Acetyl-lysine Binding Site Research Articles

Structural and biochemical insights into purine-based drug molecules in hBRD2 delineate a unique binding mode opening new vistas in the design of inhibitors of the BET family.

The bromodomain and extra-terminal (BET) family proteins, which are involved in chromatin function, have been shown to be promising drug targets in several pathological conditions, including cancer and inflammation. There is considerable interest in the development of BET inhibitors with novel scaffolds to modulate the epigenesis of such diseases. Here, high-resolution crystal structures of the purine class of FDA-approved drugs (theophylline, doxophylline and acyclovir) and non-FDA-approved compounds (3-methyl-7-propylxanthine and theobromine) complexed with hBRD2 bromodomains BD1 and BD2 are reported. Remarkably, a new binding site is exhibited by stacking the compounds against the WPF shelf of BD1 and BD2. This serendipitous binding, in addition to the known acetyl-lysine binding site, sufficiently anchors the ligands in the solvent-exposed region. In addition, slight variations in the lipophilicity of these molecules significantly affected the in vitro binding affinity and selectivity towards BD1 compared with BD2. This idiosyncratic binding provides a new structural framework to link these sites for the development of next-generation inhibitors of the BET family.

Bromodomain Interactions with Acetylated Histone 4 Peptides in the BRD4 Tandem Domain: Effects on Domain Dynamics and Internal Flexibility.

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.

Toward more potent imidazopyridine inhibitors of Candida albicans Bdf1: Modeling the role of structural waters in selective ligand binding.

Novel agents to treat invasive fungal infections are urgently needed because the small number of established targets in pathogenic fungi makes the existing drug repertoire particularly vulnerable to the emergence of resistant strains. Recently, we reported that Candida albicans Bdf1, a bromodomain and extra-terminal domain (BET) bromodomain with paired acetyl-lysine (AcK) binding sites (BD1 and BD2) is essential for fungal cell growth and that an imidazopyridine (1) binds to BD2 with selectivity versus both BD1 and human BET bromodomains. Bromodomain binding pockets contain a conserved array of structural waters. Molecular dynamics simulations now reveal that one water molecule is less tightly bound to BD2 than to BD1, explaining the site selectivity of 1. This insight is useful in the performance of ligand docking studies to guide design of more effective Bdf1 inhibitors, as illustrated by the design of 10 new imidazopyridine BD2 ligands 1a-j, for which experimental binding and site selectivity data are presented.

Bivalent BET Bromodomain Inhibitors Confer Increased Potency and Selectivity for BRDT via Protein Conformational Plasticity.

Bromodomain and extraterminal domain (BET) proteins are important regulators of gene transcription and chromatin remodeling. BET family members BRD4 and BRDT are validated targets for cancer and male contraceptive drug development, respectively. Due to the high structural similarity of the acetyl-lysine binding sites, most reported inhibitors lack intra-BET selectivity. We surmised that protein-protein interactions induced by bivalent inhibitors may differ between BRD4 and BRDT, conferring an altered selectivity profile. Starting from nonselective monovalent inhibitors, we developed cell-active bivalent BET inhibitors with increased activity and selectivity for BRDT. X-ray crystallographic and solution studies revealed unique structural states of BRDT and BRD4 upon interaction with bivalent inhibitors. Varying spacer lengths and symmetric vs unsymmetric connections resulted in the same dimeric states, whereas different chemotypes induced different dimers. The findings indicate that the increased intra-BET selectivity of bivalent inhibitors is due to the differential plasticity of BET bromodomains upon inhibitor-induced dimerization.

