Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity.

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.

Cyanobacterial cell-wall components as emerging environmental toxicants - detection and holistic monitoring by cellular signaling biosensors.

Cyanobacterial blooms constitute a recognized danger to aquatic environment and public health not only due to presence of main group of cyanotoxins, such as microcystins, cylindrospermopsin or anatoxin-a, but also other emerging bioactivities. An innovative approach identifying such bioactivities is the application of cellular biosensors based on reporter genes which detect the impact of cyanobacterial cells and components on actual human cells in a physiological-like setting. In the present study biosensor cell lines detecting four different types of bioactivities (ARE - oxidative stress, NFKBRE - immunomodulatory pathogen-associated molecular patterns, AHRE - persistent organic pollutants, GRE - endocrine disruptors) were exposed to concentrated cyanobacterial cells from 21 environmental bloom samples and from eight cultures (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii and Raphidiopsis raciborskii). The AHRE and GRE biosensors did not detect any relevant bioactivity. In turn, ARE biosensors were significantly activated by bloom samples from Jeziorsko (180-250%) and Sulejów (250-400%) reservoirs with the highest cyanobacterial biomass, while activation by cultures was weak/undetectable. The same biosensors were stimulated by microcystin-LR (250%) and anatoxin-a (150%). The NFKBRE biosensors were activated to varying extent (140-650%) by most bloom and culture samples, pointing to potential immunomodulatory toxic effects on humans. Lipopolysaccharide and lipoproteins were identified as responsible for NFKBRE activation (probably via pattern recognition receptors), while peptidoglycan had no bioactivity in this assay. Thus, the holistic approach to sample analysis with the application of cellular biosensors geared towards 4 separate pathways/bioactivities was validated for identification of novel bioactivities in organisms with recognized public health significance (e.g. this study is the first to describe cyanobacterial lipoproteins as potential environmental immunomodulators). Moreover, the ability of cellular biosensors to be activated by intact cyanobacterial cells from blooms provides proof of concept of their direct application for environmental monitoring, especially comparison of potential threats without need for chemical analysis and identification of toxicants.

Application of cellular biosensors for detection of atypical toxic bioactivity in microcystin-containing cyanobacterial extracts.

Despite the focus of most ecotoxicological studies on cyanobacteria on a select group of cyanotoxins, especially microcystins, a growing body of evidence points to the involvement of other cyanobacterial metabolites in deleterious health effects. In the present study, original, self-developed reporter gene-based cellular biosensors, detecting activation of the main human xenobiotic stress response pathways, PXR and NFkappaB, were applied to detect novel potentially toxic bioactivities in extracts from freshwater microcystin-producing cyanobacterial blooms. Crude and purified extracts from cyanobacteria containing varying levels of microcystins, and standard microcystin-LR were tested. Two cellular biosensor types applied in this study, called NHRTOX (detecting PXR activation) and OXIBIOS (detecting NFkappaB activation), successfully detected potentially toxic or immunomodulating bioactivities in cyanobacterial extracts. The level of biosensor activation was comparable to control cognate environmental toxins. Despite the fact that extracts were derived from microcystin-producing cyanobacterial blooms and contained active microcystins, biosensor-detected bioactivities were shown to be unrelated to microcystin levels. Experimental results suggest the involvement of environmental toxins (causing a response in NHRTOX) and lipopolysaccharides (LPS) or other cell wall components (causing a response in OXIBIOS) in the potentially harmful bioactivity of investigated extracts. These results demonstrate the need for further identification of cyanobacterial metabolites other than commonly studied cyanotoxins as sources of health risk, show the usefulness of cellular biosensors for this purpose and suggest a novel, more holistic approach to environmental monitoring.