The stereoselectivity and hydrolysis efficiency of recombinant d-hydantoinase from Vigna angularis against 5-benzylhydantoin derivatives with halogen and methyl substituents

The researches on d-hydantoinase activity and substrate specificity towards dihydropyrimidine and hydantoin derivatives have been carried out intensively over the last few decades. So far, the major efforts have focused on (R,S)-5-phenylhydantoin and (R,S)-5-(4-hydroxyphenyl)hydantoin, the most desirable d-hydantoinase substrates from pharmaceutical industry point of view. However, it was shown that d-hydantoinase is a substrate-dependent enzyme, and its activity and stereoselectivity towards 5-monosubstituted hydantoins varied significantly with the type of substrate and the source of the enzyme. The aim of this study was to estimate the substrate specificity of d-hydantoinase towards series of 5-benzylhydantoin derivatives with halogen and methyl substituents in the phenyl ring. The biotransformations were carried out by using commercial enzyme: immobilized, recombinant, cloned, and expressed in Escherichia colid-hydantoinase from Vigna angularis (rD-HYD). All reactions were monitored by capillary electrophoresis (CE), and the conversion yields were calculated. Additionally, enantiomeric ratios of the obtained d-phenylalanine derivatives were estimated by chiral high-performance liquid chromatography (HPLC). Interestingly, the differences in the activities of examined enzyme towards particular 5-benzylhydantoin derivatives were observed. CE was also shown as a promising method for monitoring the hydrolysis of new substrates by d-hydantoinase and further analyzing of enzyme substrate specificity

Development of novel cellular model for affinity studies of histamine H(4) receptor ligands

The G protein-coupled histamine H4 receptor (H4R) is the last member of histamine receptors family discovered so far. Its expression pattern, together with postulated involvement in a wide variety of immunological and inflammatory processes make histamine H4 receptor an interesting target for drug development. Potential H4R ligands may provide an innovative therapies for different immuno-based diseases, including allergy, asthma, pruritus associated with allergy or autoimmune skin conditions, rheumatoid arthritis and pain. However, none of successfully developed selective and potent histamine H4 receptor ligands have been introduced to the market up to date. For that reason there is still a strong demand for pharmacological models to be used in studies on potent H4R ligands. In current work we present the development of novel mammalian cell line, stably expressing human histamine H4 receptor, with use of retroviral transduction approach. Obtained cell line was pharmacologically characterized in radioligand binding studies and its utility for affinity testing of potent receptor ligands was confirmed in comparative studies with the use of relevant insect cells expression model. Obtained results allow for statement that developed cellular model may be successfully employed in search for new compounds active at histamine H4 receptor

The study of cellular cytotoxicity of argireline® - an anti-aging peptide

Argireline® is well know, innovative anti-aging product used in the cosmetic market. This short chain peptide is used as active ingredient in dermal ointment and creams. Argireline® prevents formation of skin lines and wrinkles in a very similar way to the botulinum toxin (Botox), inhibiting neurotransmitter release at the neuromuscular junction. Argireline® does not require under skin muscle injections and it is believed to be relatively safe. However, despite the fact that some toxicity data has been provided by the product manufacturer, there is an evident lack of reliable information about cytotoxicity of argireline® in the literature. The aim of the presented study was to estimate the antiproliferation effect of argireline® solution in several concentrations. The influence of argireline® on cellular proliferation was examined against: human embryonic kidney HEK-293 cell line, human neuroblastoma IMR-32 cell line, and human primary skin fibroblasts. Tests were performed using formazan-based cell proliferation assay: EZ4U, which allows to measure the efficiency of mitochondrial oxidative activity in living cells. The argireline® inhibitory concentration, IC50 values were calculated and the results were compared to the IC50 value of the reference compound: doxorubicin. In conclusion, the considered method resulted in dose-dependent argireline® anti-proliferation effects. However, the significant cytotoxicity of argireline® solution was observed under 18 to 10 000 fold higher concentrations (depending on cells that were examined) in comparison to doxorubicin