Design, synthesis, and anticancer evaluation of ammosamide B with pyrroloquinoline derivatives as novel BRD4 inhibitors

Bromodomain-containing protein 4 (BRD4), which is a member of the bromodomain and extra-terminal domain (BET) family, plays an important role in the regulation of gene expression as the “reader” of epigenetic regulation. BRD4 has become a promising target to treat cancer, because the up-regulation of BRD4 expression is closely associated with the occurrence and development of various cancers. At present, several BRD4 inhibitors are in clinical trials for cancer therapy, but no BRD4 inhibitors are on the market. Here, we designed and synthesized a series of compounds bearing pyrrolo[4,3,2-de]quinolin-2(1H)-one scaffold through structural modification of natural products ammosamide B, which is a natural pyrroloquinoline derivative reported for its potential antitumor activity. All target compounds were evaluated for their BRD4 BD1 inhibition activities via the protein thermal shift assays or AlphaSceen assay. The representative compound 49 showed potent activity (IC50 = 120 nM). The co-crystal of compound 49 with BRD4 BD1 was solved to study the structure activity relationship, which showed that 49 could combine with the acetyl lysine binding site and formed a hydrogen bond with the conserved residue Asn140. The results demonstrate that compound 49 is worthy of further investigation as a promising BRD4 inhibitor.

Discovering and Exploiting Selectivity in Bromodomain Recognition of Epigenetic Lysine Acylation

Bromodomains are promising epigenetic targets in cancer, diabetes, and other inflammatory disorders, and bromodomain inhibitors are in clinical trials for multiple cancers and cardiovascular disease. Bromodomains bind sites of lysine acetylation on histones and transcription factors. Interestingly, nearly half of human bromodomains exist in tandem with at least one other bromodomain on a single polypeptide. These tandem bromodomain-containing proteins are localized disproportionately at super-enhancers, genomic regions with large clusters of elements that enhance gene transcription. The basis of this localization is unknown but important given that super-enhancers are enriched at loci with oncogenic potential. Here, we show that tandem bromodomains can scaffold nucleosomes in an acetylation-dependent manner using biochemical, structural, biophysical, cellular, bioinformatic, and other computational approaches. This scaffolding is consistent with our hypothesis that tandem bromodomains enact acetylation-dependent reorganization of chromatin; for instance, joining promotors with their corresponding distal enhancers to facilitate enhancer-driven oncogenic gene transcription. This mechanism of chromatin reorganization is paradigm-shifting with broad implications on the cellular role of tandem bromodomain proteins. We also hypothesize that changes in acyl-CoA metabolism induce distinct lysine acylations on histones that are selectively recognized by bromodomains, thereby linking metabolism to transcription. Yet, the broader lysine acylation binding specificity of bromodomains is only beginning to be characterized. Here, we address this knowledge gap by determining how metabolically-derived acylations tune bromodomain binding to histones using biophysical, structural biology, and proteomic techniques. To aid mechanistic inquiries, we are removing a critical barrier in the study of bromodomain biology: the lack of inhibitors and chemical probes that selectively target individual bromodomains. This lack of selectivity may be responsible for the dose-limiting toxicity reported in clinical trials of existing bromodomain inhibitors. Here, we report the identification of a novel ligandable site on a bromodomain outside the acetyl-lysine binding site and novel inhibitors that target individual bromodomains. These chemical tools will be necessary to distinguish the differential activities of bromodomain-containing proteins in cell and rodent models of disease and may lead to therapeutics targeting the bromodomain axis in cancer, diabetes, and cardiovascular disease. Overall, the insight gained through these studies will pave the way for safer and more effective therapies targeting bromodomains.

Design, Synthesis, and Biological Evaluation of 8-Mercapto-3,7-Dihydro-1H-Purine-2,6-Diones as Potent Inhibitors of SIRT1, SIRT2, SIRT3, and SIRT5

