Assay principle

The HTRF XIAP BIR3 Binding Kit is a competitive assay format which uses XIAP BIR3 Red Ligand (based on A410099.1 derivative), a GST tagged human XIAP BIR3 binding domain, and an anti GST Terbium Cryptate-labeled antibody. XIAP binding compounds compete with the Red Ligand and thereby prevent FRET from occurring.

Assay protocol

The XIAP BIR3 binding domain assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standards are dispensed directly into the assay plate. The human GST-tagged XIAP BIR3 binding domain is then added, followed by the dispensing of the HTRF reagents: the anti GST antibody labeled with Terbium cryptate, and the ligand 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 either rely on SMAC mimetics (orthosteric XIAP compounds) or PROTAC/SNIPER compounds. These compounds have been shown to display lower binding affinity for BIR2 domain than for BIR3 domain or Full-length XIAP protein.

Figure 1 : Various well known pharmacological compounds were characterized. Orthosteric compounds include LCL161 (used as assay standard), GDC-0152, A410099.1 and the high affinty SM164 XIAP ligand. They display Ki values in the nM range, with a lower Ki value for the SM164, demonstrating accurate determination of compounds’ potencies as well as pharmacological ranking for the XIAP BIR3 domain as expected. SNIPER-BRD4 compound (containing the LCL161 XIAP and the (+)-JQ-1 BRD4 ligands) shows potency in the nM range. As expected, an irrelevant compound Thalidomide (Cereblon E3 ligase orthosteric ligand) does 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 indicate that the assay window as well as the IC50 do not significantly change with the increasing percentages of DMSO (at least up to 2% final).