HTRF Cereblon Binding Kit, 500 Assay Points

Assay principle

The HTRF Cereblon binding assay is a competitive assay format which uses Thalidomide-Red as Cereblon ligand, a GST tagged human Cereblon protein, and an anti GST Cryptate-labeled antibody. Your compound competes with the Thalidomide-Red and thereby prevents FRET from occurring.

Assay protocol

The Cereblon 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 plate, the human GST-tagged Cereblon protein is then added followed by the dispensing of the HTRF reagents: the anti GST antibody labeled with Europium cryptate and the Thalidomide-Red labeled with XL665. 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.

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.

Role of Cereblon in the CRL4-CRBN E3 ubiquitin ligase complex

Cereblon, also known as CRBN, is a ubiquitously expressed protein of 442 amino-acids (55 kDa).

CRBN interacts with DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4), and the regulator of Cullins 1 (RoC1) to form a functional E3 ubiquitin ligase complex. In this complex, CRBN functions as a substrate recognition subunit, and targets proteins for proteolysis through a ubiquitin-proteasome system (UPS). This E3 ubiquitin ligase complex can therefore achieve different cellular responses by targeting different substrates.

CRBN expression in the brain is associated with memory and learning processes. The nonsense mutation (R419X) is responsible for nonsyndromic mental retardation.

CRBN also ensures normal cell proliferation and metabolism by regulating the function of ion channels.

Cereblon is also the primary direct target of the immunomodulatory drugs (IMiDs) Thalidomide, Pomalidomide, and Lenalidomide, that are all FDA approved drugs for the treatment of multiple myeloma. These drugs induce ubiquitination and degradation of the lymphoid transcription factors IKZF1 and IKZF3 via the CRBN E3 ubiquitin ligase.