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Applications: Discover and optimize PROTACs targeting BRD9Design novel molecules targeting CRBNCompare the activities of different PROTACs
Background: CRBN (cereblon) is the substrate-binding component of the E3 protein ligase complex DDB1-CUL4A-RBX1 involved in the ubiquitination and proteasomal degradation of target proteins. Cereblon binds to DDB1 (Damaged DNA binding protein 1), to the scaffolding protein CUL4A (Cullin 4A), and its regulator RBX1 (RING-Box protein 1). Binding of CRBN to a substrate protein engages the E3 ligase activity of the complex and results in the ubiquitination and ultimate degradation of the protein substrate. Many proteins are known targets of CRBN, including several transcription factors, growth factors, kinases and more. CRBN has become a target of choice for the development of many therapeutic PROTACs.BRD9 (Bromodomain-containing protein 9) functions as a transcriptional regulator and is a component of a chromatin remodeling complex. It also regulates the formation of the RAD51-RAD54 complex, involved in homologous recombination. BRD9 plays a role in cancer and is a potential therapeutic target for cancer drugs.
Contraindications: Green and blue dyes, such as Trypan Blue, absorb light in the AlphaScreen™ signal emission range (520-620 nm). Avoid the use of the 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. MEM, which lacks these components, does not affect AlphaScreen™ assays.
Description: The PROTAC Optimization Kit for BRD9-Cereblon Binding is designed for the testing and profiling of PROTACs directed against BRD9 and Cereblon (CRBN). The BRD9 inhibitor BI-7273 is included as a control inhibitor of PROTAC binding to BRD9. With this kit, three simple steps are required for the measurement of PROTAC activity. First, the PROTAC of interest is incubated with CRBN and BRD9. Next, acceptor beads are added, then donor beads, followed by reading of the Alpha-counts.Illustration of the assay principle: a PROTAC of interest or positive control dBRD9 (PROTAC) interacts with both BRD9 and CRBN, bringing them in close proximity. BRD9 contains a GST tag, recognized by the GSH donor bead, while CRBN contains a FLAG tag that binds to the AlphaLISA™ acceptor bead conjugated with an anti-FLAG antibody. Upon excitation of the donor bead a singlet oxygen is generated by the donor bead, which excites the acceptor bead and emits light proportionally to the level of interaction. AlphaLISA™ immunoassays are a no-wash alternative to ELISA immunoassays. These assays are robust and ideal for a minimal hands-on approach.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Supplied As: This Kit comes in a convenient AlphaLISA™ format, with the BRD9 Degrader dBRD9 (PROTAC) added as positive control, BRD9 buffer, and purified BRD9 and CRBN proteins for 384 reactions.
Warnings: Avoid freeze/thaw cycles. dBRD9 is a pomalidomide-derivative, which is known to cause severe birth defects in humans. Use all appropriate precautions when handling this compound!
Biosafety Level: Not applicable (BSL-1)
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