Biological activity of 3-(2-benzoxazol-5-yl)alanine derivatives.

Searching for new drugs is still a challenge for science, mainly because of civilization development and globalization which promote the rapid spread of diseases, which is particularly dangerous in the case of infectious ones. Moreover, readily available already known antibiotics are often overused or misused, possibly contributing to the increase in the number of multidrug-resistant microorganisms. A consequence of this is the need for new structures of potential drugs. One of them is a benzoxazole moiety, a basic skeleton of a group of fluorescent heterocyclic compounds already widely used in chemistry, industry, and medicine, which is also present in naturally occurring biologically active compounds. Moreover, synthetic benzoxazoles are also biologically active. Considering all of that, a large group of non-proteinogenic amino acids based on 3-(2-benzoxazol-5-yl)alanine skeleton was studied in search for new antimicrobial and anticancer agents. Screening tests revealed that antibacterial potential of 41 compounds studied is not very high; however, they are selective acting only against Gram-positive bacteria (B. subtilis). Moreover, almost half of the studied compounds have antifungal properties, also against pathogens (C. albicans). Most of studied compounds are toxic to both normal and cancer cells. However, in a few cases, toxicity to normal cells is much lower than for cancer cells indicating these compounds as future anticancer agents. The research carried out on such a large group of compounds allowed to establish a structure-activity relationship which enables to select candidates for further modifications, necessary to improve their biological activity and obtain a new lead structure with potential for therapeutic use.

Simultaneous determination of non-steroidal anti-inflammatory drugs and natural estrogens in the mussels Mytilus edulis trossulus.

In this study, an ASE (Accelerated Solvent extraction)-SPE (Solid Phase Extraction)-GC/MS(SIM) method for the simultaneous determination of five non-steroidal anti-inflammatory drugs (NSAIDs: ibuprofen (IBU), paracetamol (PAR), diclofenac (DIC), naproxen (NAP) and ketoprofen (KET)) and three natural estrogens (estrone (E1), 17ß-estradiol (E2), estriol (E3)) in mussels was proposed, which is simple, cheap and easy for use in environmental laboratories. For the first time, the sorbents: PSA (Primary Secondary Amine) and FLORISIL®, placed directly inside the extraction cells, were used for the improvement of the ASE procedure for the isolation of these pollutants from mussel samples. The application of FLORISIL® especially allowed the purification of the extract to increase recovery, without the loss of analytes, or prolongation of the extraction time. The proposed ASE-SPE-GC/MS(SIM) method was validated (the method detection limit values were in the range of 1 ng/g dry weight (d.w.) for IBU to 7 ng/g d.w. for E1; the measurement intermediate precision was between 0.24% and 7.85%; the mean recovery was in the range of 80-118%) and used for the determination of NSAIDs and estrogenic hormones in whole tissue of the mussels Mytilus edulis trossulus collected from the Gulf of Gdansk (southern Baltic Sea). IBU, NAP, DIC and E1 were determined in these samples; however, the pharmaceuticals were found only in smaller (with a length of 2-3 cm) individuals. The observed differences in the concentrations of CEC in smaller and older mussel organisms were fully discussed. Summarizing, this method could be used for monitoring these CEC in such organisms in order to expand our knowledge of their influence on the water ecosystem, however, in such investigations smaller mussel organisms should be used.

Synthesis and SAR Studies of Antibacterial Peptidyl Derivatives Based upon the Binding Site of Human Cystatin C.

BACKGROUND: Antibacterial peptidyl derivative - Cystapep 1, was previously found to be active both against antibiotic-resistant staphylococci and streptococci as well as antibioticsusceptible strains of these species. Therefore, it is a promising lead compound to search for new antimicrobial peptidomimetics. OBJECTIVES: We focused on identifying structural elements that are responsible for the biological activity of Cystapep 1 and its five analogues. We tried to find an answer to the question about the mechanism of action of the tested compounds. Therefore, we have investigated in details the possibility of interacting these compounds with biological membrane mimetics. METHODS: The subject compounds were synthesized in solution, purified and characterized by HPLC and mass spectrometry. Then, the staphylococci susceptibility tests were performed and their cytotoxicity was established. The results of Cystapep 1 and its analogues interactions with model target were examined using the DSC and ITC techniques. At the end the spatial structures of the tested peptidomimetics using NMR technique were obtained. RESULTS: Antimicrobial and cytotoxicity tests show that Cystapep 1 and its peptidomimetic V are good drug candidates. DSC and ITC studies indicate that disruption of membrane is not the only possible mechanism of action of Cystapep 1-like compounds. For Cystapep 1 itself, a multi-step mechanism of interaction with a negatively charged membrane is observed, which indicates other processes occurring alongside the binding process. The conformational analysis indicated the presence of a hydrophobic cluster, composed of certain side chains, only in the structures of active peptidomimetics. This can facilitate the anchoring of the peptidyl derivatives to the bacterial membrane. CONCLUSION: An increase in hydrophobicity of the peptidomimetics improved the antimicrobial activity against S. aureus, however there is no simple correlation between the biological activity and the strength of interactions of the peptidyl with bacterial membrane.

