Abstract 5084: Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action
Authors: Fernández-Montalván Amaury E. , Berger Markus , Kuropka Benno , Koo Seong Joo , Badock Volker , Weiske Joerg , Holton Simon J. , Chaikuad Apirat , Díaz-Sáez Laura , Bennett James , Federov Oleg , Huber Kilian , Centrella Paolo , Clark Matthew A. , Dumelin Christoph E. , Sigel Eric A. , Soutter Holly S. , Troast Dawn M. , Zhang Ying , Cuozzo John W. , Keefe Anthony D. , Roche Didier , Rodeschini Vincent , Hübner Jan , Weinmann Hilmar , Hartung Ingo V. , Gorjanacz Matyas 
Published: July 2017

ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein.

Citation Format: Amaury E. Fernández-Montalván, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Díaz-Sáez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hübner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5084. doi:10.1158/1538-7445.AM2017-5084