Abstract LB-135: Selective small molecule inhibition of the N-terminal BET bromodomain
Authors: Seitzberg Jimmi G. , Hansen Margit H. , Kronborg Tine T. Akinleminu , Underwood Christina R. , Friberg Gitte , Tonnesen Berit , Teuber Lene , Franch Thomas , Nielsen Søren J. 
Published: May 2015

Abstract
Introduction:
The bromodomain acetyl-lysine ‘readers’ is an emerging target class with particular interest centered around the BET family proteins BRD2, 3, and 4. Pre-clinical target validation experiments of BET proteins using genetic tools and/or small molecule bromodomain inhibitors have demonstrated strong disease linkage in several cancers, and a number of BET inhibitors are currently in early clinical trials within oncology. BET proteins contain a unit of tandem bromodomains (BD1 and BD2), each capable of binding to acetylated lysines within partner proteins. However, the contribution of individual bromodomains and of specific BET isoforms to biological activity is currently unclear, and domain-specific inhibition (intra- and/or inter-BET) of BET proteins has been insufficiently explored. Here we report and characterize the first BD1-selective BET inhibitors.
Methods:
Chemetics DNA-encoding small molecule libraries was used to identify small molecule BET inhibitors. Binding of hit compounds to bromodomains was investigated using AlphaScreen and FRET assays. Compounds were then assayed for AML cell growth inhibition using MTS and CellTiter-Glo assays, and for inhibition of cytokine release from human PBMCs following LPS stimulation.
Results:
To discover novel inhibitors of BET bromodomains, we screened ∼300 million small molecule compounds for binding towards BD1 of human BRD4. Representative hits from several chemical series were resynthesized and confirmed for binding to BRD4 in the high nM range. Chemical optimization of one series yielded compounds with low nM affinity against BRD4 BD1 with comparable affinity against the BD1s of other BET proteins. Interestingly, the compounds display marked intra-BET BD selectivity, with a strong preference (10-40 fold) for binding to BD1. In cellular growth inhibition assays, the compounds selectively target MLL-translocated AML cell lines. LPS-induced cytokine release from human PBMCs is strongly inhibited by both the pan-BET inhibitors JQ-1/I-BET762 and our BD1-selective compounds. In contrast, the BD2-selective compound RVX-208 show limited activity in the cell models tested here.
Conclusions:
Using DNA-encoding technology, we discovered low nM small molecule compounds that selectively inhibit the first bromodomain of BET proteins. The compounds strongly inhibit BET-dependent cellular processes. Furthermore, our data suggest that a number of BET biological activities are mediated pimarily by BD1, and that BD1 blockade is sufficient to elicit BET inhibition. Efforts to understand the mechanism and potential therapeutic utility of BD1-selectivity are ongoing.
Citation Format: Jimmi G. Seitzberg, Margit H. Hansen, Tine T. Akinleminu Kronborg, Christina R. Underwood, Gitte Friberg, Berit Tonnesen, Lene Teuber, Thomas Franch, Søren J. Nielsen. Selective small molecule inhibition of the N-terminal BET bromodomain. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-135. doi:10.1158/1538-7445.AM2014-LB-135