Reimar Krieg

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor 2-mediated hepatocellular carcinoma cell invasion

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.

Novel (N-Ferrocenylmethyl)amines and (N-Ferrocenylmethylen)imines derived from vicinal steroid amino alcohols and amines: synthesis, molecular structure, and biological activity

Novel steroidal (N-ferrocenylmethyl)amines with potential biologic activity and of potential interest as chiral ligands for metal complexation were synthesized. The new compounds were screened in vitro for their potential as antimicrobial agents. The synthesis of the new steroidal ferrocenes, including two X-ray crystal structures and biologic assays, are described. The 16-(ferrocenylmethyl)amino-estratrienes 4a-d, 7b, and 10b exhibited outstanding broad antimicrobial activity particularly against mycobacteria and multi-resistant staphylococci. Thus, they can be considered as new lead structures. In contrast, the analogous 3 alpha-(ferrocenylmethyl)amino-cholestanes 12 possessed only weak activity. The reaction of the four isomeric amino alcohols 1a-d (Scheme 1) with ferrocenecarbaldehyde was studied. 1b and 1c with 16/17-trans configuration yielded nearly quantitatively the (E)-Schiff bases 2b and 2c (Scheme 2). In contrast to the trans-compounds, condensation of the cisconfigurated amino alcohols 1a and 1d furnished tautomeric mixtures of the Schiff bases (2a and 2d, respectively) and their corresponding 1,3-oxazolidines (3a and 3d, respectively). The novel (N-ferrocenylmethyl)amines 4a-d were obtained in excellent yields by reduction of the tautomer mixtures and the uniform Schiff bases with sodium borohydride in ethanol. Starting with the 16 beta-hydroxy compound 5a, the synthesis of 16 beta- and 16 alpha-amino-3-methoxy-estra-1,3,5(10)-triene (6b, 9b) is described. The corresponding 16-(N-ferrocenylmethyl)amines 7b and 10b and the 3 alpha-(N-ferrocenylmethyl)amino-cholestanes 12 were synthesized (Scheme 3) for comparison in biologic tests.

Reactions of the four diastereomeric 16-amino-17-hydroxy-3-methoxy-estra-1,3,5(10)-trienes with aromatic ortho-hydroxy and heteroaromatic α-aldehydes and with 1,3-dicarbonyl compounds—molecular structures of condensation products and of copper(II) complexes ☆

Vicinal amino alcohols of steroids have been used as starting materials for the synthesis of chiral ligands with defined arrangements of functional groups. Condensation of the four diastereomeric 16,17-steroid amino alcohols 1a-1d with aromatic o-hydroxy and heteroaromatic alpha-aldehydes afforded the Schiff bases 2-6. When the 16,17-substituted compounds 2d, 5d, 6a, and 6d were in solution, the isomeric oxazolidines were detectable by (1)H NMR spectroscopy. The formation of oxazolidines could be avoided by using bulky aldehydes. Reduction of the Schiff bases (also in mixtures with oxazolidines) with NaBH(4) yielded the new N-substituted amino alcohols 12-15. The condensation products of 1a-1d with 1,3-dicarbonyl compounds (7 and 8) exhibited the 1-enamino-3-oxo structure ((1)H NMR spectroscopy). By means of X-ray analysis of 2a-2d, 3d, 7a, and 7c, the torsion angles for the 16N, 17O substituents, which are important for a participation of the 17O substituent in the complexation of metal ions, have been determined. Furthermore, a preferred arrangement between the chelate ring and the steroid plane existed in all investigated condensation products attributable to torsion angles 16H-C16-16N-C of 5-61 degrees. This arrangement was also preserved in the copper(II) complex 11 with 16alpha,17beta-trans configuration of the bidentate steroid ligand and a ratio of 2:1 for ligand: copper in contrast with dimeric copper(II) complexes with a tridentate steroid ligand of 16beta, 17beta-cis configuration (ratio of 1:1 for ligand:copper). The crystal structures of the condensation products are also discussed. In most cases, intermolecular hydrogen bonds between 17-hydroxy groups and the chelate oxygen caused polymeric strands.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol–ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Tailoring and histochemical application of fluorescent homo-dimeric styryl dyes using frozen sections: from peroxidase substrates to new cytochemical probes for mast cells, keratin, cartilage and nucleic acids.

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.

Detection of endogenous and immuno-bound peroxidase--the status quo in histochemistry.

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.

Arylmethylamino steroids as antiparasitic agents

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.