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Applications: Identify and optimize functional activity of PROTACs targeting bromodomains of the BET family.Design novel molecules linking bromodomains of the BET family and the Cereblon complex.Directly compare ubiquitination of target proteins by different PROTACs.Identify potential inhibitors of ubiquitination.
Background: Cereblon is the substrate-binding component of the E3 protein ligase complex Cereblon-CUL4A (cullin-4A)-RBX1 (RING-box protein 1) that targets proteins for degradation through the proteasome system. The binding of Cereblon to a substrate protein engages the E3 ligase activity of the complex and results in the ubiquitination and ultimate degradation of the substrate protein. PROTACs are bifunctional molecules that bring together a therapeutic target and an E3 ligase to promote the ubiquitination and ultimately the degradation of the target. Efficiency of ubiquitination is an important factor for optimization of heterobifunctional degraders. BRD3, also known as RING3L (RING3-like protein) belongs to the BET family of proteins and is involved in transcription by associating with acetylated lysines present in histones and transcription factors. Chromosomal translocations of BRD3 with NUT (nuclear protein in testis) can lead to cancer, and it has been shown that the use of BET inhibitors or depletion of BRD3 can slow down cancer progression in models of prostate cancer. The use of PROTAC technology for degradation of BRD proteins is an active field of research. Development of novel PROTAC molecules involved in the recruitment of BRD3 (BD2) provides a framework for future efforts to utilize Cereblon to degrade other proteins of interest.
Contraindications: The final concentration of DMSO in the assay should not exceed 1%.Avoid green and blue dyes that absorb light in the AlphaScreen signal emission range (λ=520-620 nm), such as Trypan Blue.Avoid the use of potent singlet oxygen quenchers such as sodium azide (NaN3) or metal ions (Fe2+, Fe3+, Cu2+, Zn2+ and Ni2+).The presence of >1% RPMI 1640 culture medium leads to a signal reduction due to the presence of excess biotin and iron in this medium. Media like MEM, which lacks these components, does not affect AlphaScreen assays.
Description: The PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains is designed for the testing and profiling of PROTACs targeting the bromodomains on the BET (bromodomain and extraterminal) protein family. After tertiary complex formation between the E3 protein, the PROTAC and the protein of interest, ubiquitination of the target protein is a critical step preceding its degradation. The PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains comes in an AlphaLISA® format with enough optimized assay buffer, purified recombinant E1 (UBE1), E2 (UbcH5b), and E3 enzymes (Cereblon complex), BRD3 (BD2) (bromodomain-containing protein 3) (amino acids 305-417) as target protein, ATP, and biotinylated ubiquitin for 384 reactions. This kit also contains the PROTAC dBET1 as the control, and JQ1 as control inhibitor. dBET1 or a PROTAC® of interest is incubated with Cereblon complex and BRD3 (BD2), bringing them in close proximity. Ubiquitination reaction occurs following the addition of E2 enzyme, ATP, biotinylated ubiquitin, and, finally, E1 enzyme. Once ubiquitination has taken place, AlphaLISA® donor and acceptor beads are added. BRD3 (BD2) contains a GST-tag, which is recognized by GSH-AlphaLISA™ acceptor beads. Ubiquitinated BRD3 (BD2) contains biotin-ubiquitin, that binds to Streptavidin donor beads. Upon excitation of the donor bead, a singlet oxygen is generated. The singlet oxygen excites the acceptor bead, which emits light. The resulting signal is directly proportional to the level of ubiquitination of BRD3 (BD2).
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
References: Kaur, S.D., et al. 2023 Cancer Lett; 556: 216065.
Wang C., et al., 2022 J Enzyme Inhib Med Chem 37(1): 1694-1703.
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