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.

Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.

Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.

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.

Disrupting the interaction of BRD4 with diacetylated Twist suppresses tumorigenesis in basal-like breast cancer.

Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we report a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a "histone H4-mimic" GK-X-GK motif that is diacetylated by Tip60. The diacetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructing an activated Twist/BRD4/P-TEFb/RNA-Pol II complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.

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.

Lipid-bound factor Xa regulates tissue factor activity.

The activation of coagulation factor X by tissue factor (TF) and coagulation factor VIIa (VIIa) on a phospholipid surface is thought to be the key step in the initiation of blood coagulation. In this reaction, the product, fXa, is transiently and reversibly bound to the TF-VIIa enzyme complex. This in effect leads to a probabilistic inhibition of subsequent fX activations; a new fX substrate molecule cannot be activated until the old fXa molecule leaves. In this study, we demonstrate that benzamidine and soybean trypsin inhibitor-conjugated Sepharose beads, which bind fXa and sequester it away from the reaction, serve to enhance fX activation by the TF-VIIa complex. Thus, removal of fXa from the reactive zone, by either flow, fXa sequestration, or binding to distant lipid surfaces, can serve to enhance the levels of TF-VIIa activity. Using resonance energy transfer, we found the dissociation constants of fX and fXa for 100 nm diameter phospholipid vesicles to be on the order of 30-60 nM, consistent with previous measurements employing planar lipid surfaces. On the basis of the measurements of binding of fXa to phospholipid surfaces, we demonstrate that the rates of fX activation by the TF-VIIa complex under a variety of experimental conditions depend inversely on the amount of product (fXa) bound to the TF-phospholipid surface. These data support an inhibitory role for the reaction product, fXa, and indicate that models previously employed in understanding this initial coagulation reaction must now be re-evaluated to account for both the product occupancy of the phospholipid surface and the binding of the product to the enzyme. Moreover, the inhibitory properties of fXa can be described on the basis of the estimated surface density of fXa molecules on the TF-phospholipid surface.

Phospholipid surfaces regulate the delivery of substrate to tissue factor:VIIa and the removal of product.

For many years, the essential role of tissue factor (TF) in coagulation and thrombogenesis has been recognized. The catalytic complex of TF and VIIa (TF:VIIa) is membrane-bound whereas its substrate, factor X (FX), is distributed between a phospholipid-bound fraction and one that is in true solution in 3-dimensional space. This complicates analytical solutions for the kinetic mechanisms describing this reaction because dimensionality must be preserved. We believe that, at the time of activation, FX is simultaneously bound to TF:VIIa and the phospholipid surface. The hydrolysis of a peptide bond activates FX and the product, Xa, is yet bound to the catalytic complex in a manner such that it must leave before a new molecule of X encounters the complex. This means that, in principle, the classically defined Vmax does not apply because on a surface, infinite substrate and its attendant infinite collision frequency do not apply. We show that increasing the lipid surface area available to each TF:VIIa increases the apparent k(cat) and that it approaches a maximum asymptotically, exhibiting a K(1/2) at a 40 nm lipid radius. Notably, this is of the same order as transient confinement zones that have been identified on the surface of living cells. We believe the increased lipid surface area allows the Xa to easily diffuse away from the enzyme complex along the 2D lipid surface, thereby allowing new substrate to approach the enzyme and minimizing collisions between the product and the enzyme complex (product inhibition). Thus, after Xa leaves the vicinity of the enzyme, a new FX molecule is able to bind TF:VIIa and the rate at which this complex forms cannot exceed the leaving rate of Xa from the TF:VIIa and phospholipid sites. Thus, this parameter is of critical interest. Starting with the off-rate of Xa from appropriate phospholipid surfaces, we note that the literature values differ by a factor of approximately 500. Using energy transfer techniques between 30% phosphatidylserine/70% phosphatidylcholine vesicles and human F.Xa, we measured this off rate and found it agrees closely with the Biacore generated data. We have determined the binding parameters of Xa to vesicles and a continuous supported bilayer. Our data are in excellent agreement with the data derived using a lipid coated Biacore chip.

Phospholipid regulates the activation of factor X by tissue factor/factor VIIa (TF/VIIa) via substrate and product interactions.

Although the phospholipid requirement for tissue factor (TF) activity has been well-established, the mechanism by which the surface regulates enzymatic activity remains unclear. We added phospholipid vesicles to already relipidated TF (30/70 PS/PC) and found that added lipid can both enhance and inhibit the rate of factor X (F.X) activation. Using active-site-inhibited F.Xa we demonstrate that F.Xa is a more potent inhibitor of TF/VIIa at lower lipid concentrations, and that this inhibition is attributable to high surface occupancy by F.Xa near the enzyme. We also find that exactly twice as many F.Xa molecules are bound to a lipid surface at saturation as F.X, and that a dimer model of F.Xa binding to the lipid can account for the experimentally observed, preferential binding of F.Xa (compared to F.X) to phospholipid surfaces. We manipulated the amount of phospholipid available to each TF molecule by controlling vesicle size and the number of TF molecules per vesicle and found that, as the 2D radius of phospholipid available to each TF molecule was increased, the observed k(cat) increased hyperbolically toward a maximum or "true k(cat)". At a 2D lipid radius of approximately 37 nm, the observed k(cat) was 50% of the "true k(cat)". Thus, phospholipid surface serves as a conduit for F.X presentation and F.Xa removal, and the rate at which F.Xa leaves the vicinity of the enzyme, either by lateral diffusion or desorption from the surface, regulates the rate of F.X activation. We argue that these findings require reevaluation of existing models of coagulation.

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics.

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.

Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.

Quantitative analysis of tryptophan analogue incorporation in recombinant proteins.

Three different methods to quantitate tryptophan (Trp) analogue incorporation into recombinant proteins are described: first, spectroscopic analysis based on a linear combination of the absorption spectra of the aromatic residues in the denatured Trp-containing or analogue-substituted protein; second, chromatographic separation of analogue-substituted and Trp-containing proteins by HPLC; and third, mass spectrum analysis of the mixture of analogue-substituted and Trp-containing proteins. An accurate estimate of analogue incorporation in single-Trp proteins can be obtained directly by either analysis of the absorption spectrum or HPLC chromatography. While analysis of the absorption spectrum or HPLC chromatogram can provide an assessment of the average level of analogue incorporation for proteins that contain two or more Trp residues, mass spectroscopy analysis of peptides generated by protease digestion and separated by HPLC provides a general method for a complete quantitative description of the distribution of analogue incorporation. The more complex analysis by mass spectroscopy becomes important for multi-Trp proteins because the distribution of analogue versus Trp-containing polypeptide chains may not be the same as that predicted on the basis of average level of analogue incorporation.