Search

Infections with enteropathogenic Escherichia coli (EPEC) cause severe diarrhea in children. The noninvasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir (translocated intimin receptor). Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. In addition, these substances reduced bacterial binding to host cells, effector-dependent cell detachment, and abolished attaching and effacing lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and enterohemorrhagic E. coli infections.

Der Oberflachenrezeptor DC-SIGN ermoglicht die Aufnahme von Krankheitserregern wie HIV, Ebolavirus oder Mycobacterium tuberculosis. Daher gab es zahlreiche Versuche, Inhibitoren gegen die Bindung dieses Rezeptors an Kohlenhydratstrukturen dieser Pathogene zu entwickeln. Wegen der hydrophilen und wenig markanten Glykan-Bindestelle kennt man nur wenige Wirkstoff-ahnliche Liganden fur DC-SIGN. Parallele Fragment-Screenings mit SPR-Spektroskopie und einem Reporter Displacement Assay erganzen vorherige Screenings mit 19F-NMR-Spektroskopie und chemischen Mikroarrays. Die Validierung aktiver Substanzen mit SPR- und 1H-15N-HSQC-NMR-Spektroskopie zeigte, dass kein Fragment in die primare Kohlenhydrattasche bindet, dafur jedoch funf sekundare Bindestellen vorhanden sind, die mit Wirkstoff-ahnlichen Molekulen interagieren konnen. Mithilfe der Schlusselinteraktionen der hier gezeigten Fragmente konnen diese Regionen gezielt erkundet und zukunftige Ansatze zur Entwicklung von DC-SIGN-Inhibitoren beschleunigt werden.

Background Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. Methods Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. Results Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. Conclusion The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites’ redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up. Electronic supplementary material The online version of this article (10.1186/s12936-019-2651-z) contains supplementary material, which is available to authorized users.

DC-SIGN is a cell-surface receptor for several pathogenic threats, such as HIV, Ebola virus, or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate–protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site. Herein, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using 19F NMR spectroscopy and chemical fragment microarrays. Hit validation by SPR and 1H–15N HSQC NMR spectroscopy revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbor drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors.

The unique synthesis of 3-amino-4-nitrothiophenes 2 provides easy access to reference species of a new family of push-pull substituted thiophenes. The chemoselective modification of 2 into suitable derivatives was accomplished by selective S-oxidation of the vinylsulfanyl unit, followed by substitution of the vinylsulfinyl group of 3 by nucleophiles. Dihalothiophenes were formed this way in very good yields. A subsequent selective dehalogenation at the 2, 5 or 2 and 5 positions also proved feasible, and the push-pull unit was untouched. The new 2,5-dihalothiophenes could be interesting monomers for conducting polymers.

Type I Interferons (IFNs-I) are species-specific glycoproteins which play an important role as primary defence against viral infections and that can also modulate the adaptive immune system. In some autoimmune diseases, interferons (IFNs) are over-produced. IFNs are widely used as biopharmaceuticals for a variety of cancer indications, chronic viral diseases, and for their immuno-modulatory action in patients with multiple sclerosis; therefore, increasing their therapeutic efficiency and decreasing their side effects is of high clinical value. In this sense, it is interesting to find molecules that can modulate the activity of IFNs. In order to achieve that, it was necessary to establish a simple, fast and robust assay to analyze numerous compounds simultaneously. We developed four reporter gene assays (RGAs) to identify IFN activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them present a Z´ factor higher than 0.7. By using these RGAs, natural and synthetic compounds were analyzed simultaneously. A total of 442 compounds were studied by the Low Throughput Screening (LTS) assay using the four RCLs to discriminate between their inhibitory or enhancing effects on IFN activity. Some of them were characterized and 15 leads were identified. Finally, one promising candidate with enhancing effect on IFN-α/-β activity and five compounds with inhibitory effect were described. Fil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Comisión Nacional de Investigación Científica y Tecnológica; Chile Fil: Zapol´skii, Victor. Technical University Of Clausthal; Alemania Fil: Hinkelmann, Bettina. Helmholtz Centre For Infection Research; Alemania Fil: Köster, Märio. Helmholtz Centre For Infection Research; Alemania Fil: Kaufmann, Dieter. Technical University Of Clausthal; Alemania Fil: Sasse, Florenz. Helmholtz Centre For Infection Research; Alemania Fil: Hänsjorg, Hauser. Helmholtz Centre For Infection Research; Alemania Fil: Etcheverrigaray, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina Fil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina Fil: Bollati Fogolín, Mariela. Instituto Pasteur de Montevideo; Uruguay Fil: Oggero Eberhardt, Marcos Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina

A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .

The nitration reaction of trichloroethene to its three main products is comprehensively investigated and optimized.

The nitropolychlorobutadienes 3, 4 are valuable building blocks for various amination and successive heterocyclization products. Nucleophilic substitution reactions of the partially protected, bioactive amines 1, 2 with either vinyl, imidoyl or carbonyl chlorides result in the formation of the enamines 11, 12, 13, 16, 25, the amidine 6, and the amides 20, 21, respectively. In the following, cyclization to the highly functionalized pyrazoles 27, 28, pyrimidine 26 and pyridopyrimidine 24 succeeded. Deprotection of 21, 12 and 28 proved to be only partially feasible.

