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 nM range, demonstrating accurate determination of compounds’ potencies as well as pharmacological ranking for the cIAP1 BIR3 domain as expected. SNIPER-BRD4 compound (containing the cIAP1 LCL161 and the (+)-JQ-1 BRD4 ligand) shows potency around 100nM. 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.

 

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 ligands) demonstrate expected selectivity profile for BIR3 (in the nM range)  versus BIR2 domain (in the µM range).

 

 

 

 

 

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 change significantly with the increasing percentages of DMSO (at least 2% final).