Methods for measuring small molecule affinity to cereblon

1. A method of measuring the affinity of a compound to cereblon (CRBN) or determining if a compound binds to cereblon (CRBN), the method comprising:

contacting the compound with a complex, wherein the complex comprises

(i) a CRBN having a europium-anti-his antibody on the N-terminus of the CRBN, and

(ii) a Cy5-conjugated small molecule, wherein the Cy5-conjugated small molecule binds the CRBN;

measuring fluorescence resonance energy transfer (FRET) after contacting the compound with the complex; and

determining the binding and/or affinity of the compound to CRBN based on the measured FRET; and

wherein the Cy5-conjugated small molecule has a structure of Formula (I)

wherein the Cy5-conjugated small molecule binds in a hydrophobic tri-tryptophan pocket formed by Trp380, Trp386, and Trp400 on the CRBN.

2. The method of claim 1 , wherein the complex further comprises a DNA damage-binding protein 1 (DDB1).

3. The method of claim 1 , wherein the method further comprises measuring FRET prior to contacting the compound with the complex.

4. The method of claim 3 , further comprises determining that the compound binds to the CRBN if FRET is reduced after contacting the compound with the complex.

5. The method of claim 1 , wherein FRET is observed by exciting at 340 nm and monitoring emission at 615 nm (non-FRET emission) and 665 nm (FRET emission).

6. The method of claim 5 , wherein a first FRET efficiency is determined prior to contacting the compound with the complex, and a second FRET efficiency is determined after contacting the compound with the complex, and wherein FRET efficiency is the ratio of FRET emission at 665 nm to non-FRET emission at 615 nm.

7. The method of claim 6 , further comprises determining that the compound binds to the CRBN if the second FRET efficiency is less than the first FRET efficiency.