The bromodomain containing proteins TRIM24 (Tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethyl benzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor, 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

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.

Here we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg 2؉ ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/ structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases.With the emergence of antibiotic resistance worldwide, the search for new antibacterial compounds has become increasingly important (1). NADKs 3 represent an attractive and novel drug target for antibiotic discovery. Indeed, NADKs (EC 2.7.1.23) were recently shown to be essential for growth in many bacteria (2-4) including various human pathogens such as Mycobacterium tuberculosis (5), Staphylococcus aureus (6), Streptococcus pneumoniae (7), Salmonella enterica (8), and Pseudomonas aeruginosa. 4 NADKs are ubiquitous enzymes involved in the last step of the biosynthesis of NADP catalyzing the transfer of a phosphate group on the substrate NAD to produce NADP in the presence of both ATP and Mg 2ϩ . NADKs are also involved in the tight regulation of the NADH/NADPH ratio (8). Both NAD(P) and NAD(P)H are essential cofactors for a large number of enzymes involved in various metabolic pathways such as oxidoreductases. This activity of NADKs has been well known for decades, but their genes were cloned only recently (9), subsequently leading to rapid identification of NADK orthologs in many organisms from bacteria, Archaea, plants, and human (Pfam PF01513). Bacterial and human enzymes show significant functional and sequence divergences, and the NADK activity in human cells is low (10).However, despite recent advances in the characterization of NADKs, the reaction mechanism is not well understood, and some original features of NADKs remain unexplained. First, during catalysis the phosphorylation occurs specifically on 2Ј-hydroxyl of the adenosine moiety of NAD and not on the neighboring 3Ј-hydroxyl. Second, the specificity for the catalytic di-cation is low (Mn 2ϩ , Ca 2ϩ , Zn , and Fe 2ϩ appear equivalent to Mg 2ϩ ) (11), whereas it is generally high in other well characterized kinases. Furthermore, the di-cation binding motif is yet unknown. Finally, most NADKs use as a phospho-donor various nucleoside triphosphates (9, 12, 13). Polyphosphates can also ...