HTRF cIAP1 BIR2 Binding Kit HTRF®

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The cIAP1 BIR2 Binding Kit is designed to identify, characterize, and profile compounds that bind to the BIR2 domain of the E3 ligase cIAP1 protein.

No-wash
Low sample consumption
All inclusive kit

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The cIAP1 BIR2 Binding Kit is designed to identify, characterize, and profile compounds that bind to the BIR2 domain of the E3 ligase cIAP1 protein.

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Overview

A fast and easy way to identify and characterize new binders to cIAP1 protein targeting the BIR2 domain.

cIAP1, also called BIRC2 , is a member of the Inhibitor of Apoptosis Proteins (IAP) family. IAP proteins are involved in multiple biological processes, such as innate immunity, and play an important role in apoptosis inhibition.

cIAP1 overexpression has been associated with cancer resistance, making it an attractive target in cancer therapy where several therapeutic strategies have been investigated, such as SMAC mimetics.

Moreover, cIAP1 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. SNIPER molecules (Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) can induce the degradation of both the targeted proteins and IAPs, and are expected to harness cancer cell killing.

Therefore, new compounds targeting cIAP1 which exhibit dual roles: i) inhibition of cIAP1 anti-apoptotic function and ii) induction of targeted protein degradation, represent a promising therapeutic approach.

cIAP1, 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.

cIAP1 compound characterization on the BIR2 and BIR3 domains enables accurate profiling and selectivity studies.

Benefits

Discover cIAP1 targeting compounds
Identify cIAP1 ligand-based PROTAC compounds
Compound profiling (cIAP1 BIR2 versus BIR3 domains)

Assay principle

The HTRF cIAP1 BIR2 Binding Kit is a competitive assay format which uses Streptavidin-d2 /LCL161-Biotin as cIAP1 BIR2 ligand, a GST tagged human cIAP1 BIR2 binding domain, and an anti GST Cryptate-labeled antibody. cIAP1 binding compounds compete with the LCL161-Biotin Ligand and thereby prevent FRET from occurring.

Assay protocol

The cIAP1 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 standards are dispensed directly into the assay plate. The human GST-tagged cIAP1 BIR2 binding domain is then added, followed by the dispensing of the HTRF reagents: the anti GST antibody labeled with Terbium cryptate, and the LCL161-Biotin/ 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 cIAP1 Orthosteric and PROTAC ligands

cIAP1 anti-cancer drugs either rely on SMAC mimetics (orthosteric cIAP1 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 cIAP1 protein.

Figure 1 : Various cIAP1 compounds were characterized. Orthosteric compounds include LCL161 (assay standard), GDC-0152 and A4110099.1 and display similar Ki values in the µM range, demonstrating accurate determination of compounds’ potencies as well as pharmacological ranking for the cIAP1 BIR2 domain according to the literature (ref). SNIPER-BRD4 compound (containing the cIAP1 LCL161 and the (+)-JQ-1 BRD4 ligands) shows potency in the µM range (ref). As expected, an irrelevant compound Thalidomide (Cereblon E3 ligase orthosteric ligand) does not compete with the binding of cIAP1 fluorescent ligand, demonstrating the specificity of the kit for cIAP1 binders.

Figure 2 : Low affinity bestatin based PROTAC Compounds were tested. Bestatin is a cIAP1 orthosteric ligand known to display low binding affinity ranged between 100 µM – 1 mM. As expected Erα PROTAC Degrader and RAR PROTAC Degrader compounds, which include bestatin as cIAP1 Ligand, exhibit very low potencies with FRET competition detected from 100 µM.

Screening of cIAP1 Orthosteric and PROTAC ligands

Bestatin-based PROTAC low affinity compounds

Compounds profiling for cIAP1 BIR3 versus BIR2 domain

Various cIAP1 ligands were characterized. Orthosteric compounds : LCL161 (assay standard), GDC-0152 and A4110099.1 and SNIPER-BRD4 compound (containing the cIAP1 LCL161 and the (+)-JQ-1 BRD4 ligand) demonstrate expected selectivity profile for BIR3 (in the nM range) versus BIR2 domain (in the µM range).

Compounds profiling for cIAP1 BIR3 versus BIR2 domain-GDC-0152

Compounds profiling for cIAP1 BIR3 versus BIR2 domain-A4100099.1

Compounds profiling for cIAP1 BIR3 versus BIR2 domain-SNIPER BRD4

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 2% final).

DMSO effect on the cIAP1 BIR2 Binding Assay
% DMSO in final assay volume (20 µl)	0%	0.4%	0.8%	1%	1.5%	2%
% DMSO in compound working solution (5 µl)	0%	1.6%	3.2%	4%	6%	8%
IC50 (µM)	1.5	1.5	1.6	1.5	1.6	1.6

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