Abstract 4691: Identification and characterization of BRPF1 bromodomain inhibitors
Authors: Haendler Bernard , Bouché Léa , Siegel Stephan , Fernandez-Montalvan Amaury E. , Sugawara Tatsuo , Meier Julia , Knapp Stefan , Gamble Vicki 
Published: July 2016

BRPF (bromodomain and PHD finger-containing) proteins represent a subgroup of the bromodomain protein family with three closely related members, namely BRPF1, -2, and -3. They are found in different histone acetyltransferase complexes and regulate development and cell differentiation. Despite their high similarity, they have unique, non-redundant functions. Knock-out of BRPF1 in mice leads to embryonic lethality at E9.5 due to vascular defects whereas BRPF2 deficiency leads to embryonic lethality at E15.5 due to anemia. BRPF proteins contain multiple functional regions including a PZP domain which binds to unmodified H3K4, a PWWP domain which binds to H3K36me3 and a single bromodomain which recognizes acetyl-lysines in histones. BRPF1 is overexpressed in a variety of tumors, suggesting a role in cell proliferation. As first steps towards understanding the role of BRPF1, we performed knock-down studies in different tumor cell lines and initiated the search for potent and selective inhibitors of the BRPF1 bromodomain.

BRPF1-specific siRNAs were designed and used for gene silencing experiments in bladder and ovarian cancer cell lines. Following BRPF1 knock-down, marked inhibition of proliferation was observed in the CAL-29, 5637 and JMSU-1 bladder cancer cell lines, and in the ES-2 and OVCAR-8 ovarian cancer cell lines. In order to further delineate the function of BRPF1, we sought to identify selective BRPF1 bromodomain inhibitors. A collection of 5000 compounds was selected by virtual screening based on the BRPF1 bromodomain crystal structure (4LC2) and tested in a biochemical TR-FRET assay for inhibition of the interaction between BRPF1 bromodomain and an acetylated histone H4-derived peptide. Two potent hit clusters (IC50 &lt; 150 nM) were identified and subsequent optimization led to BAY-140 and BAY-496 which were selected for further analyses. The IC50 values of BAY-140 and BAY-496 for inhibition of BRPF1 bromodomain binding were 30 and 20 nM, and for BRPF2 bromodomain binding 380 and 160 nM, respectively. Importantly, no interaction was seen for either BRD4 bromodomain (IC50 &gt;20 μM). This binding and selectivity profile was confirmed in cellular NanoBRET assays where IC50 values for BAY-140 and BAY-496 were 1300 and 810 nM for BRPF1 bromodomain, 3600 and 2310 nM for BRPF2 bromodomain, and &gt;10 μM for full-length BRD4. In vitro proliferation assays performed with these compounds showed single digit micromolar inhibition for the ovarian cancer ES-2, small cell lung cancer DMS-53 and acute myeloid leukemia THP-1 cell lines. However, no anti-proliferative activity was observed in a number of other tumor cell lines derived from a variety of hematological and solid tumors.

Altogether, these data show that inhibiting BRPF1 bromodomain has only a limited anti-proliferative effect on tumor cell lines, suggesting that other regions than the bromodomain of the protein account for the anti-proliferative effects seen in knock-down experiments.

Citation Format: Bernard Haendler, Léa Bouché, Stephan Siegel, Amaury E. Fernandez-Montalvan, Tatsuo Sugawara, Julia Meier, Stefan Knapp, Vicki Gamble. Identification and characterization of BRPF1 bromodomain inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4691.