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1.
Viruses ; 13(8)2021 07 29.
Article in English | MEDLINE | ID: mdl-34452354

ABSTRACT

The hepatic bile acid transporter Na+/taurocholate co-transporting polypeptide (NTCP) was identified in 2012 as the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Since then, this carrier has emerged as promising drug target for HBV/HDV virus entry inhibitors, but the synthetic peptide Hepcludex® of high molecular weight is the only approved HDV entry inhibitor so far. The present study aimed to identify small molecules as novel NTCP inhibitors with anti-viral activity. A ligand-based bioinformatic approach was used to generate and validate appropriate pharmacophore and QSAR (quantitative structure-activity relationship) models. Half-maximal inhibitory concentrations (IC50) for binding inhibition of the HBV/HDV-derived preS1 peptide (as surrogate parameter for virus binding to NTCP) were determined in NTCP-expressing HEK293 cells for 150 compounds of different chemical classes. IC50 values ranged from 2 µM up to >1000 µM. The generated pharmacophore and QSAR models were used for virtual screening of drug-like chemicals from the ZINC15 database (~11 million compounds). The 20 best-performing compounds were then experimentally tested for preS1-peptide binding inhibition in NTCP-HEK293 cells. Among them, four compounds were active and revealed experimental IC50 values for preS1-peptide binding inhibition of 9, 19, 20, and 35 µM, which were comparable to the QSAR-based predictions. All these compounds also significantly inhibited in vitro HDV infection of NTCP-HepG2 cells, without showing any cytotoxicity. The best-performing compound in all assays was ZINC000253533654. In conclusion, the present study demonstrates that virtual compound screening based on NTCP-specific pharmacophore and QSAR models can predict novel active hit compounds for the development of HBV/HDV entry inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Hepatitis Delta Virus/drug effects , User-Computer Interface , Virus Internalization/drug effects , Drug Discovery , HEK293 Cells , Hep G2 Cells , Hepatocytes/virology , Humans , Inhibitory Concentration 50 , Organic Anion Transporters, Sodium-Dependent , Quantitative Structure-Activity Relationship , Virus Attachment/drug effects
2.
J Mol Med (Berl) ; 99(2): 261-272, 2021 02.
Article in English | MEDLINE | ID: mdl-33330947

ABSTRACT

Developing resistance mechanisms of pathogens against established and frequently used drugs are a growing global health problem. Besides the development of novel drug candidates per se, new approaches to counteract resistance mechanisms are needed. Drug candidates that not only target the pathogens directly but also modify the host immune system might boost anti-parasitic defence and facilitate clearance of pathogens. In this study, we investigated whether the novel anti-parasitic steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni, might exhibit immunomodulatory properties. Our results reveal that 50 µM sc1o amplified the inflammatory potential of M1 macrophages and shifted M2 macrophages in a pro-inflammatory direction. Since M1 macrophages used predominantly glycolysis as an energy source, it is noteworthy that sc1o increased glycolysis and decreased oxidative phosphorylation in M2 macrophages. The effect of sc1o on the differentiation and activation of dendritic cells was ambiguous, since both pro- and anti-inflammatory markers were regulated. In conclusion, sc1o has several immunomodulatory effects that could possibly assist the immune system by counteracting the anti-inflammatory immune escape strategy of the parasite P. falciparum or by increasing pro-inflammatory mechanisms against pathogens, albeit at a higher concentration than that required for the anti-parasitic effect. KEY MESSAGES: • The anti-parasitic steroid compound 1o (sc1o) can modulate human immune cells. • Sc1o amplified the potential of M1 macrophages. • Sc1o shifts M2 macrophages to a M1 phenotype. • Dendritic cell differentiation and activation was ambiguously modulated. • Administration of sc1o could possibly assist the anti-parasitic defence.


