Observed bromodomain flexibility reveals histone peptide- and small molecule ligand-compatible forms of ATAD2

Abstract

Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2A in cancer cells is associated with poor prognosis making the bromodomain of ATAD2A a promising epigenetic therapeutic target. In the development of an invitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2A bromodomain. Structural studies on apo-, peptide and smallmolecule-ATAD2A complexes (by co-crystalization) revealed the bromodomain adopts a “closed”, histone-compatible conformation, and a more “open” ligand-compatible conformation of the binding-site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2A binders which aided in the validation of the invitro screen and in the analysis of these conformational studies.

Fragments in bromodomain drug discovery

“…56 Despite this, the resulting dimethyl isoxazole hit 59 had Kd for ATAD2 of 200 μM (LE 0.28), comparable to that reported for the similar isoxazole fragments 7 and 14 to the BET bromodomain (see above) It also found that known inhibitors of other bromodomains were relatively weak against ATAD2.…”

“…Some fragments bind to ATAD2 and other bromodomains with unexpected displacement of some or all of the waters of the conserved network. 56 The optimisation of selective CREBBP inhibitors was assisted by a pocket created by the movement of a flexible arginine sidechain, which was not visible in the unbound crystal structure (Fig. In addition, the KAc-site environment is formed by the ZA and BC loop regions between α-helices, which have been seen to be mobile by solution NMR spectroscopy.…”

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

“…Equally encouraging are inhibitors that have now been identified for more difficult to drug bromodomains, 18 such as BAZ2B 19, 20 and ATAD2A. 21-23 Unlike the BET sub-family, which contain two bromodomains that primarily drive their binding and therefore function, a large proportion of the other BCPs contain auxiliary domains, many with diverse and sometimes unknown functions. These multi-domain containing BCPs can also be part of multi-protein complexes.…”

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain

“…15 One such bromodomain-containing protein is the ATPase family, AAA domain-containing protein 2 (ATAD2, UniProtKB Q6PL18, also called ANCCA/PRO 2000). 16 ATAD2 has been shown to recognize histone H4 that is acetylated at lysine 5 (H4K5ac) and lysine 12 (H4K12ac), 17,18 and has recently been proposed to function as a reader of newly synthesized H4K5acK12ac di-acetyllysine marks during DNA replication. 19 Several studies suggest a role for ATAD2 in the pathogenesis of cancer where it functions as a co-regulator of oncogenic transcription factors including E2F, 20,21 the estrogen receptorα, 16 the androgen receptor 22 , and MYC.…”

“…25,26,30,35,43 This pattern across multiple cancers makes the ATAD2 bromodomain an attractive target for cancer therapy, and several inhibitors are currently in development. 17,[47][48][49][50] To further investigate structural features of the ATAD2 bromodomain that contribute to histone ligand and small molecule inhibitor Invitrogen) cells (ATAD2 CO). An ATAD2 bromodomain C1101A mutant protein was created using site-directed mutagenesis with the QuikChange II kit (Agilent).…”