Description
A fast and easy way to identify new binders to MDM2 protein.
MDM2, also known as murine double minute 2, is involved in many biological processes and is closely associated with DNA repair, cell cycle arrest, apoptosis, and the occurrence of many diseases. MDM2 is one of the most popular E3 ligases which is recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a targeted protein. MDM2 interacts with several proteins to form the functional E3 ubiquitin ligase complex, in which MDM2 functions as a substrate receptor of the E3 ubiquitin ligase complex and targets various proteins to proteolysis.
Therefore, identifying new PROTAC MDM2 ligands can improve selective proteasomal-dependent degradation of proteins of interest, involved in disease onset such as cancers, neurodegenerative, and metabolic disorders.
Assay principle
The HTRF MDM2 Binding Kit is a competitive assay format which uses MI-1061 Red Ligand as MDM2 ligand, a GST tagged human MDM2 protein, and an anti GST Europium Cryptate-labeled antibody. MDM binding compounds compete with the HTRF MI-1061 Red Ligand and thereby prevent FRET from occurring.
Assay protocol
The MDM2 binding 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 plateT the MDM2 GST-tagged protein is then added, followed by the dispensing of the HTRF reagents: the anti GST antibody labeled with Europium cryptate and the MI-1061 Ligand labeled with a Red HTRF acceptor. The reagents labeled 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 MDM2 orthosteric ligands
A panel of MDM2 ligands were characterized. AMG-232 (assay standard), Nutlin-3a and MI-773 display the expected potency and pharmacological ranking in good correlation with the literature. An irrelevant compound Lenalidomide (Cereblon ligand) did not compete with the MI-1061 Red-Ligand binding, demonstrating the specificity of the HTRF MDM2 Binding Kit.
Screening of PROTAC compounds (MDM2-ligand based)
Two MDM2-ligand based PROTAC compounds (MD-224 and A-1874) were characterized. Both compound display the right potency in good correlation with the literature.
DMSO effect on assay performance
Different percentages of DMSO were tested, from 0.4% to 2% (final in the wells). The results indicate that the assay window is reduced with the increasing percentages of DMSO, while the pharmacology remains stable.
