title: Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery.
abstract: BACKGROUND
  Proteins that 'read' the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases).
  
  
  RESULTS
  Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform.
  
  
  CONCLUSIONS
  In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies.
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Yanai
authors.initials: Y
authors.lastname: Zhan
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Kost-Alimova
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Shi
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Leo
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: Bardenhagen
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Hannah E
authors.initials: HE
authors.lastname: Shepard
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Srikanth
authors.initials: S
authors.lastname: Appikonda
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Bhavatarini
authors.initials: B
authors.lastname: Vangamudi
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Shuping
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Trang N
authors.initials: TN
authors.lastname: Tieu
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Shiming
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Heffernan
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Marszalek
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Carlo
authors.initials: C
authors.lastname: Toniatti
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Giulio
authors.initials: G
authors.lastname: Draetta
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Tyler
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Barton
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Jones
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Wylie S
authors.initials: WS
authors.lastname: Palmer
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Geck Do
authors.affiliation: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77230 USA.|XTuit Pharmaceuticals, 700 Main Street, Cambridge, MA 02139 USA.
authors.forename: Jannik N
authors.identifier: 0000-0003-4528-2120
authors.initials: JN
authors.lastname: Andersen
keywords: AlphaLISA; AlphaScreen; Bromodomain inhibitor; Bromodomain–histone-binding assays; Chromatin drug-target displacement; IACS-6558; IACS-9571; In situ cell extraction; TRIM24