Subject(s)
Antiparasitic Agents/pharmacology , Dendritic Cells/drug effects , Immunologic Factors/pharmacology , Macrophages/drug effects , Steroids/pharmacology , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dendritic Cells/metabolism , Energy Metabolism/drug effects , Humans , Macrophages/metabolism
3.
Sci Rep ; 10(1): 7534, 2020 05 05.
Article in English | MEDLINE | ID: mdl-32371995

ABSTRACT

Parasite-mediated diseases like malaria and schistosomiasis are growing health problems worldwide and novel drug candidates are urgently needed. In this study, the in-vitro safety profile of steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni with an IC50 value of 5 nM, was characterized. We assessed viability/proliferation, apoptosis and cell cycle tests to determine the cytotoxic profile of sc1o in cancer cells. The mutagenic potential was determined with the AMES test. To identify off-target effects we investigated whether sc1o interacts with safety-relevant molecules such as cytochrome P450 (CYP) enzymes, phosphodiesterases (PDE), histone deacteylases (HDAC) and human ether-a-go-go related gene (hERG). Furthermore, to predict the potential bioavailability of sc1o, its effect on Caco-2 cell barrier integrity, by measurement of the transepithelial electrical resistance (TEER), was determined. Sc1o at 25 µM reduced cell viability, probably through cell-cycle arrest, but did not induce apoptosis in cancer cells. No adverse off-target effects nor mutagenic potential of sc1o were observed. Furthermore, sc1o did not disturb the integrity of the cell barrier, but exhibited low membrane permeability, apparently due to cell adherence. In conclusion, sc1o up to 10 µM showed a good in-vitro safety profile.


Subject(s)
Antiparasitic Agents/pharmacology , Steroids/pharmacology , Animals , Antimalarials/pharmacology , Apoptosis , Caco-2 Cells , Cell Proliferation , Cell Survival , HCT116 Cells , Humans , In Vitro Techniques , Inhibitory Concentration 50 , Mitochondria/metabolism , Parasites/drug effects , Parasitic Sensitivity Tests , Patient Safety , Permeability , Plasmodium falciparum/drug effects , Schistosoma mansoni/drug effects , Schistosomiasis
5.
Nat Commun ; 8: 14478, 2017 02 17.
Article in English | MEDLINE | ID: mdl-28211535

ABSTRACT

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC50 1-5 nM) as well as against gametocytes. In P. berghei-infected mice, oral administration of 1o drastically reduces parasitaemia and cures the animals. Furthermore, 1o efficiently blocks parasite transmission from mice to mosquitoes. The steroid compounds show low cytotoxicity in mammalian cells and do not induce acute toxicity symptoms in mice. Moreover, 1o has a remarkable activity against the blood-feeding trematode parasite Schistosoma mansoni. The steroid and the hydroxyarylmethylamino moieties are essential for antimalarial activity supporting a chelate-based quinone methide mechanism involving metal or haem bioactivation. This study identifies chemical scaffolds that are rapidly internalized into blood-feeding parasites.


Subject(s)
Amines/pharmacology , Antiparasitic Agents/pharmacology , Steroids/pharmacology , Amines/chemistry , Amines/pharmacokinetics , Animals , Anopheles/parasitology , Anti-Infective Agents/pharmacology , Antiparasitic Agents/chemistry , Antiparasitic Agents/pharmacokinetics , Cell Death/drug effects , Cell Proliferation/drug effects , Female , Germ Cells/drug effects , Inhibitory Concentration 50 , Life Cycle Stages/drug effects , Malaria/parasitology , Malaria/transmission , Mice , Models, Biological , Parasites/drug effects , Plasmodium berghei/drug effects , Plasmodium berghei/growth & development , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development , Schistosoma mansoni/drug effects , Schistosoma mansoni/ultrastructure , Steroids/chemistry , Steroids/pharmacokinetics , Toxicity Tests, Acute
6.
Acta Histochem ; 113(7): 682-702, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21477847

