Antibody Recognition of Cancer Cells via Glycan Surface Engineering.

Stimulation of the body's immune system toward tumor cells is now well recognized as a promising strategy in cancer therapy. Just behind cell therapy and monoclonal antibodies, small molecule-based strategies are receiving growing attention as alternatives to direct immune response against tumor cells. However, the development of small-molecule approaches to modulate the balance between stimulatory immune factors and suppressive factors in a targeted way remains a challenge. Here, we report the cell surface functionalization of LS174T cancer cells with an abiotic hapten to recruit antibodies to the cell surface. Metabolic glycoengineering followed by covalent reaction with the hapten results in antibody recognition of the target cells. Microscopy and flow cytometry studies provide compelling evidence that metabolic glycoengineering and small molecule stimulators can be combined to direct antibody recognition.

Profiling withanolide A for therapeutic targets in neurodegenerative diseases.

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.

Biosynthesis of fragin is controlled by a novel quorum sensing signal.

Members of the diazeniumdiolate class of natural compounds show potential for drug development because of their antifungal, antibacterial, antiviral, and antitumor activities. Yet, their biosynthesis has remained elusive to date. Here, we identify a gene cluster directing the biosynthesis of the diazeniumdiolate compound fragin in Burkholderia cenocepacia H111. We provide evidence that fragin is a metallophore and that metal chelation is the molecular basis of its antifungal activity. A subset of the fragin biosynthetic genes is involved in the synthesis of a previously undescribed cell-to-cell signal molecule, valdiazen. RNA-Seq analyses reveal that valdiazen controls fragin biosynthesis and affects the expression of more than 100 genes. Homologs of the valdiazen biosynthesis genes are found in various bacteria, suggesting that valdiazen-like compounds may constitute a new class of signal molecules. We use structural information, in silico prediction of enzymatic functions and biochemical data to propose a biosynthesis route for fragin and valdiazen.

Comparative effects of nodularin and microcystin-LR in zebrafish: 2. Uptake and molecular effects in eleuthero-embryos and adult liver with focus on endoplasmic reticulum stress.

Microcystin (MC) and nodularin are structurally similar cyanobacterial toxins that inhibit protein phosphatases. Additional modes of action are poorly known, in particular for nodularin. In our associated work, we showed that active cellular uptake is mediated by the organic anion transporting polypeptide drOatp1d1 in zebrafish (Faltermann et al., 2016). Here, we assessed the transcriptional expression of three genes encoding three uptake transporters during embryonic development from 24h post fertilization (hpf) to 168 hpf. Transcripts of drOatp1d1 and drOatp2b1 are present at 24 hpf. The abundance increased after hatching and remained about constant up to 168 hpf. Transcripts of drOatp2b1 were most abundant, while drOapt1f transcripts showed very low relative abundance compared to drOatp1d1 and drOatp2b1. We further demonstrated the uptake of fluorescent labeled MC-LR in eleuthero-embryos and its accumulation in the glomerulus of the pronephros. An important molecular effect of MC-LR in human liver cells is the induction of endoplasmic reticulum (ER)-stress. Here, we investigated, whether MC-LR and nodularin similarly lead to induction of ER-stress in zebrafish by analyzing changes of mRNA levels of genes indicative of ER-stress. In zebrafish liver organ cultures short- and long-term exposures to 0.15 and 0.3 µmol L(-1) MC-LR, and 0.5 and 1 µM L(-1) nodularin led to significant transcriptional induction of several ER-stress marker genes, including the chaperone glucose regulated protein 78 (bip), the spliced form of x-box binding protein (xbp-1s), the CCAAT-enhancer-binding protein homologous protein (chop) and activating transcription factor 4 (atf4). Furthermore, strong transcriptional changes occurred for tumor necrosis factor alpha (tnfa) and dual specificity phosphatase 5 (dusp5), associated with mitogen activated protein kinase (MAPK) pathway. However, no alterations in transcript levels of pro-apoptotic genes Bcl-2 like protein 4 (bax) and p53 occurred. In contrast to adult liver, MC-LR and nodularin did not result in detectable changes of mRNA levels of selected target genes involved in ER-stress in zebrafish eleuthero-embryos, nor was the abundance of transcripts belonging to the MAPK and pro-apoptosis pathways altered. In conclusion, our data indicate that MC-LR and nodularin have similar transcriptional effects. They lead to changes in mRNA levels of genes that suggest induction of ER-stress, and furthermore, lead to increased level of tnfα mRNA in the adult liver, which suggests a novel (transcriptional) mode of action in fish. However, although taken up by eleuthero-embryos, no transcriptional changes induced by these cyanobacterial toxins were detected. This is probably due to action to specific organs such as liver and kidneys that could not be identified by whole-embryo sampling.

