Hybrids of sugars and aromatics: A Pd-catalyzed modular approach to chromans and isochromans

Herein we describe the synthesis of highly substituted chromans and isochromans using carbohydrates as starting materials. The key step of our synthetic approach is the annelation of the benzene moiety via a highly efficient Pd-catalyzed domino reaction. This powerful approach led to a small library of highly substituted chromans and isochromans by making use of a variety of different diynes and bromoglycals. We investigated several Pd-catalysts in order to improve the yields and to enlarge the scope of the domino reaction. Furthermore, we elucidated the mechanistic picture of the reaction with isotope-labelling experiments. Most probably the reaction proceeds via an oxidative addition followed by two carbopalladation steps and a final cyclization reaction.     

Virtual screening,selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K_i of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K_i 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π–π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.     

Influence of spring and autumn phenological transitions on forest ecosystem productivity

We use eddy covariance measurements of net ecosystem productivity (NEP) from 21 FLUXNET sites (153 site-years of data) to investigate relationships between phenology and productivity (in terms of both NEP and gross ecosystem photosynthesis, GEP) in temperate and boreal forests. Results are used to evaluate the plausibility of four different conceptual models. Phenological indicators were derived from the eddy covariance time series, and from remote sensing and models. We examine spatial patterns (across sites) and temporal patterns (across years); an important conclusion is that it is likely that neither of these accurately represents how productivity will respond to future phenological shifts resulting from ongoing climate change. In spring and autumn, increased GEP resulting from an ‘extra’ day tends to be offset by concurrent, but smaller, increases in ecosystem respiration, and thus the effect on NEP is still positive. Spring productivity anomalies appear to have carry-over effects that translate to productivity anomalies in the following autumn, but it is not clear that these result directly from phenological anomalies. Finally, the productivity of evergreen needleleaf forests is less sensitive to phenology than is productivity of deciduous broadleaf forests. This has implications for how climate change may drive shifts in competition within mixed-species stands.     

A biomathematical model of human thrombopoiesis under chemotherapy

Intensification of cytotoxic chemotherapy enhances the outcome of several malignancies but is limited by haematotoxicity. While neutropenia and anaemia can be treated with supportive growth factor applications, thrombocytopenia remains a dose-limiting side effect due to the lack of clinically approved pharmaceutical growth factors. Hence, it is necessary to assess the degree of thrombocytopenia of newly designed intensified regimens in the planning phase of a clinical trial.We present a simple ordinary differential equations model of thrombopoiesis under chemotherapy which maps the dynamics of stem cells, CFU-Mk, megakaryocytes and platelets in spleen and circulation. Major regulatory cytokine of thrombopoiesis is thrombopoietin (TPO) whose production and consumption is explicitly modelled. TPO acts by increasing the number of mitoses of CFU-Mk and increasing the mass and maturation of megakaryocytes. Chemotherapy is modelled by a drug-dose and cell-stage specific acute cell loss.Most of the cell kinetic parameters of the model were taken from literature. Parameters regarding TPO regulation and chemotherapy toxicity were estimated by fitting the predictions of the model to time series data of platelets received from large clinical data sets of patients under seven different chemotherapies. We obtained a good agreement between model and data for all scenarios. Parameter estimates were biologically plausible throughout. For validation, the model also explains data of TPO and platelet dynamics after thrombopheresis taken from literature.We used the model to make clinically relevant predictions. Regarding thrombocytopenia we estimated that the CHOP regimen for the treatment of high-grade non-Hodgkin's lymphoma can be time-intensified to a cycle duration of 12 days while the time-intensified CHOEP regimen would result in severe cumulative toxicity. We conclude that our proposed model proved validity for both, different chemotherapeutic regimens and thrombopheresis as well. It is useful to assess the thrombocytopenic risk in the planning phase of a clinical trial.     

Toll-Like Receptor Agonists Synergistically Increase Proliferation and Activation of B Cells by Epstein-Barr Virus

Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cells in vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection.Epstein-Barr virus (EBV), a herpesvirus, is a very successful infectious agent: it establishes and maintains latent infection in >95% of human beings worldwide. This success is related to EBV''s varied strategies to maintain itself in its preferred host cell type, the B cell, by establishing different modes of latent infection (46). Some of these modes (latency modes 0, I, and II) are characterized by a resting B-cell phenotype and expression of a very limited set of EBV proteins (from none to four). In contrast, latency III involves the expression of at least 12 EBV latent-cycle gene products (10 proteins and 2 RNAs) (30, 31), which in their combined action profoundly alter the B cell''s appearance and behavior by inducing B-cell activation associated with proliferation, altered receptor expression, and cytokine secretion, as well as causing enhanced antigen presentation (31).In these various features, EBV infection of the latency III type resembles physiological activation of B cells in germinal centers even in its molecular details, because EBV closely mimics or constitutively activates some of the B cell''s main signaling pathways. Exogenous physiological signals leading to B-cell activation have been classified as “signal 1,” the stimulation of the B-cell receptor (BCR) by antigen binding; “signal 2,” the stimulation of CD40 by the CD40 ligand molecule, expressed on activated helper T cells; and “signal 3,” the stimulation of Toll-like receptors (TLRs) by microbial components, such as unmethylated CpG DNA, or their mimics. All three signals together are required for maximal proliferation of naive B cells (47). However, stimulation with TLR ligands alone, for example, CpG DNA, is sufficient to cause transient B-cell activation, including proliferation and induction of immune effector molecules such as CD86, a T-cell-costimulatory molecule (24). Additional immune effectors, the cytokines interleukin-6 (IL-6), IL-10, and IL-12, are induced when CpG stimulation is combined with strong CD40 stimulation (55).For primary infection of B cells, it is well established that EBV''s latent membrane proteins LMP2A (10, 39) and LMP1 (22) mimic signaling by the BCR and CD40, respectively. It is less clear whether and how EBV generates a potential signal 3 in the course of primary B-cell infection. A role of the TLR7 pathway has been proposed, based on the observation that EBV infection of naive B cells elevates the expression of TLR7 and its downstream signaling mediators (40). Additional mechanisms have recently been proposed to explain how EBV might trigger TLRs or other pattern recognition receptors in other cellular systems. For example, the Epstein-Barr virus-encoded small RNAs (EBERs) were described to trigger the retinoic acid-inducible gene I (RIG-I)-encoded protein, a receptor for various viral RNAs, in Burkitt''s lymphoma cells (48, 49). TLR2 signaling in monocytes is activated by binding of EBV particles to the cells (21) or by extracellular provision of EBV dUTPase (2).However, a physiologically relevant signal 3 need not originate in EBV itself. Other microbial agents present at the site of EBV infection might influence EBV infection, B-cell transformation, and virus release. For example, infectious mononucleosis (IM), a frequent consequence of primary EBV infection in adolescents and adults, is usually accompanied by tonsillitis with characteristic massive bacterial colonization (50), a likely source of TLR agonists acting on local EBV-infected B cells. Here we investigate the effects of CpG DNA and other exogenous TLR ligands on EBV-driven B-cell proliferation, clonal outgrowth, and induction of activation-associated cellular receptors and cytokines.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

Identification of an ABCB/P-glycoprotein-specific Inhibitor of Auxin Transport by Chemical Genomics

Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC₅₀ value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.     

Atomistic simulation of nanomechanical properties of Alzheimer’s Aβ(1–40) amyloid fibrils under compressive and tensile loading

In addition to being associated with severe degenerative diseases, amyloids show exceptional mechanical properties including great strength, sturdiness and elasticity. However, thus far physical models that explain these properties remain elusive, and our understanding of molecular deformation and failure mechanisms of individual amyloid fibrils is limited. Here we report a series of molecular dynamics simulations, carried out to analyze the mechanical response of two-fold symmetric Aβ(1–40) amyloid fibrils, twisted protein nanofilaments consisting of a H-bonded layered structure. We find a correlation of the mechanical behavior with chemical and nanostructural rearrangements of the fibril during compressive and tensile deformation, showing that the density of H-bonds varies linearly with the measured strain. Further, we find that both compressive and tensile deformation is coupled with torsional deformation, which is manifested in a strong variation of the interlayer twist angle that is found to be proportional to both the applied stress and measured strain. In both compression and tension we observe an increase of the Young's modulus from 2.34 GPa (for less than 0.1% strain in compression and 0.2% strain in tension), to 12.43 GPa for compression and 18.05 GPa for tension. The moduli at larger deformation are in good agreement with experimental data, where values in the range of 10–20 GPa have been reported. Our studies confirm that amyloids feature a very high stiffness, and elucidate the importance of the chemical and structural rearrangements of the fibrils during deformation.     

Sds22 regulates aurora B activity and microtubule-kinetochore interactions at mitosis

We have studied Sds22, a conserved regulator of protein phosphatase 1 (PP1) activity, and determined its role in modulating the activity of aurora B kinase and kinetochore-microtubule interactions. Sds22 is required for proper progression through mitosis and localization of PP1 to mitotic kinetochores. Depletion of Sds22 increases aurora B T-loop phosphorylation and the rate of recovery from monastrol arrest. Phospho-aurora B accumulates at kinetochores in Sds22-depleted cells juxtaposed to critical kinetochore substrates. Sds22 modulates sister kinetochore distance and the interaction between Hec1 and the microtubule lattice and, thus, the activation of the spindle assembly checkpoint. These results demonstrate that Sds22 specifically defines PP1 function and localization in mitosis. Sds22 regulates PP1 targeting to the kinetochore, accumulation of phospho-aurora B, and force generation at the kinetochore-microtubule interface.