Sirtuins (SIRT1-7) are a family of NAD+-dependent deacetylases. They regulate many physiological processes and play important roles in inflammation, diabetes, cancers, and neurodegeneration diseases. Sirtuin inhibitors have potential applications in the treatment of neurodegenerative diseases and various cancers. Herein, we identified new sirtuin inhibitors based on the scaffold of 8-mercapto-3,7-dihydro-1H-purine-2,6-dione. To elucidate the inhibitory mechanism, the binding modes of the inhibitors in SIRT3 were established by molecular docking, showing that the inhibitors occupy the acetyl lysine binding site and interact with SIRT3, mainly through hydrophobic interactions. The interactions were validated by site-directed mutagenesis of SIRT3 and structure–activity relationship analysis of the inhibitors. Consistently, enzyme kinetic assays and microscale thermophoresis showed that these compounds are competitive inhibitors to the acetyl substrate, and mix-type inhibitors to NAD+. Furthermore, we demonstrated that the compounds are potent SIRT1/2/3/5 pan-inhibitors. This study provides novel hits for developing more potent sirtuin inhibitors.

Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.

BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to nonhomologous cysteine residues within the C-terminal BRD4 bromodomain (BRD4-BD2), we performed a midthroughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify BRD4. Subsequent mass spectrometry, NMR, and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to BRD4 among human bromodomains. This site is orthogonal to the BRD4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays nor an acetylated histone peptide in AlphaScreen assays. Finally, we tethered our top-performing covalent fragment to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace BRD4 from chromatin.

In silico design and molecular basis for the selectivity of Olinone toward the first over the second bromodomain of BRD4.

Bromodomains (BrDs), a conserved structural module in chromatin-associated proteins, are well known for recognizing ε-N-acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem BrD containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow-up of our previous work, we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydropyrido indole-based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations, and conformational analyses of the apo-BRD4-BrD1|2 and BRD4-BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Val49 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl-lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity toward BrD1 over BrD2. Altogether, our computer-aided molecular design techniques can effectively guide the development of small-molecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically improved derivatives.

Substrate Affinity and Specificity of the ScSth1p Bromodomain Are Fine-Tuned for Versatile Histone Recognition

Bromodomains recognize a wide range of acetylated lysines in histones and other nuclear proteins. Substrate specificity is critical for their biological function and arises from unique acetyl-lysine binding sites formed by variable loop regions. Here, we analyzed substrate affinity and specificity of the yeast ScSth1p bromodomain, an essential component of the "Remodels the Structure of Chromatin" complex, and found that the wild-type bromodomain preferentially recognizes H3K14ac and H4K20ac peptides. Mutagenesis studies-guided by our crystal structure determined at 2.7-Å resolution-revealed loop residues Ser1276 and Trp1338 as key determinants for such interactions. Strikingly, point mutations of each of these residues substantially increased peptide binding affinity and selectivity, respectively. Our data demonstrate that the ScSth1p bromodomain is not optimized for binding to an individual acetylation mark, but fine-tuned for interactions with several such modifications, consistent with the versatile and multivalent nature of histone recognition by reader modules such as bromodomains.

In silico study directed towards identification of novel high-affinity inhibitors targeting an oncogenic protein: BRD4-BD1

ABSTRACTBromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) family of proteins. It epigentically regulates the transcription of growth-promoting genes and has become an attractive target for the development of anticancer and anti-inflammatory agents. In the current study, we performed an in silico screening of a small-molecule chemical library against the acetyl–lysine binding site of the first bromodomain (BD1) in BRD4 protein. Potential inhibitors identified through virtual screening were further studied through molecular dynamics simulations, water entrapment analysis and Molecular Mechanics (MM)/Poisson–Boltzmann surface area (PBSA) binding free energy calculations. Many of the identified compounds exhibit better G-score (–11.64 kcal∙mol−1 to –10.31 kcal∙mol−1) and predicted binding affinity (–9.66 kcal∙mol−1 to –6.63 kcal∙mol−1) values towards BRD4-BD1 than that of the reference compound (+)-JQ1. Molecular dynamics simulation studies show that in free-form BRD4 the reported conserved water molecules are not retained at their specific positoins due to flexibiliy in the ZA-loop. In BRD4–ligand complexes the number and positions of conserved water molecules depends on the bound ligand. Identified potential inhibitors bind stably at the acetyl–lysine binding pocket of BRD4 and form direct and water-mediated hydrogen bonds with higher occupancy which may contribute to ligand specificity towards BRD4-BD1. Further, through MM/PBSA we calculated the binding free energies of selected compounds, which shows that they have comparable energies to that of (+)-JQ1, while NSC744713 shows better binding free energy.

