Molecular Glue/PROTAC® Optimization Kit for RBM39-DCAF15
The Molecular Glue/PROTAC® Optimization Kit for RBM39-DCAF15 Binding is designed for the testing and profiling of Molecular Glues (MG)/PROTACs targeting the RBM39 (RNA-binding protein 39) and DCAF15 (DDB1 and CUL4 associated factor 15) interaction. The Molecular Glue/PROTAC® Optimization Kit for RBM39-DCAF15 Binding comes in a convenient AlphaLISA® format, with enough optimized assay and detection buffer, purified recombinant RBM39 (amino acids 245-530) and Rbx1/CUL4B/DDB1/DCAF15/DDA1 complex for 384 reactions. This kit also contains the molecular glue E7820 and Arginine as controls.
Figure 1. Schematic representation of the Molecular Glue/PROTAC® Optimization Kit for RBM39-DCAF15 Binding assay kit principle.
The Molecular Glue/PROTAC® of interest is incubated with DCAF15 complex and RBM39, bringing them in close proximity. DCAF15 contains a His-tag, which is recognized by the donor beads. RBM39 contains a GST tag that binds to the GSH- AlphaLISA™ acceptor beads. Upon excitation of the donor bead, a singlet oxygen is generated by the bead. The singlet oxygen excites the acceptor bead, which emits light proportionally to the level of interaction between RBM39 and DCAF15
- AlphaLISA GSH acceptor beads, 250 µg (PerkinElmer #AL109C)
- Alpha Nickel donor beads, 5 mg/ml (PerkinElmer #AS101D)
- Optiplate 384 (PerkinElmer #6007290)
- AlphaScreen microplate reader
- Adjustable micropipettor and sterile tips
| Catalog # | Name | Amount | Storage |
| 101497 | Rbx1/CUL4B/DDB1/ DCAF15/DDA1 Complex* | 2 x 100 µg | -80°C |
| 102059 | RBM39, GST-Tag* | 2 x 50 µg | -80°C |
| E7820 (MW=336 Da) | 100 µg | -20°C | |
| 5x PP-02 Assay Buffer | 4 ml | -20°C | |
| 4x Detection Buffer 3D | 2 ml | -20°C | |
| L-Arginine | 10 mg | Room Temp | |
| Plate sealer | 1 | Room Temp |
*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.
DCAF15 is the substrate-binding component of the E3 protein ligase complex DDB1-DDA1-CUL4-Rbx1, involved in the ubiquitination and proteasomal degradation of target proteins. Binding of DCAF15 to a substrate protein engages the E3 ligase activity of the complex and results in the ubiquitination and ultimate degradation of the protein substrate. One of the most important targets of DCAF15 is RBM39 (RNA-binding motif protein 39). RBM39 is a key factor in tumor-targeted mRNA and protein expression. Recent studies have revealed that RBM39 is the unexpected target of aryl sulphonamides, which act as molecular glues between RBM39 and the DCAF15-associated E3 ubiquitin ligase complex leading to selective degradation of the target. E7820 is an example of aryl sulfonamides that recruit RBM39 to Rbx-CUL4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. Development of novel MG and PROTACS involved in the recruitment of RBM39 provides a framework for future efforts to utilize DCAF15 to degrade other proteins of interest.
Nunes, J., et al. 2019 ACS Med Chem Lett; 10(7): 1081-1085
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