Process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4

Patent metadata

Field	Value
Publication number	US-9360482-B2
Application number	US-201013505474-A
Country	US
Kind code	B2
Filing date	Nov 3, 2010
Priority date	Nov 5, 2009
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

A process for the identification of compounds with a molecular weight in the range 100 to 750 which inhibit the binding of the first and/or second bromodomains of human BRD-2 to 4 to acetylated lysine residues of their physiological partner proteins which comprises selecting those compounds which are able to:
a) form a hydrogen bonding interaction in which the compound accepts a hydrogen bond from the sidechain NH2 group of the asparagine residue found at:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 ASN156 ASN429 ASN116 ASN391 ASN140 ASN433
or
b) accept a water-mediated hydrogen bond in which the compound accepts a hydrogen bond from a water that is itself hydrogen-bonded to the sidechain hydroxyl of the tyrosine residue found at

BRD-2 BRD-3 BRD-4 BD1 BRD-2 BD2 BD1 BRD-3 BD2 BD1 BRD-4 BD2 TYR113 TYR386 TYR73 TYR348 TYR97 TYR390
and
c) which are also able to form a Van der Waals interaction with a lipophilic binding region of a binding pocket such that one or more heavy atoms of the said compounds lie within a 5 Å range of any of the heavy atoms of the following bromodomain residues which define the binding pocket:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 TRP97 TRP370 TRP57 TRP332 TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161 ASP434 ASP121 GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146 VAL439 MET165 MET438 MET125 MET400 MET149 MET442
pharmaceutical compositions containing such compounds, and their use in therapy.

First claim

The invention claimed is:

1. A process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4 to acetylated lysine residues of their physiological partner proteins which process comprises the steps of:
1) selecting compounds with a molecular weight in the range 100 to 750 Da; and
2) determining which of those selected compounds is an interacting compound, wherein an interacting compound will:
a) form a hydrogen bonding interaction in which the compound accepts a hydrogen bond from the sidechain NH 2 group of the asparagine residue found at: ASN429 of BRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2

and
b) form a Van der Waals interaction with a lipophilic binding region of a binding pocket such that one or more heavy atoms of the said compounds lie within a 7.5 Å range of at least one heavy atom of each of the 3 residues of the second bromodomain wherein said bromodomain residues are selected from the group consisting of:
PRO371, ASP434, and VAL435 of BRD-2 BD2;
PRO333, GLU396, and VAL397 of BRD-3 BD2; and
PRO375, GLU438, and VAL439 of BRD-4 BD2
and

3) testing the ability of the interacting compound to inhibit the binding of the second bromodomain to a fluorescent ligand in a fluorescent anisotropy binding assay; wherein the fluorescent ligand is a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH 2 ) of the compound; and wherein the interacting compound which is able to inhibit the binding of the second bromodomain to the fluorescent ligand in the fluorescent anisotropy binding assay is identified as the compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3 and BRD-4 to the acetylated lysine residues of their physiological partner proteins.

2. A process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4 to acetylated lysine residues of their physiological partner proteins which process comprises the steps of:
1) selecting compounds with a molecular weight in the range 100 to 750 Da;
2) determining which of those selected compounds is an interacting compound, wherein an interacting compound will:
a) form a hydrogen bonding interaction in which the compound accepts a hydrogen bond from the sidechain NH 2 group of the asparagine residue found at: ASN429 of BRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2

and
b) accept a water-mediated hydrogen bond in which the compound accepts a hydrogen bond from a water that is itself hydrogen-bonded to the sidechain hydroxyl of the tyrosine residue found at the TYR386 of BRD-2 BD2, or TYR348 of BRD-3 BD2, or TYR390 of BRD-4 BD2

and
c) form a Van der Waals interaction with a lipophilic binding region of a binding pocket such that one or more heavy atoms of the said compounds lie within a 7.5 Å range of at least one heavy atom of each of the 3 residues of the second bromodomain wherein said second bromodomain residues are selected from the group consisting of:
PRO371, ASP434, and VAL435 of BRD-2 BD2;
PRO333, GLU396, and VAL397 of BRD-3 BD2; and
PRO375, GLU438, and VAL439 of BRD-4 BD2; and

