Abstract 5137: Parallel approaches to the discovery of novel BRD4 inhibitors
Authors: Jones David H. , Groot Marcel J. de , Heald Robert , Arhin Ebenezer , Goodwin Ria , Burton Brenda , Kulagowski Jan , Brown David , Irving Steven , Bazin Richard , Bruce Deborah , Devos Rene , Price Steven , Ray Nick , Lockey Peter , Montana John , Albertella Mark R. , Green Simon R. 
Published: May 2015

Epigenetic signalling is essential for the regulation of gene expression and cellular development, and disruption of these pathways has been identified in a wide range of tumours. The ϵ-N-acetylation of lysine residues (Kac) within histone tails is one of the most abundant epigenetic modifications and bromodomain containing proteins (BRD) represent an important class of reader protein that recognize and bind such residues. BRD4 is a critical mediator of transcription, functioning to recruit the positive transcription elongation factor complex (P-TEFb) and resulting in increased expression of growth-promoting genes. Inhibition of BRD4 has been shown to result in tumour growth inhibition both in vitro and in vivo. In addition, BRD4 has been shown to be a proto-oncogene which is mutated by chromosomal translocations in the rare form of NUT midline carcinoma.

Although the interaction between bromodomains and acetylated histone represents a protein-protein interaction, co-crystal structures have demonstrated that Kac is recognized by a central deep hydrophobic cavity, which represents “an attractive druggable pocket”. We performed a virtual screen based on modelling known BRD4 inhibitors in crystal structures of both BRD4 domain 1 and domain 2. The virtual screen used a combination of substructure, shape, electrostatic and docking methods combining the strengths of each of these approaches to select compounds from commercially available sources. In addition, we screened BRD4 binding domain 1 against an Argenta fragment library using surface plasmon resonance. A number of novel hits were identified from these screening approaches (up to 18µM in potency) and crystal structures obtained for key hits.

We developed a comprehensive screening cascade validated with tool compounds for profiling hits, starting with an in vitro Brd4 AlphaScreen binding assay and comprising a number of cellular pharmacodynamic assays evaluating effects on established biomarkers of BRD4 inhibition. These screening assays included mRNA analyses for c-myc, p21 and bcl2, a c-myc promoter reporter assay and were confirmed by Western analyses. Phenotypic assays based on growth inhibition of multiple myeloma and acute myeloid leukemia cell lines were also established.

Citation Format: David H. Jones, Marcel J. de Groot, Robert Heald, Ebenezer Arhin, Ria Goodwin, Brenda Burton, Jan Kulagowski, David Brown, Steven Irving, Richard Bazin, Deborah Bruce, Rene Devos, Steven Price, Nick Ray, Peter Lockey, John Montana, Mark R. Albertella, Simon R. Green. Parallel approaches to the discovery of novel BRD4 inhibitors. [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 5137. doi:10.1158/1538-7445.AM2014-5137