Assay principle

The HTRF XIAP BIR2 Binding Kit is a competitive assay format which uses Streptavidin-d2 /XIAP BIR2-biotin ligand (based on A410099.1 derivative), a GST tagged human XIAP BIR2 binding domain, and an anti GST Terbium Cryptate-labeled antibody. XIAP binding compounds compete with the biotinylated ligand and thereby prevent FRET from occurring.

Assay protocol

The XIAP BIR2 binding domain assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standard are dispensed directly into the assay plate. The human GST-tagged XIAP BIR2 binding domain is then added, followed by the dispensing of the HTRF reagents: the anti GST antibody labeled with Terbium cryptate, and the biotinylated ligand / Streptavidin labeled with d2. The reagents labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Screening of XIAP Orthosteric and PROTAC ligands

XIAP anti-cancer drugs rely either on SMAC mimetics (orthosteric XIAP compounds) or on PROTAC/SNIPER compounds. These compounds have been shown to display lower binding affinity for the BIR2 domain than for BIR3 domain or Full-length XIAP protein.

Figure 1 : Various well known pharmacological compounds were characterized. Orthosteric compounds included LCL161 (used as an assay standard), GDC-0152, A410099.1, and the high affinity XIAP SM164 ligand. While the others displayed Ki values in the µM range, SM164 showed a higher affinity of 50 nM, demonstrating accurate determination of the compound potencies as well as pharmacological ranking for the XIAP BIR2 domain, as expected. The SNIPER-BRD4 compound (containing the LCL161 XIAP and the (+)-JQ-1 BRD4 ligands) showed potency in the µM range. Again as expected, an irrelevant compound, Thalidomide (Cereblon E3 ligase orthosteric ligand), did not compete with the binding of XIAP fluorescent ligand, demonstrating the specificity of the kit for XIAP binders.

DMSO effect on assay performance

Different percentages of DMSO were tested, from 0.4% to 2% (final per well). The results indicated that the assay window as well as the IC50 did not significantly change with the increasing percentages of DMSO, at least to 1.5% final. Although the assay window was reduced at 2% DMSO, it did not dramatically impact the  pharmacological parameter.