Chemo- and regio-selective enzymatic lipophilisation of rutin, and physicochemical and antioxidant properties of rutin ester derivatives.

Enzymes are one of the most powerful tools in organic Green Chemistry and enzymatic reactions offer numerous advantages like regio- and enantio-selectivity along with their eco-friendly and sustainable nature. More specifically, lipases can catalyse both ester hydrolysis and formation depending on the nature of the substrate and water content. Herein, the focus is on the development of an enzymatically catalysed lipophilisation of natural compounds using lipases of microbial origin and the investigation of the optimal reaction conditions, aiming ultimately to ameliorate the compounds' properties. The flavonoid disaccharide rutin (quercetin-3-O-rutinoside) was the model compound on which the acylation protocol was built, allowing an efficient procedure to be established, while simultaneously offering the possibility of developing rapid, clear and robust methodologies, using state-of-the-art techniques, for analysis and purification of the synthesized compounds. An optimal 72 h reaction at 55 °C, using Candida antarctica lipase B immobilized on acrylic resin, combined with silicon dioxide as dehydrating agent, followed by product purification, achieved conversion ratios up to 50%. Full characterization and evaluation of the physicochemical and antioxidant properties of the esterified compounds was obtained. The lipophilicity of the rutin esters produced increased with increasing alkyl chain length, yet antioxidant properties were unaffected in comparison with the parent compound. A preparatively useful acylation protocol was established, allowing full investigation into the properties of the acylated compounds. It is also applicable for use on mixtures of compounds as most natural products are found in nature in mixtures and such a development greatly enhances the potential of this method for future commercial applications.

A Biomimetic, One-Step Transformation of Simple Indolic Compounds to Malassezia-Related Alkaloids with High AhR Potency and Efficacy.

Malassezia furfur isolates from diseased skin preferentially biosynthesize compounds which are among the most active known aryl-hydrocarbon receptor (AhR) inducers, such as indirubin, tryptanthrin, indolo[3,2-b]carbazole, and 6-formylindolo[3,2-b]carbazole. In our effort to study their production from Malassezia spp., we investigated the role of indole-3-carbaldehyde (I3A), the most abundant metabolite of Malassezia when grown on tryptophan agar, as a possible starting material for the biosynthesis of the alkaloids. Treatment of I3A with H2O2 and use of catalysts like diphenyldiselenide resulted in the simultaneous one-step transformation of I3A to indirubin and tryptanthrin in good yields. The same reaction was first applied on simple indole and then on substituted indoles and indole-3-carbaldehydes, leading to a series of mono- and bisubstituted indirubins and tryptanthrins bearing halogens, alkyl, or carbomethoxy groups. Afterward, they were evaluated for their AhR agonist activity in recombinant human and mouse hepatoma cell lines containing a stably transfected AhR-response luciferase reporter gene. Among them, 3,9-dibromotryptanthrin was found to be equipotent to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an AhR agonist, and 3-bromotryptanthrin was 10-times more potent than TCDD in the human HG2L7.5c1 cell line. In contrast, 3,9-dibromotryptanthrin and 3-bromotryptanthrin were ∼4000 and >10,000 times less potent than TCDD in the mouse H1L7.5c3 cell line, respectively, demonstrating that they are species-specific AhR agonists. Involvement of the AhR in the action of 3-bromotryptanthrin was confirmed by the ability of the AhR antagonists CH223191 and SR1 to inhibit 3-bromotryptanthrin-dependent reporter gene induction in human HG2L7.5c1 cells. In conclusion, I3A can be the starting material used by Malassezia for the production of both indirubin and tryptanthrin through an oxidation mechanism, and modification of these compounds can produce some highly potent, efficacious and species-selective AhR agonists.

Antifungal activity of selected Malassezia indolic compounds detected in culture.

BACKGROUND: Malassezia yeasts produce bioactive indolic substances when grown on L-tryptophan agar. A panel of these substances was tested against commensal and opportunistic fungi, the Minimum Inhibitory Concentration (MIC) was determined and the potential for in loco antifungal activity on the skin was assessed. MATERIALS AND METHODS: Eight indoles were included (malassezin, pityriacitrin, indirubin, indolo[3,2-b]carbazole, 6-formylindolo[3,2-b]carbazole, tryptanthrin, 6-hydroxymethylindolo[3,2-b]carbazole and 6-methylindolo[3,2-b]carbazole) and were tested against 40 fungal strains [yeasts: Malassezia spp.(N = 9); Cryptococcus spp.(N = 10); Candida spp.(N = 7); Yarrowia lipolytica(N = 1); Exophialla dermatitidis (N = 2); moulds: Aspergillus spp.(N = 7); Fusarium spp.(N = 2); Rhizopus oryzae(N = 2)]. The concentration of 5/8 of the tested indoles on diseased skin was calculated from published data. Kruskal-Wallis and Mann-Whitney U tests were employed for group susceptibility evaluation in 33 strains. RESULTS: The MIC range was 0.125-32 µg/mL, and the median log2 MIC was four. Indirubin was the most potent antifungal agent and differed significantly from the others. The highest median MIC was found for FICZ. Malassezia with Candida strains were more susceptible compared to Cryptococcus and Aspergillus, and this inhibitory activity was predicted to be valid also on human skin. CONCLUSIONS: Malassezia yeasts produce indolic species that inhibit an array of clinically significant yeasts and moulds.

Discovery and Optimization of a Selective Ligand for the Switch/Sucrose Nonfermenting-Related Bromodomains of Polybromo Protein-1 by the Use of Virtual Screening and Hydration Analysis.

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein-ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 µM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5).

Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast.

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

Malassezia yeasts produce a collection of exceptionally potent activators of the Ah (dioxin) receptor detected in diseased human skin.

Malassezia yeasts are commensal microorganisms, which under insufficiently understood conditions can become pathogenic. We have previously shown that specific strains isolated from diseased human skin can preferentially produce agonists of the aryl hydrocarbon receptor (AhR), whose activation has been linked to certain skin diseases. Investigation of skin scale extracts from patients with Malassezia-associated diseases demonstrated 10- to 1,000-fold higher AhR-activating capacity than control skin extracts. Liquid chromatography-tandem mass spectrometry analysis of the patients' extracts revealed the presence of indirubin, 6-formylindolo[3,2-b]carbazole (FICZ), indolo[3,2-b]carbazole (ICZ), malassezin, and pityriacitrin. The same compounds were also identified in 9 out of 12 Malassezia species culture extracts tested, connecting their presence in skin scales with this yeast. Studying the activity of the Malassezia culture extracts and pure metabolites in HaCaT cells by reverse transcriptase real-time PCR revealed significant alterations in mRNA levels of the endogenous AhR-responsive genes Cyp1A1, Cyp1B1, and AhRR. Indirubin- and FICZ-activated AhR in HaCaT and human HepG2 cells with significantly higher, yet transient, potency as compared with the prototypical AhR ligand, dioxin. In loco synthesis of these highly potent AhR inducers by Malassezia yeasts could have a significant impact on skin homeostatic mechanisms and disease development.