Total Cereblon Cellular Kit
Simple, all-in-one kit for robust detection of Total Cereblon
The XIAP BIR3 Binding Kit is designed to identify, characterize, and profile compounds that bind to the BIR3 domain of the E3 ligase XIAP protein.
A fast and easy way to identify and characterize new binders to XIAP protein targeting the BIR3 domain.
X-linked inhibitor of apoptosis protein (XIAP) protein belongs to the inhibitor of apoptosis protein (IAP) family, which also includes cIAP1. It is the most potent caspase inhibitory IAP family member and a negative regulator of various apoptotic stimuli and death pathways.
XIAP overexpression in tumor cells is a well-described mediator of resistance to chemotherapy in many cancers and has been linked to tumor aggressiveness, making it an attractive target in cancer therapy where several therapeutic strategies have been investigated, such as SMAC mimetics.
Moreover, XIAP displays E3 ligase activity and leads to targeted proteins' ubiquitination and their subsequent degradation. This property can be exploited through a Proteolysis-targeting chimera (PROTAC) strategy. Therefore, new compounds targeting XIAP which exhibit dual roles: i) inhibition of XIAP anti-apoptotic function and ii) induction of targeted protein degradation, represent a promising therapeutic approach.
XIAP, like other IAP family proteins, contains BIR domains which interact with the IAP-binding motif of partners such as caspases. This interaction has been suggested to control pro- and anti-apoptotic activities.
XIAP compound characterization on the BIR2 and BIR3 domains enables accurate profiling and selectivity studies.
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.
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.
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.
Various XIAP ligands were characterized. Orthosteric compounds : LCL161, GDC-0152, A410099.1 and SM164 compounds demonstrate expected selectivity profile with higher afinity for BIR3 domain versus BIR2 domain.
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).
|DMSO effect on the HTRF Human XIAP BIR3 Binding kit|
|% in final assay volume (20 µl)||0||0.004||0.008||0.01||0.015||0.02|
|% in compound working solution (5 µl)||0||0.004||0.008||0.01||0.015||0.02|
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