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains.

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.

Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability.

Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding.

Twenty Crystal Structures of Bromodomain and PHD Finger Containing Protein 1 (BRPF1)/Ligand Complexes Reveal Conserved Binding Motifs and Rare Interactions

BRPF1 plays a scaffolding role in transcription. We report on fragment screening by high-throughput docking to the BRPF1 bromodomain which resulted in six chemotypes with very favorable ligand efficiency (0.45-0.50 kcal/mol per non-hydrogen atom). Twenty crystal structures of BRPF1/ligand complexes show structural conservation in the acetyllysine binding site, common binding motifs, and unusual interactions (e.g., the replacement of a conserved water molecule). The structural information is useful for the design of chemical probes.

The "Gatekeeper" Residue Influences the Mode of Binding of Acetyl Indoles to Bromodomains.

Small-molecule hits for the bromodomains of CREBBP and BAZ2B have been identified by scaffold hopping followed by docking of a set of ∼200 compounds containing the acetyl indole scaffold. Chemical synthesis of nearly 30 derivatives has resulted in ligands of representatives of three subfamilies of human bromodomains with favorable ligand efficiency. The X-ray crystal structures of three different bromodomains (CREBBP, BAZ2B, and BRPF1b) in complex with acetyl indole derivatives reveal the influence of the gatekeeper residue on the orientation of small-molecule ligands in the acetyl lysine binding site.

Abstract 3643: Targeting the acetyl-lysine binding site of BRD4 with dual nanomolar BET-JAK2 inhibitors: A new anticancer therapeutic strategy

Abstract Bromodomain (BRD)-containing proteins are essential for the recognition of acetylated lysine residues of histones during transcriptional activation. The BRD-containing proteins have emerged as promising drug targets for a number of diseases, including many cancers, that are characterized by changes in the epigenetic cell signature. Recent reports have shown that targeting BRD4 with small molecules may represent a new way to treat prostate and breast cancer, acute myeloid leukemia and melanoma. We have identified many type 1 and type 2 kinase inhibitors which also inhibit BRD4 by robotic co-crystallization screening of kinase inhibitor libraries against BRD4. In each case the co-crystal structure unambiguously revealed the inhibitor bound to the acetyl lysine site of BRD4-1. The identified BRD4 ligands were subjected to differential scanning fluorimetry (DSF) and AlphaScreen assay to assess their binding and inhibitory potentials against BRD4. As shown previously for other BRD-inhibitor-protein complexes, the melting temperatures of BRD4-kinase inhibitor complexes were logarithmically proportional to their IC50 values. We now report the design, synthesis, structural analysis and biological evaluation of next-generation nanomolar BET-selective and nanomolar dual-activity BET-JAK2 inhibitors, based on the initial co-crystallization screening hits. Structure activity relationships were developed using both DSF and co-crystallization of the ligands with BRD4, to assess binding potential and binding modes, respectively. We report initial evaluation of the anticancer potential of compounds possessing dual potent BRD4 and JAK2 inhibitory properties. In addition to myeloma cell lines, this includes the evaluation of dual BRD4-JAK2 inhibitor compounds against JAK2-driven myeloproliferative neoplasm cell lines and primary cells from patients. Citation Format: Steven Gunawan, Ayaz Muhammad, Stuart W. J. Ember, Jin-Yi Zhu, Rebecca A. Jacobsen, Norbert Berndt, Que T. Lambert, Gary W. Reuther, Harshani R. Lawrence, Ernst Schonbrunn, Nicholas J. Lawrence. Targeting the acetyl-lysine binding site of BRD4 with dual nanomolar BET-JAK2 inhibitors: A new anticancer therapeutic strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3643. doi:10.1158/1538-7445.AM2015-3643

Acetyl-lysine Binding Site of Bromodomain-Containing Protein 4 (BRD4) Interacts with Diverse Kinase Inhibitors

Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50 = 25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors.