Preparation of Fluorescent Microcystin Derivatives by Direct Arginine Labelling and Their Biological Evaluation.

Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract.

Direct arginine modification in native peptides and application to chemical probe development.

An efficient method for the direct labeling of the Arg guanidinium group in native peptides is reported. This straightforward procedure allows modifying the arginine moiety in peptides with various reporter groups, such as fluorophores, biotin, etc., under mild conditions in an operationally simple procedure. The scope of this method tolerates various functionalized amino acids such as His, Ser, Trp, Tyr, Glu, etc., while the only limitations uncovered so far are restricted to cysteine and free amine residues. The utility of this late-stage diversification method was demonstrated in direct labeling of leuprolide, a clinically used drug, for distribution monitoring in Daphnia, and in labeling of microcystin, a cyanobacterial toxin.

Functionally Optimized Neuritogenic Farinosone C Analogs: SAR-Study and Investigations on Their Mode of Action.

Several natural products derived from entomopathogenic fungi have been shown to initiate neuronal differentiation in the rat pheochromocytoma PC12 cell line. After the successful completion of the total synthesis program, the reduction of structural complexity while retaining biological activity was targeted. In this study, farinosone C served as a lead structure and inspired the preparation of small molecules with reduced complexity, of which several were able to induce neurite outgrowth. This allowed for the elaboration of a detailed structure-activity relationship. Investigations on the mode of action utilizing a computational similarity ensemble approach suggested the involvement of the endocannabinoid system as potential target for our analogs and also led to the discovery of four potent new endocannabinoid transport inhibitors.

Neuritogenic surfaces using natural product analogs.

Neuritogenic surfaces are generated by a simple dip-coating procedure, as glass slides are coated with a neurotrophin-like small organic molecule in the presence of a collagen matrix. The surfaces retain their biological activity for multiple cycles and the protocol is suitable for various substrates and coating conditions.

Caged retinoids as photoinducible activators: implications for cell differentiation and neurite outgrowth.

Aiming to control neurite formation and navigate the axonal growth by an extrinsic guidance, we report on the design, synthesis and biological evaluation of caged retinoids. Agonists of RARß have been temporarily blocked either by the [(α-methyl-2-nitropiperonyl)oxy]carbonyl (MeNPOC) group or by immobilization using nitrocatechol linkers on TiO2 particles. Release on demand has been achieved by release under UV irradiation, leading to the biologically active compounds, which have been shown to induce neuronal differentiation and neurite outgrowth.

Enantioselective total synthesis of virosaine A and bubbialidine.

The first enantioselective total syntheses of virosaine A and bubbialidine are described. Key transformations include the formation of a tetracyclic intermediate via an intramolecular aza-Michael addition, generation of a N-hydroxy-pyrrolidine through a Cope elimination and an intramolecular [1,3]-dipolar cycloaddition to generate a complex 7-oxa-1-azabicyclo[3.2.1]octane ring system.

Controlling protein transport by small molecules.

Many proteins are transported from the nucleus to the cytoplasm by the exportin CRM1, which recognizes cargo proteins through a leucine rich nuclear export signal (NES). This nuclear export process can be inhibited by several small molecules, both natural products and fully synthetic compounds. The structural basis for the inhibition of nuclear export by leptomycin (LMB) based on disruption of the protein/protein interaction between CRM1 and cargo proteins is discussed. The chemistry and inhibition of nucleocytoplasmic transport of leptomycin, anguinomycin and derivatives, goniothalamin, JBIR-02, valtrate, dihydrovaltrate, ACA, peumusolide A and several synthetic compounds is presented. Consequences for the design of nuclear export inhibitors are discussed, and the potential of these compounds as anticancer agents is evaluated.

Spatial isolation favours the divergence in microcystin net production by Microcystis in Ugandan freshwater lakes.

It is generally agreed that the hepatotoxic microcystins (MCs) are the most abundant toxins produced by cyanobacteria in freshwater. In various freshwater lakes in East Africa MC-producing Microcystis has been reported to dominate the phytoplankton, however the regulation of MC production is poorly understood. From May 2007 to April 2008 the Microcystis abundance, the absolute and relative abundance of the mcyB genotype indicative of MC production and the MC concentrations were recorded monthly in five freshwater lakes in Uganda: (1) in a crater lake (Lake Saka), (2) in three shallow lakes (Lake Mburo, George, Edward), (3) in Lake Victoria (Murchison Bay, Napoleon Gulf). During the whole study period Microcystis was abundant or dominated the phytoplankton. In all samples mcyB-containing cells of Microcystis were found and on average comprised 20+/-2% (SE) of the total population. The proportion of the mcyB genotype differed significantly between the sampling sites, and while the highest mcyB proportions were recorded in Lake Saka (37+/-3%), the lowest proportion was recorded in Lake George (1.4+/-0.2%). Consequently Microcystis from Lake George had the lowest MC cell quotas (0.03-1.24 fg MC cell(-1)) and resulted in the lowest MC concentrations (0-0.5 microg L(-1)) while Microcystis from Lake Saka consistently showed maximum MC cell quotas (14-144 fg cell(-1)) and the highest MC concentrations (0.5-10.2 microg L(-1)). Over the whole study period the average MC content per Microcystis cell depended linearly on the proportion of the mcyB genotype of Microcystis. It is concluded that Microcystis populations differ consistently and independently of the season in mcyB genotype proportion between lakes resulting in population-specific differences in the average MC content per cell.

