title: Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target.
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.
authors.affiliation: Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA. Electronic address: charu.chaudhry@bms.com.
authors.forename: Charu
authors.initials: C
authors.lastname: Chaudhry
authors.affiliation: Molecular Structure and Design, Bristol-Myers Squibb, Waltham, MA 02453, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Davis
authors.affiliation: Discovery Chemistry, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Molecular Structure and Design, Bristol-Myers Squibb, Waltham, MA 02453, USA.
authors.forename: Shana
authors.initials: S
authors.lastname: Posy
authors.affiliation: Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.affiliation: Discovery Biology, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Henry
authors.initials: H
authors.lastname: Shen
authors.affiliation: Protein Sciences, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Yan
authors.affiliation: Hybridoma Research, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Devaux
authors.affiliation: Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Litao
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Blat
authors.affiliation: Lead Discovery and Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: William
authors.initials: W
authors.lastname: Metzler
authors.affiliation: Discovery Chemistry, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Borzilleri
authors.affiliation: Discovery Biology, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Randy L
authors.initials: RL
authors.lastname: Talbott
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008807:Mice, Inbred BALB C; D057786:Peptidomimetics / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D057786:Peptidomimetics; D051636:X-Linked Inhibitor of Apoptosis Protein; D053148:Caspase 3
keywords: Apoptosis; Assay; HTRF; Off-rate; Smac; XIAP