Quinic acids from Aster caucasicus and from transgenic callus expressing a beta-amyrin synthase

Several different classes of secondary metabolites, including flavonoids, triterpenoid saponins and quinic acid derivatives, are found in Aster spp. (Fam. Asteraceae). Several Aster compounds revealed biological as well as pharmacological activities. In this work, a phytochemical investigation of A. caucasicus evidenced the presence of quinic acid derivatives, as well as the absence of triterpene saponins. To combine in one species the production of different phytochemicals, including triterpenes, an Agrobacterium-mediated transformation of A. caucasicus was set up to introduce A. sedifolius beta-amyrin synthase (AsOXA1)-encoding gene under the control of the constitutive promoter CaMV35S. The quali-quantitative analysis of transgenic calli with ectopic expression of AsOXA1 showed, in one sample, a negligible amount of triterpene saponins combined with higher amount of quinic acid derivatives as compared with the wild type callus.     

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral–chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.     

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells

Background: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. Methods: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. Results: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD’s catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. Conclusions: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.     

Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAF^V600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAF^V600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC₅₀ values in the 40–88 nM range. Selected compounds inhibited BRAF^V600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.     

Thermal behaviour of hydrated lysozyme in the presence of sucrose and trehalose by EINS

The main aim of the present work is to investigate the dynamical effects of the addition of disaccharides to hydrated lysozyme. The Self-Distribution-Function procedure is at first applied on Elastic Incoherent Neutron Scattering data obtained by the IN13 spectrometer on trehalose/H₂O and sucrose/H₂O where it highlights a different Q-dependence for the two disaccharides. Then a quantitative analysis of the Mean Square Displacement of lysozyme/trehalose/H₂O and of lysozyme/sucrose/H₂O from Elastic Incoherent Neutron Scattering data obtained by the IN10 spectrometer is presented. It is shown how the resolution function gives rise to a time integration of a given time-dependent Mean Square Displacement function. Furthermore the analysis shows that the protein dynamical transition, registered at a temperature value of about T = 200-220 K in the H₂O hydrated lysozyme sample, is inhibited by the addition of the disaccharides.     

Glaucopine C, a new diterpene from the fruiting bodies of Sarcodon glaucopus

In this work the mushroom Sarcodon glaucopus was studied. A new cyathane, glaucopine C (1), was isolated from the hexane extract and identified by 1H and 13C NMR spectra analysis. Glaucopine C showed anti-inflammatory acitvity.     

Double decarboxylative Claisen rearrangement reactions: microwave-assisted de novo synthesis of pyridines

Microwave-assisted double decarboxylative Claisen rearrangement of bis(allyl) 2-tosylmalonates provides substituted 1,6-heptadienes, which may be alkylated, and then converted into pyridines by ozonolysis followed by reaction with ammonia generated in situ under microwave conditions.     

Strategies to control the particle size distribution of poly-epsilon-caprolactone nanoparticles for pharmaceutical applications

In this work turbulent precipitation through solvent displacement for the production of poly-epsilon-caprolactone (PCL) nanoparticles is investigated; two different PCL molecular weights have been employed, using acetone and water as solvent and anti-solvent, respectively. The main important thermodynamic and kinetic parameters, such as solubility and interfacial tension of PCL in water-acetone mixtures, are determined and the effect of the process operating conditions on the final particle size distribution is also investigated. Particles produced under different conditions into a Confined Impinging Jets Reactor (CIJR) were characterized by Dynamic Light Scattering, Zeta potential measurements and Scanning Electronic Microscopy. Results clearly show the strong effect of mixing on the particle size distribution and how mixing must be controlled in order to obtain a product with particular characteristics. Eventually the measured thermodynamic and kinetic parameters are used to interpret the obtained experimental data.