In vivo engineered cereblon protein

Patent metadata

Field	Value
Publication number	US-10781239-B2
Application number	US-201916536227-A
Country	US
Kind code	B2
Filing date	Aug 8, 2019
Priority date	Dec 28, 2018
Publication date	Sep 22, 2020
Grant date	Sep 22, 2020

Abstract

Disclosed herein are in vivo engineered cereblon protein and methods of making the same. The in vivo engineered cereblon protein can include a site-specific non-naturally occurring modification at cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO:2 or 3, the modification comprising a moiety resulting from an in vivo Michael addition reaction between an exogenous Michael acceptor and the cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO:2 or 3.

First claim

What is claimed is:

1. An in vivo engineered cereblon protein consisting of a non-naturally occurring covalent modification at cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO: 2 or 3, the modification resulting from an in vivo Michael addition reaction between an exogenous Michael acceptor and the cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO: 2 or 3, wherein a sulfur atom at the cysteine residue undergoes the Michael reaction with a double bond of the exogenous Michael acceptor, and wherein the engineered cereblon has reduced engagement potency at cysteine 318 as set forth in SEQ ID NO:1 or at cysteine 317 as set forth in SEQ ID NO: 2 or 3.

2. The in vivo engineered cereblon of claim 1 , wherein the IMiD pocket-dependent binding on the engineered cereblon protein is prevented by the modification.

3. The in vivo engineered cereblon of claim 1 , wherein the engineered cereblon protein has an inhibited or reduced binding for immunomodulatory drugs as compared to unmodified cereblon.

4. The in vivo engineered cereblon of claim 1 , wherein the amino acid residue at cysteine 318 as set forth in SEQ ID NO:1 or cysteine 317 as set forth in SEQ ID NO: 2 or 3 is not modified.

5. The in vivo engineered cereblon of claim 1 , wherein the amino acid residues at cysteine 234, cysteine 205, cysteine 219, cysteine 366, cysteine 188, and cysteine 343 as set forth in SEQ ID NO:1 are not modified.

6. The in vivo engineered cereblon of claim 1 , wherein the engineered cereblon protein is only modified at cysteine 287 as set forth in SEQ ID NO:1 or cysteine 286 as set forth in SEQ ID NO: 2 or 3.

7. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor has a global promiscuity that is no greater than the global promiscuity of KB02.

8. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor has a global promiscuity that is no greater than ⅓ of the global promiscuity of KB02.

9. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor has a global promiscuity of no greater than 20% at 500 μM.

10. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor has a global promiscuity of no greater than 10% at 500 μM.

11. The in vivo engineered cereblon protein of claim 1 , wherein the exogenous Michael acceptor has a TE 50 of no greater than 80 μM.

12. The in vivo engineered cereblon protein of claim 1 , wherein the exogenous Michael acceptor has a TE 50 of no greater than 60 μM.

13. The in vivo engineered cereblon of claim 12 , wherein the exogenous Michael acceptor has a global promiscuity no greater than 5% at the TE 50 .

14. The in vivo engineered cereblon of claim 12 , wherein the exogenous Michael acceptor has a global promiscuity no greater than 2.5% at the TE 50 .

15. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor has a higher TE 50 than

16. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor is an acrylamide.

17. The in vivo engineered cereblon of claim 1 , wherein the cereblon has a sequence as set forth in SEQ ID NO: 1 and the modification is at the cysteine residue C287.

18. The in vivo engineered cereblon of claim 1 , wherein the cereblon has a sequence as set forth in SEQ ID NO: 2 or 3 and the modification is at the cysteine residue C286.

19. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor forms a hydrophobic interaction with the one or more residues selected from Pro 93, Ile 160, Met 246, Pro 274, Tyr 282, Ala 286, Cys 287, Cys 343, Ala 347, Ala 348, or a combination thereof.

20. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor forms a hydrophobic interaction with the one or more residues selected from Met 345, Leu 321, Leu 422, Leu 423, Pro 424, Leu 360, or a combination thereof.

21. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor forms a charged interaction with the one or more residues selected from Asp279, Asp 265, Arg 162, Arg 283, Lys 324, Arg 419, or a combination thereof.

22. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor forms a polar interaction with the one or more residues selected from Gln 95, Thr 96, or Ser 420.

23. The in vivo engineered cereblon of claim 1 , wherein the exogenous Michael acceptor comprises at least one double bond.

24. The in vivo engineered cereblon of claim 1 , wherein the cereblon is a human cereblon protein.

Assignees

• Vividion Therapeutics Inc
• Scripps Research Inst

Inventors

• Weinstein David
• Cravatt Benjamin F
• Patricelli Matthew
• Stamos Dean
• Simon Gabe
• Horning Benjamin
• Vinogradova Ekaterina
• Nordin Brian
• Baltgalvis Kristen
• Kinsella Todd

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K14/4702

  Regulators; Modulating activity · CPC title

• C07D211/94 Primary

  Oxygen atom, e.g. piperidine N-oxide · CPC title

• C07K14/4703 Primary

  Inhibitors; Suppressors · CPC title