TR-FRET Cereblon-4C Binding Assay Kit

Background

E3 ubiquitin ligases are a large family of enzymes that catalyze the critical final step in the ubiquitination cascade. Cereblon (CRBN) serves as the substrate-recognition component of the E3 ubiquitin ligase complex CRBN/DDB1/CUL4A/RBX1 (CRBN-4C). CRBN is one of the most widely utilized E3 ligases in the design of PROTACs (Proteolysis-Targeting Chimeras) for targeted protein degradation (TPD) in drug discovery.

Assay Principle

The TR-FRET CRBN Binding Assay Kit is designed to measure the binding affinity between CRBN and its ligands. The kit includes Tag1-CRBN-4C (a complex of CRBN/CUL4A/DDB1/RBX1), a terbium- labeled anti-Tag1 antibody, and a fluorescently labeled CRBN ligand, thalidomide. When CRBN binds to thalidomide, the terbium donor (on the anti-Tag1 antibody) is brought into close proximity to the fluorescent acceptor (fluorophore-labeled thalidomide), resulting in fluorescence resonance energy transfer (FRET). The binding interaction can be quantitatively measured as an HTRF signal, calculated from the ratio of the emission intensities at 665 nm (acceptor) and 620 nm (donor). If a test compound competes with thalidomide for CRBN binding, the HTRF signal will decrease, indicating inhibition of CRBN–thalidomide interaction.

Application

High throughput screening of compounds that bind to Cereblon for drug discovery. -4510 or 858453-5700 Fax: 855-898-3979 1

Instrument Required

A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.

Kit Components

Materials Not Supplied

• Microplate reader, HTRF® certified microplate reader
• Adjustable micro-pipettor
• Sterile Tips
• Prepare the inhibitor compound solution
• Prepare CRBN-4C solution
• Add inhibitor
• Prepare FL-Thalidomide solution
• Prepare dye solution
• Incubate the reaction at room temperature for 20 minutes.
• Measure fluorescent intensity
• Excitation wavelength at 340 nm and emission at 620 nm.
• Excitation wavelength at 340 nm and emission at 665 nm.
• Calculate sample HTRF signal of each well.
• Calculate percentage activity

Assay Protocol

1. Step 1. Prepare CRBN-4C solution Thaw CRBN-4C protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: CRBN-4C protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the CRBN-4C protein 20-fold (1µL CRBN-4C + 19 µL assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.
2. Step 2. Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of assay buffer to each of negative and positive control wells. If the compound is diluted in 10% DMSO, add 2 µl of assay buffer containing 10% DMSO to each of negative and positive control wells.
3. Step 3. Prepare FL-Thalidomide solution Dilute FL-Thalidomide 125-fold (1 µL FL-Thalidomide + 124 µL of assay buffer). Add 4 µl of diluted FL-Thalidomide solution to each well.
4. Step 4. Prepare dye solution Dilute Terbium-labeled anti-Tag1 antibody 1:200 in assay buffer. For example: 1 µl of Terbium- labeled anti-Tag1 antibody + 199 µl of assay buffer. Add 10 µl of this dye mixture to each well. -4510 or 858453-5700 Fax: 855-898-3979 3
5. Step 5. Incubate the reaction at room temperature for 20 minutes.
6. Step 6. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
7. Step 7. Excitation wavelength at 340 nm and emission at 620 nm.
8. Step 8. Excitation wavelength at 340 nm and emission at 665 nm.

Data Analysis

Calculate sample HTRF signal of each well. Calculate percentage activity In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the following equation: % activity = (S-N)/(P-N) X100, where S= the sample signal in the presence of the compound.

Biological Pathway / Process

Ubiquitin-Proteasome / Targeted Protein Degradation

Therapeutic / Disease Area

Oncology; Hematology; PROTAC Drug Discovery