhong Yan; Brigitte Devaux; Litao Zhang; Yuval Blat; William Metzler; Robert M Borzilleri; Randy L Talbott

doi:10.1016/j.ab.2015.11.026

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

Anal Biochem. 2016 Mar 15:497:8-17. doi: 10.1016/j.ab.2015.11.026. Epub 2015 Dec 30.

Authors

Charu Chaudhry¹, Jonathan Davis², Yong Zhang³, Shana Posy², Ming Lei⁴, Henry Shen⁵, Chunhong Yan⁶, Brigitte Devaux⁷, Litao Zhang⁴, Yuval Blat⁴, William Metzler⁴, Robert M Borzilleri³, Randy L Talbott⁵

Affiliations

¹ Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA. Electronic address: [email protected].
² Molecular Structure and Design, Bristol-Myers Squibb, Waltham, MA 02453, USA.
³ Discovery Chemistry, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
⁴ Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
⁵ Discovery Biology, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
⁶ Protein Sciences, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
⁷ Hybridoma Research, Bristol-Myers Squibb, Princeton, NJ 08543, USA.

PMID: 26743718
DOI: 10.1016/j.ab.2015.11.026

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.

Keywords: Apoptosis; Assay; HTRF; Off-rate; Smac; XIAP.

MeSH terms

Animals
Caspase 3 / metabolism
Cell Line
Fluorescence Resonance Energy Transfer / methods*
Humans
Mice, Inbred BALB C
Peptidomimetics / chemistry
Peptidomimetics / pharmacology*
Protein Binding
Protein Interaction Domains and Motifs
X-Linked Inhibitor of Apoptosis Protein / antagonists & inhibitors*
X-Linked Inhibitor of Apoptosis Protein / chemistry
X-Linked Inhibitor of Apoptosis Protein / metabolism*

Substances

Peptidomimetics
X-Linked Inhibitor of Apoptosis Protein
Caspase 3