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Optimization of a “bump-and-hole” approach to allele-selective BET bromodomain inhibition

Optimization of a “bump-and-hole” approach to allele-selective BET bromodomain inhibition
Optimization of a “bump-and-hole” approach to allele-selective BET bromodomain inhibition
Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins – BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin via their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound – 9-ME-1 – shows ~200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of in vitro and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression , likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and in vivo target validation studies.
1478-6524
Runcie, A.C.
58997fb5-6bd6-4eea-973c-d799ac380966
Zengerle, M.
6843f52d-8e5d-4bd1-b67a-ec7c2c7c6093
Chan, K.-H.
87d77c74-fcc0-4255-b0a9-e97a4b695e9f
Testa, A.
f5dd0605-dc5f-426f-ba05-12704d080fb2
van Beurden, L.
1961e85b-ee49-49c9-9830-0ab015dbf8fa
Baud, M.G.J.
8752d519-3d33-43b6-9a77-ab731d410c2e
Epemolu, O.
12bacb78-bb3d-4960-888b-1b4295d9feb6
Ellis, L.C.J.
a28ac259-bbc5-46ff-878c-9afad9b3ca1d
Read, K.D.
cba5dec6-1b5a-4176-a0c0-f0a15dc81d60
Coulthard, V.
df1bc8a1-ace4-4198-98d7-f57da679cfe4
Ciulli, A.
8a04c43e-58ae-49e3-9c2f-72aae3b2e014
Runcie, A.C.
58997fb5-6bd6-4eea-973c-d799ac380966
Zengerle, M.
6843f52d-8e5d-4bd1-b67a-ec7c2c7c6093
Chan, K.-H.
87d77c74-fcc0-4255-b0a9-e97a4b695e9f
Testa, A.
f5dd0605-dc5f-426f-ba05-12704d080fb2
van Beurden, L.
1961e85b-ee49-49c9-9830-0ab015dbf8fa
Baud, M.G.J.
8752d519-3d33-43b6-9a77-ab731d410c2e
Epemolu, O.
12bacb78-bb3d-4960-888b-1b4295d9feb6
Ellis, L.C.J.
a28ac259-bbc5-46ff-878c-9afad9b3ca1d
Read, K.D.
cba5dec6-1b5a-4176-a0c0-f0a15dc81d60
Coulthard, V.
df1bc8a1-ace4-4198-98d7-f57da679cfe4
Ciulli, A.
8a04c43e-58ae-49e3-9c2f-72aae3b2e014

Runcie, A.C., Zengerle, M., Chan, K.-H., Testa, A., van Beurden, L., Baud, M.G.J., Epemolu, O., Ellis, L.C.J., Read, K.D., Coulthard, V. and Ciulli, A. (2018) Optimization of a “bump-and-hole” approach to allele-selective BET bromodomain inhibition. Chemical Science. (doi:10.1039/C7SC02536J).

Record type: Article

Abstract

Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins – BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin via their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound – 9-ME-1 – shows ~200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of in vitro and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression , likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and in vivo target validation studies.

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Accepted/In Press date: 23 January 2018
e-pub ahead of print date: 24 January 2018

Identifiers

Local EPrints ID: 417754
URI: https://eprints.soton.ac.uk/id/eprint/417754
ISSN: 1478-6524
PURE UUID: f8c52c06-a453-4f99-a6c4-692ba9bda47c
ORCID for M.G.J. Baud: ORCID iD orcid.org/0000-0003-3714-4350

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Date deposited: 13 Feb 2018 17:30
Last modified: 17 Sep 2019 04:26

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