Acute aquatic toxicity assessment of six anti-cancer drugs and one metabolite using biotest battery - Biological effects and stability under test conditions.

Available ecotoxicological data for anti-cancer drugs and their metabolites are incomplete, and only some studies have been accompanied by chemical analysis. Therefore, the main aim of this study was to evaluate the acute toxicity of the six most commonly used cytostatics, namely cyclophosphamide (CF), ifosfamide (IF), 5-fluorouracil (5-FU), imatinib (IMT), tamoxifen (TAM) and methotrexate (MET) and its metabolite - 7-hydroxymethotrexate (7-OH-MET), towards selected aquatic organisms, namely bacteria Vibrio fischeri, algae Raphidocelis subcapitata, crustaceans Daphnia magna and duckweed Lemna minor. All ecotoxicological tests were accompanied by chemical analysis to determine the differences between nominal and actual concentrations of investigated compounds and their stability under test conditions. For unstable compounds, tests were performed in static and semi-static conditions. It was observed that L. minor was the most sensitive organism. The compounds that were most toxic to aquatic organisms were 5-FU (highly toxic to algae, EC50 = 0.075 mg L-1), MET and TAM (very toxic to highly toxic to duckweed depending on the test conditions; EC50MET 0.08-0.16 mg L-1, EC50TAM 0.18-0.23 mg L-1). It is suspected that MET and 5-FU mainly affected algae and plants most probably because the exposure time was long enough for them to cause a specific effect (they inhibit DNA replication and act predominantly on actively dividing cells). Furthermore, the obtained results also suggest that the toxicity of the metabolites/potentially produced degradation products of MET towards duckweed is lower than that of the parent form, whereas the toxicity of TAM degradation products is in the same range as that of TAM.

Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I.

The Thermus sp. family of bifunctional type IIS/IIG/IIC restriction endonucleases (REase)-methyltransferases (MTase) comprises thermo-stable TaqII, TspGWI, TspDTI, TsoI, Tth111II/TthHB27I enzymes as well as a number of putative enzymes/open reading frames (ORFs). All of the family members share properties including a large protein size (ca. 120kDa), amino acid (aa) sequence homologies, enzymatic activity modulation by S-adenosylmethionine (SAM), recognition of similar asymmetric cognate DNA sites and cleavage at a distance of 11/9 nt. Analysis of the enzyme aa sequences and domain/motif organisation led to further Thermus sp. family division into the TspDTI and TspGWI subfamilies. The latter exhibits an unprecedented phenomenon of DNA recognition change upon substitution of SAM by its analogue, sinefungin (SIN), towards a very frequent DNA cleavage. We report cloning in Escherichia coli (E. coli), using a two-stage procedure and a putative tthHB27IRM gene, detected by bioinformatics analysis of the Thermus thermophilus HB27 (T. thermophilus) genome. The functionality of a 3366 base pair (bp)-/1121 aa-long, high GC content ORF was validated experimentally through the expression in E. coli. Protein features corroborated with the reclassification of TthHB27I into the TspDTI subfamily, which manifested in terms of aa-sequence/motif homologies and insensitivity to SIN-induced specificity shift. However, both SAM and SIN stimulated the REase DNA cleavage activity by at least 16-32 times; the highest was observed for the Thermus sp. family. The availability of TthHB27I and the need to include SAM or SIN in the reaction in order to convert the enzyme from "hibernation" status to efficient DNA cleavage is of practical significance in molecular biotechnology, extending the palette of available REase specificities.

Analysis of structure-cytotoxicity in vitro relationship (SAR) for perfluorinated carboxylic acids.

Perfluorinated carboxylic acids (PFAs) represent derivatives of naturally occurring compounds and have been widely used in various industrial fields for decades. They are known to be environmentally persistent. Thus far numerous reports have been focused on reproductive toxicity of PFAs in animals but few studies have been carried out on toxicity towards human cells. Viability tests were performed here at varying time-exposures on C6-C18 PFAs with human colon carcinoma (HCT116) cells. These cells were found earlier as the most useful line for in vitro assays. A chain length-EC50 dependence has been clearly observed. Estimated values of EC50 decreased with elongation of fluorocarbon chain (PFHxA > PFHpA > PFOA > PFNA > PFDA > PFDoA > PFTeDA). Further elongation (C16 and C18) did not deepen the effect but even partially reversed it. The effect was intensified after longer exposure (72 h); at relatively low 40 microM PFTeDA, the viability decreased to approximately 50%. It seems that PFAs are not acutely toxic at the cellular level. Even so, however, they can trigger cell apoptosis, which is prominent in the case of myristic acid perfluorinated analogue.