Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity-dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium-induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

Biological Activity of Newly Synthesized Benzimidazole and Benzothizole 2,5-Disubstituted Furane Derivatives.

Newly designed and synthesized cyano, amidino and acrylonitrile 2,5-disubstituted furane derivatives with either benzimidazole/benzothiazole nuclei have been evaluated for antitumor and antimicrobial activity. For potential antitumor activity, the compounds were tested in 2D and 3D cell culture methods on three human lung cancer cell lines, A549, HCC827 and NCI-H358, with MTS cytotoxicity and BrdU proliferation assays in vitro. Compounds 5, 6, 8, 9 and 15 have been proven to be compounds with potential antitumor activity with high potential to stop the proliferation of cells. In general, benzothiazole derivatives were more active in comparison to benzimidazole derivatives. Antimicrobial activity was evaluated with Broth microdilution testing (according to CLSI (Clinical Laboratory Standards Institute) guidelines) on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Additionally, Saccharomyces cerevisiae was included in testing as a eukaryotic model organism. Compounds 5, 6, 8, 9 and 15 showed the most promising antibacterial activity. In general, the compounds showed antitumor activity, higher in 2D assays in comparison with 3D assays, on all three cell lines in both assays. In natural conditions, compounds with such an activity profile (less toxic but still effective against tumor growth) could be promising new antitumor drugs. Some of the tested compounds showed antimicrobial activity. In contrast to ctDNA, the presence of nitro group or chlorine in selected furane-benzothiazole structures did not influence the binding mode with AT-DNA. All compounds dominantly bound inside the minor groove of AT-DNA either in form of monomers or dimer and higher-order aggregates.

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles.

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.

Antiproliferative activity of amino substituted benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles explored by 2D and 3D cell culture system.

Benzimidazo[1,2-a]quinolines and benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles with amino chains on the different positions have been evaluated by 2D and 3D assays on the human breast cancer cells. Pentacyclic derivatives were synthesized by microwave assisted amination to study the influence of the thiophene substructure on antitumor activity in comparison to tetracyclic analogues. The results obtained from 2D assay reveals that the antitumor activity is strongly dependent on the nature and position of amino chains. Tetracyclic derivatives displayed selective activity on SK-BR-3 with the 2-amino substituted derivatives as the most active ones while pentacyclic derivatives 6-16 and 21-25 showed more pronounced activity on T-47D. The evaluation of antitumor activity in the 3D assay pointed out that some of the tetracyclic and pentacyclic amino substituted derivatives showed selective activity on the MDA-MB-231 cell line. Influence of physico-chemical properties of the compounds on antiproliferative activity have been investigated by multivariate statistical methods. As a measure of lipophilicity, experimental Chrom LogD values have been determined and number of structural parameters have been calculated for investigated compounds. Main factors contributing to the antiproliferative effect for both 2D and 3D cell cultures are found to be basicity, lipophilicity, molecular weight and number of H-bond donors.

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems.

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure-activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.

Satellite DNA as a driver of population divergence in the red flour beetle Tribolium castaneum.

Tandemly repeated satellite DNAs are among most rapidly evolving sequences in eukaryotic genome, usually differing significantly among closely related species. By inducing changes in heterochromatin and/or centromere, satellite DNAs are expected to drive population and species divergence. However, despite high evolutionary dynamics, divergence of satellite DNA profiles at the level of natural population which precedes and possibly triggers speciation process is not readily detected. Here, we characterize minor TCAST2 satellite DNA of the red flour beetle Tribolium castaneum and follow its dynamics among wild-type strains originating from diverse geographic locations. The investigation revealed presence of three distinct subfamilies of TCAST2 satellite DNA which differ in monomer size, genome organization, and subfamily specific mutations. Subfamilies Tcast2a and Tcast2b are tandemly arranged within pericentromeric heterochromatin whereas Tcast2c is preferentially dispersed within euchromatin of all chromosomes. Among strains, TCAST2 subfamilies are conserved in sequence but exhibit a significant content variability. This results in overrepresentation or almost complete absence of particular subfamily in some strains and enables discrimination between strains. It is proposed that homologous recombination, probably stimulated by environmental stress, is responsible for the emergence of TCAST2 satellite subfamilies, their copy number variation and dispersion within genome. The results represent the first evidence for the existence of population-specific satellite DNA profiles. Partial organization of TCAST2 satellite DNA in the form of single repeats dispersed within euchromatin additionally contributes to the genome divergence at the population level.