3) testing the ability of the interacting compound to inhibit the binding of the second bromodomain to a fluorescent ligand in a fluorescent anisotropy binding assay; wherein the fluorescent ligand is a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH 2 ) of the compound; and wherein the interacting compound which is able to inhibit the binding of the second bromodomain to the fluorescent ligand in the fluorescent anisotropy binding assay is identified as the compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3 and BRD-4 to the acetylated lysine residues of their physiological partner proteins.

3. A process according to claim 1 wherein said molecular weight is in the range 100 to 500 Da.

4. A process according to claim 2 wherein said molecular weight is in the range 100 to 500 Da.

5. A process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4 to acetylated lysine residues of their physiological partner proteins which process comprises the steps of:
1) selecting compounds with a molecular weight in the range 100 to 750 Da; and
2) determining which of those selected compounds is an interacting compound, wherein an interacting compound will:
a) form a hydrogen bonding interaction in which the compound accepts a hydrogen bond from the sidechain NH 2 group of the asparagine residue found at: ASN429 of BRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2

and
b) form a Van der Waals interaction with a lipophilic binding region of a binding pocket such that one or more heavy atoms of the said compounds lie within a 7.5 Å range of at least one heavy atom of each of the 3 residues of the second bromodomain wherein said bromodomain residues are selected from the group consisting of:
PRO371, ASP434, and VAL435 of BRD-2 BD2;
PRO333, GLU396, and VAL397 of BRD-3 BD2; and
PRO375, GLU438, and VAL439 of BRD-4 BD2
and

3) testing the ability of the interacting compound to inhibit the binding of the second bromodomain to a fluorescent ligand in a fluorescent anisotropy binding assay, wherein the fluorescent ligand is a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH 2 ) of the compound, wherein the second bromodomain is comprised in a human bromodomain protein that comprises a histidine tag; and wherein the interacting compound which is able to inhibit the binding of the second bromodomain to the fluorescent ligand in the fluorescent anisotropy binding assay is identified as the compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3 and BRD-4 to the acetylated lysine residues of their physiological partner proteins.

6. A process according to claim 5 wherein said molecular weight is in the range 100 to 500 Da.

7. A process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4 to acetylated lysine residues of their physiological partner proteins which process comprises the steps of:
1) selecting compounds with a molecular weight in the range 100 to 750 Da;
2) determining which of those selected compounds is an interacting compound, wherein an interacting compound will:
a) form a hydrogen bonding interaction in which the compound accepts a hydrogen bond from the sidechain NH2 group of the asparagine residue found at: ASN429 of BRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2

and
b) accept a water-mediated hydrogen bond in which the compound accepts a hydrogen bond from a water that is itself hydrogen-bonded to the sidechain hydroxyl of the tyrosine residue found at the TYR386 of BRD-2 BD2, or TYR348 of BRD-3 BD2, or TYR390 of BRD-4 BD2

and
c) form a Van der Waals interaction with a lipophilic binding region of a binding pocket such that one or more heavy atoms of the said compounds lie within a 7.5 Å range of at least one heavy atom of each of the 3 residues of the second bromodomain wherein said second bromodomain residues are selected from the group consisting of:
PRO371, ASP434, and VAL43

Assignees

• Bamborough Paul
• Chung Chun-Wa
• Glaxosmithkline Llc

Inventors

• Bamborough Paul
• Chung Chun-Wa

Classifications

• G01N33/566 Primary

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title

• G01N33/6803 Primary

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01N2500/10

  involving cells · CPC title

• C12N9/1205 Primary

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title

• G01N2500/02

  Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) · CPC title