Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF

TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/TRIM24 dual inhibitor that bound with a KD of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data.


Constructs and protein expression and purification
cDNA encoding human TRIM24 from synthetic sources and used as templates to amplify the phd-bromodomain region using polymerase chain reaction (PCR) in the presence of Platinum® Pfx DNA polymerase (Invitrogen TM , UK). PCR products were purified (QIAquick PCR Purification Kit, Qiagen Ltd. UK) and further subcloned into a modified pET15 vector using NdeI/XhoI sites for expression as recombinant proteins with a His6 tag fused with small ubiquitin like modifier (SUMO)-1 fusion tag at the N terminus 1

Isothermal Titration Calorimetry
Calorimetric experiments were performed on a VP-ITC micro-calorimeter (MicroCal TM , LLC Northampton, MA). Protein solutions were buffer exchanged by gel filtration or dialysis into buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP)]. All measurements were carried out at 293.15 K while stirring at 286 rpm. The micro syringe was loaded with a protein solution of 250 µM, the compound solution was prepared at 20 µM and 2mL for the cell. All injections were performed using an initial injection of 2 µl followed by 34 injections of 8 µl with a duration of 16 sec per injection and a spacing of 240 sec between injection. The data were analysed with the MicroCal ORIGIN software package employing a single binding site model. The first data point was excluded from the analysis.
Thermodynamic parameters were calculated (∆G = ∆H -T∆S = -RTlnK B where ∆G, ∆H and ∆S are the changes in free energy, enthalpy and entropy of binding, respectively).

Temperature shift assays
Temperature shift assays have been carried out as described in Fedorov et al. 2 .

Fluorescence recovery after photobleaching (FRAP)
The TRIM24 FRAP assay has been carried out as described in Philpott et al. 3 . For the development of a BRPF1B FRAP assay we cloned a construct in which the PhD bromodomain was triplicated and fused to a C-terminal nuclear localization signal.
Experiments were carried out as described 3 .

Cytotoxicity assay
The

Data Collection and Structure solution
Crystals were cryo-protected using the well solution supplemented with additional 20 % glycerol and were flash frozen in liquid nitrogen. Data were collected at Diamond Light Source beamline I02 at a wavelength of 0.9795 Å. Indexing and integration was carried out using XDS 4 and scaling was performed with AIMLESS 5 . Initial phases were calculated by molecular replacement with PHASER 6 using the apo template structure 3O33.pdb. Unique and initial solutions were improved in a total of 50 cycles of automated protein chain tracing starting from existing model and computed using ARP/wARP 7 . Further manual building with COOT 8 and refinement against maximum likelihood target using REFMAC5 9 . Thermal motions were analysed using TLSMD and hydrogen atoms were included in late refinement cycles. PRODRG 10 was used to generate compound coordinates and cif files. All model validations were carried out using MolProbity 11 . Data collection and refinement statistics are compiled in Supplemental Table 1. The models and structure factors have been deposited with PDB accession codes: 4ZQL.pdb.

Inhibitors
All compounds have been purchased from ChemDiv.