Total Cereblon Cellular Kit
Simple, all-in-one kit for robust detection of Total Cereblon
The XIAP BIR2 Binding Kit is designed to identify, characterize, and profile compounds that bind to the BIR2 domain of the E3 ligase XIAP protein.
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.
A fast and easy way to identify and characterize new binders to XIAP protein targeting the BIR2 domain.
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.
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.
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 affinty 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.
Various XIAP ligands were characterized. Orthosteric compounds, LCL161, GDC-0152, A410099.1 and SM164 demonstrated expected selectivity profiles with higher affinity for the BIR3 domain versus the BIR2 domain.
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.
|DMSO effect on the HTRF Human XIAP BIR2 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||0.016||0.032||0.04||0.06||0.08|
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