ABSTRACT

Homo-dimers of styryl dyes were chemically tailored in order to become specific cytochemical probes for use in the life sciences. Histochemical applications using fixed cryotome sections are discussed. It is concluded, that homo-dimerization of specific styryl substrates of peroxidase (PO) by way of their covalent linkage, does not necessarily lead to improved detection sensitivity of endogenous and immuno-bound peroxidase (PO) activity. In general, these dimers act less specific towards PO activity than parent monomers. Synergetic interactions of the doubled basic dye compartments with cell constituents cause a pronounced staining of further targets at the cellular level. This behavior depends on the functional groups present in each dye compartment in a crucial manner. However, by way of chemical dye tailoring centering of these initially unwanted staining properties is possible leading to novel highly fluorescent stains for mast cells, nucleic acids, keratin and cartilage tissue. Structure/staining behavior-relationships of these stains are discussed.


Subject(s)
Cartilage/metabolism , Fluorescent Dyes/analysis , Keratins/analysis , Mast Cells/metabolism , Nucleic Acids/analysis , Peroxidases/metabolism , Styrenes/chemistry , Dimerization , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Frozen Sections , Histocytochemistry , Styrenes/chemical synthesis , Substrate Specificity
7.
Prog Histochem Cytochem ; 45(2): 81-139, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20488278

ABSTRACT

The discovery of synthetic dyes goes back to 1856 and launched the development of the whole chemical and pharmaceutical industry. In life sciences synthetic dyes represent indispensable tools for the microscopic and macroscopic level. Small dyes have the advantage of their easy adaptability to various measuring equipments. By way of structural modification of the chromophore portion, dye labels can be tailored that they absorb and emit light at desired wavelengths ranging from the UV to the near infrared region of the spectrum. Assisted by the development of light measuring techniques and the commercial availability of highly sensitive equipment, today luminescent labels represent most sensitive detection tools in life sciences and dominate over chromogen based techniques. However, for detection of active sites of peroxidase (PO) so far fluorescent labels have been confined to only a few substrates while a broad variety of well-established chromogenic techniques exist. This review covers fluorescent and chromogenic approaches for the permanent detection of immuno-bound and endogenous PO-activity in fixed cells and tissues. Thereby the tailoring of suitable dye labels is additionally challenged by two demands: (1) The applied dye (or its precursor) must act as enzyme substrate specifically and (2) the enzymatic impact must furnish an insoluble dye product from easy soluble starting materials in a very quick reaction. Hence it is not surprising that among PO-substrates (and enzyme substrates generally), dye conjugates represent only an exception while most of these labels represent reactive dyes or suitable precursors. Chromogenic and fluorescent approaches for the permanent labeling of enzymatic sites are compiled. Furthermore, various area-spanning PO-detection principles are discussed ranging from transmission light (TLM) and fluorescence light (FLM) microscopy (chromogenes, flourochromes, fluorescent chromogenes, chromogenes with nonlinear optical properties) to correlated transmission electron microscopy (TEM; photoconversion of specific chromogenic reaction products, electron opaque and/or osmiophilic chromogenic substrates). Also, approaches for reflectance laser microscopy (RLM), polarization microscopy (PM), and correlative TLM, FLM, and multiphoton fluorescence microscopy (MFM) are discussed.


Subject(s)
Chromogenic Compounds/chemistry , Fluorescent Dyes/chemistry , Peroxidase/analysis , Peroxidase/chemistry , Animals , Humans , Immunohistochemistry , Microscopy, Electron, Transmission
8.
Carcinogenesis ; 30(9): 1487-96, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19546160

ABSTRACT

The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.


Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Proto-Oncogene Proteins/physiology , Receptor, PAR-2/physiology , Receptors, Growth Factor/physiology , Transcriptional Activation , Amino Acid Sequence , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/physiology , Humans , Molecular Sequence Data , Neoplasm Invasiveness , Phosphorylation , Proto-Oncogene Proteins c-met , Reactive Oxygen Species/metabolism , Receptor, PAR-2/analysis
9.
J Gen Virol ; 88(Pt 2): 536-546, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17251572

ABSTRACT

PB1-F2 is a pro-apoptotic polypeptide of many influenza A virus (FLUAV) isolates encoded by an alternative ORF of segment 2. A comprehensive GenBank search was conducted to analyse its prevalence. This search yielded 2226 entries of 80 FLUAV subtypes. Of these sequences, 87 % encode a PB1-F2 polypeptide greater than 78 aa. However, classic swine influenza viruses and human H1N1 isolates collected since 1950 harbour a truncated PB1-F2 sequence. While PB1-F2 of human H1N1 viruses terminates after 57 aa, classic swine H1N1 sequences have in-frame stop codons after 11, 25 and 34 codons. Of the avian sequences, 96 % encode a full-length PB1-F2. One genetic lineage of segment 2 sequences which is avian-like and different from the classic swine FLUAV comprises PB1-F2 sequences of porcine FLUAVs isolated in Europe (H1N1, H1N2, H3N2). Of these PB1-F2 sequences, 42 % also exhibit stop codons after 11, 25 and 34 codons. These amino acid positions are highly conserved among all FLUAV isolates irrespective of their origin. Molecular genetic analyses reveal that PB1-F2 is under constraint of the PB1 gene. The PB1-F2 polypeptide of FLUAVs isolated from European pigs is expressed in host cells as demonstrated by immunohistochemistry. Using different PB1-F2 versions fused to an enhanced GFP, mitochondrial localization is demonstrated for those PB1-F2 polypeptides which are greater than 78 aa while a truncated version (57 aa) shows a diffuse cytoplasmic distribution. This indicates similar properties and function of porcine and human FLUAV PB1-F2.


Subject(s)
Influenza A virus/genetics , Viral Proteins/genetics , Animals , HeLa Cells , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H2N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/metabolism , Influenza A virus/classification , Influenza A virus/metabolism , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Subcellular Fractions/metabolism , Swine , Viral Proteins/metabolism
10.
Histochem Cell Biol ; 126(4): 507-15, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16673095

ABSTRACT

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 x 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman's layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.


Subject(s)
Cornea/ultrastructure , Lasers , Microscopy, Fluorescence, Multiphoton/methods , Retina/ultrastructure , Animals , Epithelial Cells/ultrastructure , Keratinocytes/ultrastructure , Microscopy, Fluorescence, Multiphoton/instrumentation , Nerve Fibers/ultrastructure , Tomography, Optical
11.
J Mol Histol ; 35(5): 471-87, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15571325

ABSTRACT

Some 2-(2-styryl)-benzothiazole derivatives have been synthesized as novel fluorescent substrates for the localization of peroxidase activity. Excellent localization, high staining sensitivity and exceptionally low background staining were achieved by optimizing the choice of substrate. Multiple step-by-step anchoring of enzymatically-activated individual substrate molecules to surrounding nucleophiles, related to the catalysed reporter deposition (CARD) technique, is discussed. In contrast to tyramine conjugates, as employed in the CARD technique, the separation between reporting and anchoring function is eliminated, thus yielding a new fluorochrome with altered fluorescence properties after enzymatic cross-linking. (E)-2-(2-[4-hydroxyphenyl] vinyl)-3-ethyl-1,3-benzothiazolium iodide has been found to the best substrate so far. This was demonstrated in histochemical applications for the localization of endogenous and immunobound peroxidase activity using fixed cryostat, paraffin or semi-thin Epon sections. The specific final reaction product is efficiently excitable over a wide spectrum from green to violet, providing an outstanding sensitive localization of sites of enzymatic activity with high photo stability. In a comparative study with the Alexa Fluor 546-tyramine conjugate, endogenous and immunobound peroxidase activity was visualized and the results compared using an epi-fluorescence confocal laser scanning microscope. The novel substrate provided an improved specificity and very low background staining whereas the Alexa Fluor-tyramide exhibited a strong overall background staining. FITC-labelled secondary antibodies also yielded very low background staining but the staining was less specific compared with the biotin-based ABC amplification systems labelled with the selected substrate or the Alexa-tyramide. In conclusion, multiple fluorochrome generation close to sites of peroxidase activity, by enzymatic cross-linking of styrene-related substrates, is a promising alternative to the fluorochrome-labelled tyramine ('tyramide') deposition technique.


