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Fragment-based screening of the bromodomain of ATAD2.

ABSTRACT: Cellular and genetic evidence suggest that inhibition of ATAD2 could be a useful strategy to treat several types of cancer. To discover small-molecule inhibitors of the bromodomain of ATAD2, we used a fragment-based approach. Fragment hits were identified using NMR spectroscopy, and ATAD2 was crystallized with three of the hits identified in the fragment screen.

SUBMITTER: Harner MJ

PROVIDER: S-EPMC4255743 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Fragment-based screening of the bromodomain of ATAD2.

Harner Mary J MJ Chauder Brian A BA Phan Jason J Fesik Stephen W SW

Journal of medicinal chemistry 20141111 22

Cellular and genetic evidence suggest that inhibition of ATAD2 could be a useful strategy to treat several types of cancer. To discover small-molecule inhibitors of the bromodomain of ATAD2, we used a fragment-based approach. Fragment hits were identified using NMR spectroscopy, and ATAD2 was crystallized with three of the hits identified in the fragment screen. ...[more]

PMID: 25314628

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Chemical synthesis of the ATAD2 bromodomain.

Project description:Due to the emerging importance of the bromodomain binding region in the study of epigenetic effectors and the vast implications for a wide variety of human disease, the bromodomain region of human ATPase family AAA+ (ATPases associated with diverse cellular activities) domain-containing protein 2 (ATAD2) was targeted for chemical synthesis. The ATAD2 bromodomain (130 aa) was divided into five strategic fragments to be assembled using native chemical ligation with a focus on maximal convergency and efficiency. The fragments were assembled with one cysteine and three thioleucine ligations, unveiling the native alanine and leucine amino acids at the ligation points following metal-free dethiylation. Synthetic highlights of the study are a photolabile dimethoxynitrobenzyl-protected glutamic acid side chain used to impede hydrolysis of the C-terminal Glu-thioester, a thiazolidine-protected thioleucine, and an efficient assembly of three fragments in a single reaction vessel with dual-mode kinetic-standard chemical ligation. With a focus on material throughput and convergency, the five peptide fragments were assembled into the native ATAD2 bromodomain region with a total of three HPLC events in 8% overall yield from the fragments.

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A Chemical Probe for the ATAD2 Bromodomain.

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Discovery of novel ATAD2 bromodomain inhibitors that trigger apoptosis and autophagy in breast cells by structure-based virtual screening.

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Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach.

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Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain.

Project description:The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

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Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain.

Project description:The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential biomarker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Site-directed mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman's assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligand-binding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module.

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Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening.

Project description:PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with Kd values from 45 μM to >2 mM. Structure-activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.

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Hitting a Moving Target: Simulation and Crystallography Study of ATAD2 Bromodomain Blockers.

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