Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.


Reagents.
All reagents for which the synthesis is not described below are either commercially available or were synthesized according to literature procedures. All final products were at least 95% pure, as determined by analytical HPLC.

5-Bromo-2-chloro-4-hydroxybenzonitrile 2
To a stirred solution of 2-chloro-4-hydroxybenzonitrile 1, 5.0 g (32.5 mmol) in acetonitrile (100 mL) was added dropwise trifluoromethanesulfonic acid, 3.17 mL (35.8 mmol, 1.1 eq.) at -30 °C. After 10 min at -30 °C, N-bromosuccinimide, 8.11 g (45.6 mmol, 1.4 eq.) was added and the resulting mixture was stirred at r.t. for 18 h. Most of the solvent was removed under reduced pressure and the residue was partitioned between aq. sat. sodium hydrogen carbonate and ethyl acetate. The layers were separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: cyclohexane/ethyl acetate, 4/1) to give the desired product, 1.26 g (16%) as a white solid, and a mixture fraction containing 50% of the desired product together with the starting material and a regioisomer, 1.
The resulting suspension was then allowed to warm to r.t., stirred for 1 h and then heated at 90 °C for 3 h. After cooling to r.t., the mixture was concentrated in vacuum and the resulting purple

Library Design
The 11 DNA-encoded chemical libraries (DELs) (3) amounting to 65 billion compounds tested were generated using a "split-and-pool" strategy (4) in three synthetic cycles. The single sublibrary of 110-million formyl acid derivatives leading to BAY-850 was prepared as follows: First, a headpiece comprised of a short DNA oligonucleotide with a primary aliphatic amine was split into 300 wells and in each well a unique Fmoc amino acid was acylated onto the primary amine and a unique encoding oligonucleotide tag was ligated onto the headpiece (cycle B). This step was followed by pooling of the 300 conjugates and the subsequent splitting of this mixture into 150 wells in which unique individual formyl acids were installed by acylation and encoded followed by pooling and splitting into 2300 wells in which unique amines were installed by reductive amination and encoded.

Affinity-Mediated Selection Protocol
The DNA-encoded chemical library and GST-ATAD2 were combined in solution and affinity- V) in order to find optimal conditions to obtain a narrow peak shape without disrupting the noncovalent protein-ligand interactions. Calibration of the instrument was performed using sodium iodide clusters (2 µg/µL in 2-propanol:water; 1:1) up to m/z 3500. In order to improve the signal-to-noise ratio mass spectra were accumulated for 1-2 min. The instrument was controlled and data were evaluated using MassLynx software version 4.1.

Size Exclusion Chromatography (SEC)
To assess ATAD2 bromodomain dimerisation by BAY-850, ATAD2 and GST-ATAD2 preparations were each equally separated into two samples; one of which was incubated with BAY-850 (50 mM DMSO stock solution) in a molar ratio of 1:1, while the other was incubated with the same amount of DMSO instead of compound (apo samples). The samples were then analysed by SEC using 16/600 HiLoad Superdex200 prep grade column and buffer B. Column calibration was performed using commercial gel filtration standards (Biorad).

Cell-based Assays
Cell cultures, proliferation inhibition and GI50 determination Cells were cultivated at 37°C and 5% CO2 in different mediums; HMEC in Mammary The normalized curves were fitted to double exponential curve and half recovery time was calculated using GraphPad Prism 6. For each groups at least 15 images were analyzed and student t-test was used to determine significant differences between the groups.
Caco2 cells purchased from the DSMZ were seeded at a density of 4.5 x 104 cells per well and grown for 15 d in DMEM with typical supplements. Cells were kept at 37 °C in a humidified 5% CO2 atmosphere. Before the permeation assay was run, the culture medium was replaced by a       .  Table   S1.