Subject(s)
Fluorescent Dyes , Staining and Labeling , Animals , Horseradish Peroxidase/chemistry , Immunoenzyme Techniques/methods , Oxidation-Reduction , Rats , Sensitivity and Specificity , Staining and Labeling/methods
12.
Cell Mol Biol (Noisy-le-grand) ; 48 Online Pub: OL297-308, 2002.
Article in English | MEDLINE | ID: mdl-12643447

ABSTRACT

The lysosomal cysteine protease cathepsin S is synthesized as inactive precursor at the rough endoplasmic reticulum (ER), further processed in the Golgi compartment and finally targeted to the lysosomes where it becomes activated by the proteolytic cleavage of the inhibitory propeptide. Biochemical studies with a non-glycosylated mutant of procathepsin S (plasma membrane binding at 2 degrees C, reuptake of secreted enzyme at 37 degrees C) led to the suggestion of an additional sorting motif in procathepsin S besides the classical Man-6-P recognition signal. In order to further confirm this suggestion on a morphological basis we performed a series of laser scanning confocal microscopy (CLSM) and electron microscopical analyses with HEK 293 cells expressing the mutant non-glycosylated procathepsin S. Immunolocalization with CLSM documented clearly a fine granular fluorescence in the paranuclear region of mutant expressing cells. Electron microscopy demonstrated the presence of cathepsin S immunoreactive deposits within cytosolic vacuoles (lysosomes), at the plasma membrane and in ER buds. These buds were also visible in the cytosol as well as in form of concentrated patches at the plasma membrane indicating the direct transport of (pro)cathepsin S from the ER to the cell surface.


Subject(s)
Cathepsins/genetics , Enzyme Precursors/genetics , Lysosomes/enzymology , Acid Phosphatase/metabolism , Base Sequence , Cathepsins/chemistry , Cathepsins/metabolism , Cell Line , DNA Primers , Enzyme Precursors/chemistry , Enzyme Precursors/metabolism , Humans , Immunohistochemistry/methods , Kidney , Lysosomes/ultrastructure , Microscopy, Confocal , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transfection
13.
Cell Mol Biol (Noisy-le-grand) ; 48 Online Pub: OL343-58, 2002.
Article in English | MEDLINE | ID: mdl-12643452

ABSTRACT

Alkaline phosphatase (AP) activity is often targeted in enzyme-related histochemistry as probe enzyme to detect neoplastic cells, as marker for primordial germ cells as well as in preimplantation studies, osteoblast differentiation, phosphate starvation in bacteria, yeast and phytoplankton. Moreover, AP-marker activity is a very useful tool in immunohistochemistry to detect gene sequences, antigens and antibodies. Here we describe a novel high resolution fluorescence method to localize AP-activity in cells and tissue sections based on a naphthol-AS azo coupling procedure (Jenfluor ap). This method provides amorphous photostable fluorescent final reaction products without any diffusion artifacts which are visible in conventional fluorescence microscopes as well as in confocal laser scanning and near infrared multiphoton laser scanning microscopes. The superiority of the Jenfluor ap method in comparison to the known Fast Red TR salt as well as the ELF stains is discussed.


Subject(s)
Alkaline Phosphatase/metabolism , Alkaline Phosphatase/analysis , Animals , Biomarkers , Drug Stability , Epididymis/enzymology , Female , Fluorescent Dyes , Immunohistochemistry , Kinetics , Male , Muscle, Smooth/enzymology , Organ Specificity , Rats , Rats, Wistar
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