Reaction of gem-dichloronitroalkenes with base leads to the formation of chlorinated nitrile oxides, probably via a cyclic in- termediate. The 1,3-dipoles can be trapped with alkenes to give di- hydroisoxazoles with a chlorinated side chain in 3-position. This novel synthetic method is fairly general.

Nitropolychlorobutadienes are valuable precursors for highly functionalized acyclic or (hetero)cyclic compounds. In this 8th part of our synthetically oriented series we focus on the application of these versatile starting materials in the synthesis of analogs of the heterocyclic insecticides imidacloprid™ and thiacloprid™, and the acyclic counterpart clothianidin™. In addition to the main synthetic part, leading to imidazolidines or oxazolidines, further promising types of compounds derived by subsequent chemical modifications, are introduced. Most of the new compounds show high insecticidal activity.

A one-pot annulation reaction of aniline and its ring-sub- stituted derivatives with 1,1,2-trichloro-2-nitroethene (TCNiE) was developed delivering 3-chloroquinoxalin-2(1H)-one 4-oxides, ex- clusively, in good yields. The structure was proved by X-ray analy- sis. The C-N cyclization, a competing reaction to the double SNVin reaction of 1,1,2-trichloro-2-nitroethene with amines, can be con- trolled by the mode of addition. Some of the resulting quinoxalin- ones are promising candidates with respect to their prospective biological, especially pharmacological, activity.

The recently reported capability of pentachloro-2-nitro-butadiene and some closely related derivatives to allow the synthesis of highly substituted acyclic as well as five-membered (hetero)cyclic compounds is herein extended to pyrimidines with an exceptional substitution pattern. Our novel approach starts from a 1,1-diamino-3,4,4-trichloro-2-nitrobuta-1,3-diene and an appropriate amidine. Some of the resulting perfunctionalized pyrimidines were subjected to further transformations to give promising candidates with respect to biological, especially pharmacological, activity.

The reaction of2-nitropentachlorobuta-l,3-diene with a variety of anilines substituted with electron-withdrawing groups generates, contrary to expectations, N-tetrachloroallylidene-N'-arylhydrazines instead of 1,1-bisaminated substitution products. The imidoyl-type chlorides are capable of undergoing nucleophilic substitution with amines or hydrides. The resulting compounds should exhibit physiological activity, especially for use in crop science.

The reaction of mercaptoacetic acid esters with pentachloro-2-nitro-1,3-butadiene (1) provides an appropriate precursor for the synthesis of special thiazolidin-4-ones. Applying different anilines as the second constituent for the requisite cyclization step, a series of (Z)-2-allylidenethiazolidin-4-ones was obtained in yields up to 81%. Some subsequent reactions have been examined too, such as the formation of perfunctionalized 1H-pyrazoles upon treatment with hydrazine. Thiazolidinones are as well known for their physiological activities as for their application in optoelectronics.

Acidic hydrolysis of chromene derivative (III) in MeOH affords a mixture of heterocyclic compounds (V), (VI), and (VII) with the novel tricyclic chromen-8-ones (V) as main products.

Substitution of the chloro group by CHO (ipso-formylation) is observed when the thiophene derivatives (I) react under Vilsmeyer—Haack conditions.

A novel synthesis of title tetrazoles (VI) and their conversion to a variety of derivatives is described.

Title compound (I) is used as potent building block for structurally interesting and physiologically promising nitro-enamines, -imines, -amidines, and hydrazines as well as ring closure reaction products.

Reaction of gem-dichloronitroalkenes with base leads to the formation of chlorinated nitrile oxides, probably via a cyclic in- termediate. The 1,3-dipoles can be trapped with alkenes to give di- hydroisoxazoles with a chlorinated side chain in 3-position. This novel synthetic method is fairly general.

An easy access to dichloromethyl nitrovinylidene ketones of five-membered heterocycles is presented starting from nitroperchlorobutadiene. These complex ketones are valuable starting materials for novel product structures in versatile reactions such as selective reductions, substitutions to give α-nitroacryl cyanides, fragmentations by means of an alkoxide, as well as ring-cleavage/ ring-closure steps by the action of hydrazine. Some of the products show biological activity in crop protection.

Background Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen. Results A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 μM and a minimal bactericidal concentration (MBC) of 3.2 μM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype. Conclusion The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target.

The regioselective ipso-formylation of electron-rich, 3,4-push-pull-substituted 2-chlorothiophenes under Vilsmeier-Haack conditions was performed in good yields. The synthetic scope of this new reaction was explored using various halothiophenes, chloroanilines, and 1-methyl-3-chloroindole. In comparison with their structural C-H analogs the chlorinated thiophenes, anilines, and the indole proved to be less reactive toward electrophilic attack by chloromethyleniminium salts.