Methods for measuring small molecule affinity to cereblon

Patent metadata

Field	Value
Publication number	US-2018224435-A1
Application number	US-201815887941-A
Country	US
Kind code	A1
Filing date	Feb 2, 2018
Priority date	Feb 3, 2017
Publication date	Aug 9, 2018
Grant date	—

Abstract

A complex comprises a CRBN having a europium-anti-his antibody on the N-terminus of the CRBN, and a Cy5-conjugated small molecule, wherein the Cy5-conjugated small molecule binds the CRBN, and uses thereof for identifying therapeutic compounds.

First claim

1 . A complex comprising (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.

2 . The complex of claim 1 , further comprising a DDB1.

3 . The complex of claim 1 , wherein the Cy5-conjugated small molecule binds in a hydrophobic tri-tryptophan pocket formed by Trp380, Trp386, and Trp400 on the CRBN.

4 . The complex of claim 1 , wherein the Cy5-conjugated small molecule has a structure of Formula (I)

5 . A method of determining if a compound binds to cereblon (CRBN), measuring the affinity of a compound to cereblon (CRBN), or identifying a compound for treating a disease, 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.

6 . The method of claim 5 , wherein the complex further comprises a DDB1.

7 . The method of claim 1 , wherein the Cy5-conjugated small molecule binds in a hydrophobic tri-tryptophan pocket formed by Trp380, Trp386, and Trp400 on the CRBN.

8 . The method of claim 5 , wherein the Cy5-conjugated small molecule has a structure of Formula (I)

9 . The method of claim 5 , wherein the method further comprises measuring fluorescence resonance energy transfer (FRET) after contacting the compound with the complex.

10 . The method of claim 9 , wherein the method further comprises measuring FRET prior to contacting the compound with the complex.

11 . The method of claim 9 , further comprises determining that the compound binds to the CRBN if FRET is reduced after contacting the compound with the complex.

12 . The method of claim 9 , wherein FRET is observed by exciting at 340 nm and monitoring emission at 615 nm (non-FRET emission) and 665 nm (FRET emission).

13 . The method of claim 12 , 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.

14 . The method of claim 13 , further comprises determining that the compound binds to the CRBN if the second FRET efficiency is less than the first FRET efficiency.

15 .- 34 . (canceled)

35 . A composition comprising the complex of anyone of claim 1 .

Assignees

• Celgene Corp

Inventors

• Chamberlain Philip Paul
• Matyskiela Mary
• Khambatta Godrej

Classifications

• G01N33/542 Primary

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• G01N2333/4703

  Regulators; Modulating activity · CPC title

• G01N2500/04

  Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A) · CPC title

• C07K14/47

  from mammals · CPC title