Structure-guided design of potent diazobenzene inhibitors for the BET bromodomains.

BRD4, characterized by two acetyl-lysine binding bromodomains and an extra-terminal (ET) domain, is a key chromatin organizer that directs gene activation in chromatin through transcription factor recruitment, enhancer assembly, and pause release of the RNA polymerase II complex for transcription elongation. BRD4 has been recently validated as a new epigenetic drug target for cancer and inflammation. Our current knowledge of the functional differences of the two bromodomains of BRD4, however, is limited and is hindered by the lack of selective inhibitors. Here, we report our structure-guided development of diazobenzene-based small-molecule inhibitors for the BRD4 bromodomains that have over 90% sequence identity at the acetyl-lysine binding site. Our lead compound, MS436, through a set of water-mediated interactions, exhibits low nanomolar affinity (estimated Ki of 30-50 nM), with preference for the first bromodomain over the second. We demonstrated that MS436 effectively inhibits BRD4 activity in NF-κB-directed production of nitric oxide and proinflammatory cytokine interleukin-6 in murine macrophages. MS436 represents a new class of bromodomain inhibitors and will facilitate further investigation of the biological functions of the two bromodomains of BRD4 in gene expression.

Structured Water Molecules in the Binding Site of Bromodomains Can Be Displaced by Cosolvent

Bromodomains are α-helical bundles of approximately 110 residues that recognize acetylated lysine side chains mainly on histone tails. Bromodomains are known to play an important role in cancer and inflammation, and as such, significant efforts are being made to identify small-molecule inhibitors of these epigenetic reader proteins. Here, explicit solvent molecular dynamics (MD) simulations of two bromodomains (BAZ2B and CREBBP) are used to analyze the water molecules that seem to be conserved at the bottom of the acetyl-lysine binding site in most crystal structures of bromodomains. The MD runs suggest that the occupancy of the structured water molecules is influenced by conformational transitions of the loop that connects helices Z and A. Additional simulations in the presence of 50 molecules of cosolvent (i.e., 440 mM of dimethylsulfoxide, methanol, or ethanol) indicate that some of the structured water molecules can be displaced transiently. The residence time in the acetyl-lysine binding site is calculated to be about 1 ns, 2-5 ns, and 10-30 ns for methanol, ethanol, and dimethylsulfoxide, respectively, while the affinity of the three cosolvents for BAZ2B and CREBBP is in the range of 50-500 mM. The results described have implications for ligand design, suggesting that only structured water molecules that do not exchange with cosolvent should be maintained in crystal structures used for docking campaigns, and that hydroxy substituents should be incorporated in the ligand so as to map the structured water molecules replaced by (m)ethanol.

Druggability Analysis and Structural Classification of Bromodomain Acetyl-lysine Binding Sites

Bromodomains are readers of the epigenetic code that specifically bind acetyl-lysine containing recognition sites on proteins. Recently the BET family of bromodomains has been demonstrated to be druggable through the discovery of potent inhibitors, sparking an interest in protein–protein interaction inhibitors that directly target gene transcription. Here, we assess the druggability of diverse members of the bromodomain family using SiteMap and show that there are significant differences in predicted druggability. Furthermore, we trace these differences in druggability back to unique amino acid signatures in the bromodomain acetyl-lysine binding sites. These signatures were then used to generate a new classification of the bromodomain family, visualized as a classification tree. This represents the first analysis of this type for the bromodomain family and can prove useful in the discovery of inhibitors, particularly for anticipating screening hit rates, identifying inhibitors that can be explored for lead hopping approaches, and selecting proteins for selectivity screening.