Poly(ethylene glycol) adlayers immobilized to metal oxide substrates through catechol derivatives: influence of assembly conditions on formation and stability.

We have investigated five different poly(ethylene glycol) (PEG, 5 kDa) catechol derivatives in terms of their spontaneous surface assembly from aqueous solution, adlayer stability, and resistance to nonspecific blood serum adsorption as a function of the type of catechol-based anchor, assembly conditions (temperature, pH), and type of substrate (SiO(2), TiO(2), Nb(2)O(5)). Variable-angle spectroscopic ellipsometry (VASE) was used for layer thickness evaluation, X-ray photoelectron spectroscopy (XPS) for layer composition, and ultraviolet-visible optical spectroscopy (UV-vis) for cloud point determination. Polymer surface coverage was influenced by the type of catechol anchor, type of the substrate, as well as pH and temperature (T) of the assembly solution. Furthermore, it was found to be highest for T close to the cloud point (T(CP)) and pH of the assembly solution close to pK(a1) (dissociation constant of the first catechol hydroxy group) of the polymer and to the isoelectric point (IEP) of the substrate. T(CP) turned out to depend on not only the ionic strength of the assembly solution, but also the type of catechol derivative and pH. PEG-coating dry thickness above 10 A correlated with low serum adsorption. We therefore conclude that optimum coating protocols for catechol-based polymer assembly at metal oxide interfaces have to take into account specific physicochemical properties of the polymer, anchor, and substrate.

Synthesis and catalysis.

Research projects of the Department of Chemistry, University of Basel are reviewed ranging from the synthesis of complex natural products to the development of metalorganic catalysts and organocatalysts.

Total synthesis and neuritotrophic activity of farinosone C and derivatives.

An efficient synthesis of all four possible stereoisomers of the neurotrophic amide farinosone C was developed. The absolute and relative configuration was assigned by comparison of synthetic material with a derivatized authentic sample of the natural product. Several derivatives allowed for the generation of preliminary structure activity relationships concerning neurite outgrowth in PC-12 cells, unravelling L-tyrosinol-amide as the pharmacophore.

Antimalarial natural products of marine and freshwater origin.

This review highlights recently discovered antimalarial natural products from marine and freshwater sources described in the literature from 2006 to 2008. The structures as well as bioactivities of compounds against the malaria parasites such as Plasmodium falciparum are discussed, including, for example, agelasine, xestoquinone, alisiaquinone, crambescidin, venturamide, dragomabin, gragonamide, viridamide, salinosporamide, chaetoxanthone, nodulisporacid, tumonoic acid, girolline, oroidin, nostocarboline, aerucyclamide, and microcylamide 7806 and its revised structure. Synthetic derivatives of natural products are presented including plakortin, isoaaptamine, curcuphenol, pseudopyronine, manzamine, and nostocarboline. Consequences of these discoveries for the development of novel natural product agents against malaria are discussed.

Directed biosynthesis of phytotoxic alkaloids in the cyanobacterium Nostoc 78-12A.

Nostocarboline, a chlorinated and N-methylated carbolinium alkaloid, displays potent and selective inhibition of photoautotrophic organisms as well as the malaria parasite Plasmodium falciparum, while showing very low toxicity to bacterial and fungal pathogens, rat myoblasts and crustaceans. New derivatives of nostocarboline incorporating Br, F or methyl substituents have been obtained through precursor-directed biosynthesis in Nostoc 78-12A (identical to Nostoc sp. ATCC 43238) by feeding this cyanobacterium with differently substituted tryptophan derivatives or 6-Br-norharmane (eudistomin N). These experiments substantiate the biosynthetic hypothesis and validate the inherent flexibility of the corresponding enzymes for metabolic engineering. The new derivatives inhibit the growth of the toxic-bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 above 1 microM. The mode of action of nostocarboline was investigated by using chlorophyll-a fluorescence imaging, and it was demonstrated that a decrease in photosynthesis precedes cell death, thus establishing the phytotoxic properties of this alkaloid.