Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α).

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,ß) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.

BET N-terminal bromodomain inhibition selectively blocks Th17 cell differentiation and ameliorates colitis in mice.

T-helper 17 (Th17) cells have important functions in adaptor immunity and have also been implicated in inflammatory disorders. The bromodomain and extraterminal domain (BET) family proteins regulate gene transcription during lineage-specific differentiation of naïve CD4+ T cells to produce mature T-helper cells. Inhibition of acetyl-lysine binding of the BET proteins by pan-BET bromodomain (BrD) inhibitors, such as JQ1, broadly affects differentiation of Th17, Th1, and Th2 cells that have distinct immune functions, thus limiting their therapeutic potential. Whether these BET proteins represent viable new epigenetic drug targets for inflammatory disorders has remained an unanswered question. In this study, we report that selective inhibition of the first bromodomain of BET proteins with our newly designed small molecule MS402 inhibits primarily Th17 cell differentiation with a little or almost no effect on Th1 or Th2 and Treg cells. MS402 preferentially renders Brd4 binding to Th17 signature gene loci over those of housekeeping genes and reduces Brd4 recruitment of p-TEFb to phosphorylate and activate RNA polymerase II for transcription elongation. We further show that MS402 prevents and ameliorates T-cell transfer-induced colitis in mice by blocking Th17 cell overdevelopment. Thus, selective pharmacological modulation of individual bromodomains likely represents a strategy for treatment of inflammatory bowel diseases.

Small-molecule modulators of methyl-lysine binding for the CBX7 chromodomain.

Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes trimethylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. In this study, we report the discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show that a lead compound, MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. These small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.

Selective chemical modulation of gene transcription favors oligodendrocyte lineage progression.

Lysine acetylation regulates gene expression through modulating protein-protein interactions in chromatin. Chemical inhibition of acetyl-lysine binding bromodomains of the major chromatin regulators BET (bromodomain and extraterminal domain) proteins has been shown to effectively block cell proliferation in cancer and inflammation. However, whether selective inhibition of individual BET bromodomains has distinctive functional consequences remains only partially understood. In this study, we show that selective chemical inhibition of the first bromodomain of BET proteins using our small-molecule inhibitor, Olinone, accelerated the progression of mouse primary oligodendrocyte progenitors toward differentiation, whereas inhibition of both bromodomains of BET proteins hindered differentiation. This effect was target specific, as it was not detected in cells treated with inactive analogs and independent of any effect on proliferation. Therefore, selective chemical modulation of individual bromodomains, rather than use of broad-based inhibitors, may enhance regenerative strategies in disorders characterized by myelin loss such as aging and neurodegeneration.

Structural insights into acetylated-histone H4 recognition by the bromodomain-PHD finger module of human transcriptional coactivator CBP.

Bromodomain functions as the acetyl-lysine binding domains to regulate gene transcription in chromatin. Bromodomains are rapidly emerging as new epigenetic drug targets for human diseases. However, owing to their transient nature and modest affinity, histone-binding selectivity of bromodomains has remained mostly elusive. Here, we report high-resolution crystal structures of the bromodomain-PHD tandem module of human transcriptional coactivator CBP bound to lysine-acetylated histone H4 peptides. The structures reveal that the PHD finger serves a structural role in the tandem module and that the bromodomain prefers lysine-acetylated motifs comprising a hydrophobic or aromatic residue at -2 and a lysine or arginine at -3 or -4 position from the acetylated lysine. Our study further provides structural insights into distinct modes of singly and diacetylated histone H4 recognition by the bromodomains of CBP and BRD4 that function differently as a transcriptional coactivator and chromatin organizer, respectively, explaining their distinct roles in control of gene expression in chromatin.

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.

Down-regulation of NF-κB transcriptional activity in HIV-associated kidney disease by BRD4 inhibition.

NF-κB-mediated inflammation is the major pathology in chronic kidney diseases, including HIV-associated nephropathy (HIVAN) that ultimately progresses to end stage renal disease. HIV infection in the kidney induces NF-κB activation, leading to the production of proinflammatory chemokines, cytokines, and adhesion molecules. In this study, we explored selective inhibition of NF-κB transcriptional activity by small molecule blocking NF-κB binding to the transcriptional cofactor BRD4, which is required for the assembly of the productive transcriptional complex comprising positive transcription elongation factor b and RNA polymerase II. We showed that our BET (Bromodomain and Extra-Terminal domain)-specific bromodomain inhibitor MS417, designed to block BRD4 binding to the acetylated NF-κB, effectively attenuates NF-κB transcriptional activation of proinflammatory genes in kidney cells treated with TNFα or infected by HIV. MS417 ameliorates inflammation and kidney injury in HIV-1 transgenic mice, an animal model for HIVAN. Our study suggests that BET bromodomain inhibition, targeting at the proinflammatory activity of NF-κB, represents a new therapeutic approach for treating NF-κB-mediated inflammation and kidney injury in HIVAN.

Structural basis for substrate specificity and catalysis of human histone acetyltransferase 1.

Histone acetyltransferase 1 is the founding member of the histone acetyltransferase superfamily and catalyzes lysine acetylation of newly synthesized histone H4. Here we report a 1.9-Šresolution crystal structure of human histone acetyltransferase 1 in complex with acetyl coenzyme A and histone H4 peptide. The crystal structure reveals that the cofactor and the side chain of lysine 12 of histone H4 peptide are placed in the canyon between the central and C-terminal domains. Histone H4 peptide adopts a well-defined conformation and establishes an extensive set of interactions with the enzyme including invariant residues Glu64 and Trp199, which together govern substrate-binding specificity of histone acetyltransferase 1. Our structure-guided enzyme kinetic study further demonstrates a cumulative effect of the active-site residues Glu187, Glu276, and Asp277 on deprotonation of the ε-amino group of reactive Lys12 for direct attack of the acetyl group of the cofactor.

Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis.

5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.

Rational design of cyclic peptide modulators of the transcriptional coactivator CBP: a new class of p53 inhibitors.

The CREB binding protein (CBP) is a human transcriptional coactivator consisting of several conserved functional modules, which interacts with distinct transcription factors including nuclear receptors, CREB, and STAT proteins. Despite the importance of CBP in transcriptional regulation, many questions regarding the role of its particular domains in CBP functions remain unanswered. Therefore, developing small molecules capable of selectively modulating a single domain of CBP is of invaluable aid at unraveling its prominent activities. Here we report the design, synthesis, and biological evaluation of conformationally restricted peptides as novel modulators for the acetyl-lysine binding bromodomain (BRD) of CBP. Utilizing a target structure-guided and computer-aided rational design approach, we developed a series of cyclic peptides with affinity for CBP BRD significantly greater than those of its biological ligands, including lysine-acetylated histones and tumor suppressor p53. The best cyclopeptide of the series exhibited a K(d) of 8.0 µM, representing a 24-fold improvement in affinity over that of the linear lysine 382-acetylated p53 peptide. This lead peptide is highly selective for CBP BRD over BRDs from other transcriptional proteins. Cell-based functional assays carried out in colorectal carcinoma HCT116 cells further demonstrated the efficacy of this compound to modulate p53 stability and function in response to DNA damage. Our results strongly argue that these CBP modulators can effectively inhibit p53 transcriptional activity by blocking p53K382ac binding to CBP BRD and promoting p53 instability by changes of its post-translational modification states, a different mechanism than that of the p53 inhibitors reported to date.

Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b.

Histone lysine acetylation and methylation have an important role during gene transcription in a chromatin context. Knowledge concerning the types of protein modules that can interact with acetyl-lysine has so far been limited to bromodomains. Recently, a tandem plant homeodomain (PHD) finger (PHD1-PHD2, or PHD12) of human DPF3b, which functions in association with the BAF chromatin remodelling complex to initiate gene transcription during heart and muscle development, was reported to bind histones H3 and H4 in an acetylation-sensitive manner, making it the first alternative to bromodomains for acetyl-lysine binding. Here we report the structural mechanism of acetylated histone binding by the double PHD fingers of DPF3b. Our three-dimensional solution structures and biochemical analysis of DPF3b highlight the molecular basis of the integrated tandem PHD finger, which acts as one functional unit in the sequence-specific recognition of lysine-14-acetylated histone H3 (H3K14ac). Whereas the interaction with H3 is promoted by acetylation at lysine 14, it is inhibited by methylation at lysine 4, and these opposing influences are important during transcriptional activation of the mouse DPF3b target genes Pitx2 and Jmjd1c. Binding of this tandem protein module to chromatin can thus be regulated by different histone modifications during the initiation of gene transcription.

Biochemical profiling of histone binding selectivity of the yeast bromodomain family.

BACKGROUND: It has been shown that molecular interactions between site-specific chemical modifications such as acetylation and methylation on DNA-packing histones and conserved structural modules present in transcriptional proteins are closely associated with chromatin structural changes and gene activation. Unlike methyl-lysine that can interact with different protein modules including chromodomains, Tudor and MBT domains, as well as PHD fingers, acetyl-lysine (Kac) is known thus far to be recognized only by bromodomains. While histone lysine acetylation plays a crucial role in regulation of chromatin-mediated gene transcription, a high degree of sequence variation of the acetyl-lysine binding site in the bromodomains has limited our understanding of histone binding selectivity of the bromodomain family. Here, we report a systematic family-wide analysis of 14 yeast bromodomains binding to 32 lysine-acetylated peptides derived from known major acetylation sites in four core histones that are conserved in eukaryotes. METHODOLOGY: The histone binding selectivity of purified recombinant yeast bromodomains was assessed by using the native core histones in an overlay assay, as well as N-terminally biotinylated lysine-acetylated histone peptides spotted on streptavidin-coated nitrocellulose membrane in a dot blot assay. NMR binding analysis further validated the interactions between histones and selected bromodomain. Structural models of all yeast bromodomains were built using comparative modeling to provide insights into the molecular basis of their histone binding selectivity. CONCLUSIONS: Our study reveals that while not all members of the bromodomain family are privileged to interact with acetylated-lysine, identifiable sequence features from those that bind histone emerge. These include an asparagine residue at the C-terminus of the third helix in the 4-helix bundle, negatively charged residues around the ZA loop, and preponderance of aromatic amino acid residues in the binding pocket. Further, while bromodomains exhibit selectivity for different sites in histones, individual interactions are of modest affinity. Finally, electrostatic interactions appear to be a primary determining factor that guides productive association between a bromodomain and a lysine-acetylated histone.

Structural biology of human H3K9 methyltransferases.

UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

The structural basis of gas-responsive transcription by the human nuclear hormone receptor REV-ERBbeta.

Heme is a ligand for the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 A crystal structure of the REV-ERBbeta LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBbeta complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.

Scaffold-based discovery of indeglitazar, a PPAR pan-active anti-diabetic agent.

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARalpha, PPARgamma, and PPARdelta. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARgamma, to circumvent the clinically observed side effects of full PPARgamma agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARgamma. Compared with full PPARgamma-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).

Structural and kinetic differences between human and Aspergillus fumigatus D-glucosamine-6-phosphate N-acetyltransferase.

Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.

Structural analysis of CYP2R1 in complex with vitamin D3.

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D3. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B'-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D3 is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.