Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition

Andrew Runcie, Michael Zengerle, Kwok Ho Chan, Andrea Testa, Lars Van Beurden, Matthias Baud, Rafiu Epemolu, Lucy Ellis, Kevin Read, V. Coulthard, A. Brien, Alessio Ciulli (Lead / Corresponding author)

Research output: Contribution to journalArticle

5 Citations (Scopus)
120 Downloads (Pure)

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 wildtype 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.
Original languageEnglish
Article number2452
Pages (from-to)2452-2468
Number of pages17
JournalChemical Science
Volume9
Issue number9
Early online date24 Jan 2018
DOIs
Publication statusPublished - 7 Mar 2018

Fingerprint

Benzodiazepines
Leucine
Proteins
Cell proliferation
Valine
Gene expression
Scaffolds
Alanine
Chromatin
Assays
Experiments
Genetics

Cite this

Runcie, Andrew ; Zengerle, Michael ; Chan, Kwok Ho ; Testa, Andrea ; Van Beurden, Lars ; Baud, Matthias ; Epemolu, Rafiu ; Ellis, Lucy ; Read, Kevin ; Coulthard, V. ; Brien, A. ; Ciulli, Alessio. / Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition. In: Chemical Science. 2018 ; Vol. 9, No. 9. pp. 2452-2468.
@article{a906f0db40e5405080e1bca015cbfec4,
title = "Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition",
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 wildtype 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.",
author = "Andrew Runcie and Michael Zengerle and Chan, {Kwok Ho} and Andrea Testa and {Van Beurden}, Lars and Matthias Baud and Rafiu Epemolu and Lucy Ellis and Kevin Read and V. Coulthard and A. Brien and Alessio Ciulli",
note = "This work was supported by awards to A.C. from the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant BB/J001201/2 and David Phillips Fellowship BB/G023123/2) and the European Research Council (ERC starting grant, ERC-2012-StG-311460 DrugE3CRLs). A.C.R. was supported by a BBSRC EASTBIO Doctoral Training Partnership award (BB/J01446X/1). K.-H.C. was supported by a European Commission Marie Skłodowska-Curie Actions Individual Fellowship (H2020-MSCA-IF-2014-655516). L.V.B. was funded by a BBSRC Research Experience Placement award (BB/J01446X/1). Biophysics and drug discovery activities are supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). We would like to acknowledge P. Fyfe of the in-house X-ray Crystallography Facility at the University of Dundee, which is supported by The Wellcome Trust (award no. 094090); the staff at the Diamond Light Source for beamtime (BAG proposal Page 15 of 18 Chemical Science 16 | J. Name., 2012, 00, 1-3 This journal is {\circledC} The Royal Society of Chemistry 20xxMX10071 ) and beamline support at beamline I04-1; and Dr. Sam Swift at the Dundee Imaging Facility, Dundee, which is supported by the 'Wellcome Trust Technology Platform' award [097945/B/11/Z] and the 'MRC Next Generation Optical Microscopy' award [MR/K015869/1].",
year = "2018",
month = "3",
day = "7",
doi = "10.1039/C7SC02536J",
language = "English",
volume = "9",
pages = "2452--2468",
journal = "Chemical Science",
issn = "2041-6520",
publisher = "Royal Society of Chemistry",
number = "9",

}

Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition. / Runcie, Andrew; Zengerle, Michael; Chan, Kwok Ho; Testa, Andrea; Van Beurden, Lars; Baud, Matthias; Epemolu, Rafiu; Ellis, Lucy; Read, Kevin; Coulthard, V.; Brien, A.; Ciulli, Alessio (Lead / Corresponding author).

In: Chemical Science, Vol. 9, No. 9, 2452, 07.03.2018, p. 2452-2468.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition

AU - Runcie, Andrew

AU - Zengerle, Michael

AU - Chan, Kwok Ho

AU - Testa, Andrea

AU - Van Beurden, Lars

AU - Baud, Matthias

AU - Epemolu, Rafiu

AU - Ellis, Lucy

AU - Read, Kevin

AU - Coulthard, V.

AU - Brien, A.

AU - Ciulli, Alessio

N1 - This work was supported by awards to A.C. from the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant BB/J001201/2 and David Phillips Fellowship BB/G023123/2) and the European Research Council (ERC starting grant, ERC-2012-StG-311460 DrugE3CRLs). A.C.R. was supported by a BBSRC EASTBIO Doctoral Training Partnership award (BB/J01446X/1). K.-H.C. was supported by a European Commission Marie Skłodowska-Curie Actions Individual Fellowship (H2020-MSCA-IF-2014-655516). L.V.B. was funded by a BBSRC Research Experience Placement award (BB/J01446X/1). Biophysics and drug discovery activities are supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). We would like to acknowledge P. Fyfe of the in-house X-ray Crystallography Facility at the University of Dundee, which is supported by The Wellcome Trust (award no. 094090); the staff at the Diamond Light Source for beamtime (BAG proposal Page 15 of 18 Chemical Science 16 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xxMX10071 ) and beamline support at beamline I04-1; and Dr. Sam Swift at the Dundee Imaging Facility, Dundee, which is supported by the 'Wellcome Trust Technology Platform' award [097945/B/11/Z] and the 'MRC Next Generation Optical Microscopy' award [MR/K015869/1].

PY - 2018/3/7

Y1 - 2018/3/7

N2 - 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 wildtype 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.

AB - 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 wildtype 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.

UR - http://www.scopus.com/inward/record.url?scp=85042727679&partnerID=8YFLogxK

U2 - 10.1039/C7SC02536J

DO - 10.1039/C7SC02536J

M3 - Article

C2 - 29732121

VL - 9

SP - 2452

EP - 2468

JO - Chemical Science

JF - Chemical Science

SN - 2041-6520

IS - 9

M1 - 2452

ER -