Ligand-directed covalent modification of protein

a covalent modification and protein technology, applied in the field of enzyme inhibitors, can solve the problems of protein target modification, other undesirable effects, and difficulty in developing reversible inhibitors that selectively inhibit one or more desired kinases, and achieve irreversible inhibition

Inactive Publication Date: 2011-11-03
CELGENE CAR LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]In one aspect, the invention provides a method for designing a ligand that covalently binds a target protein. The method comprises (a) providing a structural model of a reversible ligand docked within, or in proximity to, a ligand-binding site in a target protein, (b) identifying a lysine residue of the target protein in, or in proximity to, the ligand-binding site that is less than about 15 Å from the reversible ligand when the reversible ligand is docked in, or in proximity to, the ligand-binding site, (c) producing at least a structural model of at least one ligand-warhead compound docked within, or in proximity to, the ligand-binding site wherein the ligand-warhead compound comprises the reversible ligand in step (b) or a portion thereof, a warhead comprising a reactive chemical moiety, and optionally a Tether, and (d) identifying a ligand-warhead compound whose structural model allows the lysine residue in step (b) to readily assume a conformation that brings the side chain primary amine group of the lysine residue within bond-forming proximity of the warhead electrophile.

Problems solved by technology

Because the structure of the ATP-binding site is highly conserved among kinases, it has been very challenging to develop reversible inhibitors that selectively inhibit one or more desired kinases.
This dosing requirement may produce toxicity or result in other undesirable effects.
Second, because resistance mutations may compromise non-covalent binding, but even in the face of reduced non-covalent affinity, the inactivation mechanism will often, nonetheless, lead to protein target modification and irreversible inhibition.
However, amino acids such as lysine are usually sufficiently unreactive that ligands do not react in vivo with lysine.
And, as such, ligand-directed modification of lysine has heretofore remained unrealized.

Method used

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  • Ligand-directed covalent modification of protein
  • Ligand-directed covalent modification of protein
  • Ligand-directed covalent modification of protein

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0756](3S,6S,10aR)-6-((S)-2-(methylamino)propanamido)-5-oxo-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)decahydropyrrolo[1,2-a]azocine-3-carboxamide (Compound A) is a reversible inhibitor of XIAP (Ki 26 nM) (Sun et al., J. Med. Chem. 52, 593-596 (2009). Using the structure-based design algorithm described herein, Compound A was converted from a reversible inhibitor into Compound VII-1, a potent and irreversible inhibitor of XIAP. The process for the conversion of Compound A to Compound VII-1 is described below.

[0757]The X-ray crystal structure of XIAP complexed with Compound B, a related compound to Compound A, has been reported (Sun, H., et al., J. Med. Chem. 51, 7169-7180 (2008)) and was obtained from the protein databank (pdbcode 2JK7 at www.resb.org). The X-ray complex of Compound B bound to XIAP was used to

design covalent inhibitors of XIAP using the design algorithm described herein. The three-dimensional structure of Compound B was docked into the XIAP ligand-binding site using ...

example 1a

[0761]

[0762](3S,6S,10aS)-N-(3-acrylamidobenzyl)-6-(S)-2-(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide: The title compound was prepared according to the steps and intermediates as described below.

(S)-2-benzyl 1-tert-butyl 5-oxopyrrolidine-1,2-dicarboxylate (1a)

[0763]

[0764]To a stirred solution of L-pyroglutamic acid (75 g, 0.58 mol) and N,N-diisopropyl-ethylamine (87.3 g, 0.676 mol) in dry dichloromethane (1.0 L) at 0° C. was added benzyl bromide (98.84 g, 0.58 mol) dropwise. The reaction mixture was heated under reflux for 5 h, cooled to RT and washed with aqueous NaH2PO4. The aqueous layer was extracted with CH2Cl2; the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue obtained was then taken in acetonitrile (1.5 L) and 4-dimethylaminopyridine (7.09 g, 58.0 mmol) and Boc-anhydride (150.0 g, 0.688 mol) were added and stirred at RT for 3 hrs. The reaction mixture was concentra...

example 2

[0787]

[0788]tert-Butyl (S)-1-(3S,6S,10aS)-3-(3-acrylamidobenzylcarbamoyl)-5-oxodecahydropyrrolo[1,2-a]azocin-6-ylamino)-1-oxopropan-2-yl(methyl)carbamate: The title compound was prepared according to the steps and intermediates as described below.

[0789]To a mixture of the aniline 1k (60 mg) and triethylamine (80 μl) in dry dichloromethane (2 mL) was added acryloyl chloride (19 μl) dropwise at 0° C. The reaction mixture was concentrated and the residue was purified using semi-prep HPLC (TFA modifier) to give a white solid. LCMS: m / e 470.3 (M+1-tBu).

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Abstract

The present invention relates to enzyme inhibitors. More specifically, the present invention relates to ligand-directed covalent modification of proteins; method of designing same; pharmaceutical formulation of same; and method of use.

Description

PRIORITY CLAIM[0001]This application claims the benefit of U.S. provisional application 61 / 335,043, filed Dec. 30, 2009, the disclosure of which is relied on and incorporated by reference into the present application in its entirety. All patents, patent applications and referenced articles are hereby incorporated by reference into this application in their entireties.DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY[0002]The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: AVIL—001—01US_SeqList_ST25.txt, date recorded: Feb. 27, 2011, file size 26 kilobytes).FIELD OF THE INVENTION[0003]The present invention relates to enzyme inhibitors. More specifically, the present invention relates to ligand-directed covalent modification of lysine-containing proteins.BACKGROUND OF THE INVENTION[0004]Compounds that inhibit the activity of proteins, such as enzym...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/68G06G7/58C07K1/107G16B15/30
CPCG01N33/68C07D403/12C07D401/14C07D405/14C07D491/08C07D401/04C07D405/12C07D239/49C07D409/12C07D417/06C07D417/14C07D487/04C07D493/04C07D207/16C07D495/04C07D513/04C07D519/00G16C20/50G16B15/00C07D403/14C12N9/12C12N9/506C12N9/93C12N9/96C12Y207/11001C12Y304/21098C12Y603/02A61P31/14A61P43/00G16B15/30C12Q1/25C12Q1/37C12Q1/485
Inventor PETTER, RUSSELL C.JEWELL, CHARLES F.LEE, KWANGHOMEDIKONDA, ARAVIND PRASADNIU, DEQIANGQIAO, LIXINSINGH, JUSWINDERZHU, ZHENDONG
Owner CELGENE CAR LLC
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