alpha-Collatolic acid

Background: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. Purpose: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. Methods: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. Results: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. Conclusion: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.

Introduction: Lichens, due to the presence of own secondary metabolites such as depsidones and depsides, became a promising source of health-promoting organisms with pharmacological activities. However, lichens and their active compounds have been much less studied. Therefore, the present study aims to evaluate for the first time the antioxidant capacity and enzyme inhibitory activities of 14 lichen extracts belonging to cetrarioid clade in order to identify new natural products with potential pharmacological activity. Materials and methods: In this study, an integrated strategy was applied combining multivariate statistical analysis (principal component analysis and hierarchical cluster analysis), phytochemical identification, activity evaluation (in vitro battery of antioxidant assays FRAP, DPPH, and ORAC), and enzyme inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and molecular profiling with in silico docking studies of the most promising secondary metabolites. Results. Among fourteen lichen samples, Dactylina arctica stands out for its higher antioxidant capacities, followed by Nephromopsis stracheyi, Tuckermannopsis americana, Vulpicida pinastri, and Asahinea scholanderi. Moreover, Asahinea scholanderi and Cetraria cucullata extracts were the best inhibitors of AChE and BuChE. The major secondary metabolites identified by HPLC were alectoronic acid and α-collatolic acid for Asahinea scholanderi and usnic acid and protolichesterinic acid for Cetraria cucullata. Molecular docking studies revealed that alectoronic acid exhibited the strongest binding affinity with both AChE and BuChE with and without water molecules. Conclusions: Our results concluded that these species could be effective in the treatment of neurodegenerative diseases, being mandatory further investigation in cell culture and in vivo models.

The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, for the lichen compounds were evaluated. The MIC90 was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC90 was ranging from onefold up to twofold the MIC90 for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.