Diterpenoids are important compounds for plant survival and have beneficial properties for humans. Bioactive abietanic diterpenes are synthesized in roots of Salvia sclarea (e.g. aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol), but at a very low level (about 1 % of root dry weight). To enhance the biosynthesis of this interesting class of compounds, heterologous AtDXS (d-xylulose 5-phosphate synthase) or AtDXR (1-deoxy-d-xylulose 5 phosphate reductoisomerase) genes, encoding the up-stream enzymes of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP)-dependent terpenoid pathway, were ectopically expressed in S. sclarea hairy roots. Quantitative targeted metabolic analysis (HPLC–DAD) revealed that three independent root lines, expressing different levels of DXS or DXR transcripts and proteins, synthesized a significant higher content of abietanic diterpenes, compared to the control hairy root line transformed with the empty vector. The increase was gene-dependent, since the overexpression of the AtDXR triggered a 4.4-fold increase in aethiopinone, an abietane quinone-type tricyclic diterpene. In addition, aethiopinone was proved to be cytotoxic to different solid tumor cell lines, with the highest effect on human melanoma A375 cell line (IC50 11.4 µM). Overall these results show that it is possible to boost the metabolic flow towards the synthesis of abietanic diterpenes in S. sclarea hairy roots by overexpressing genes involved in the first steps of the MEP-pathway and provide new insights for the large-scale production of this class of compounds, with potential application in cancer treatment

The identification of inhibitors of Hsp90 is currently a primary goal in the development of more effective drugs for the treatment of various types of multidrug resistant malignancies. In an attempt to identify new small molecules modulating the activity of Hsp90, we screened a small library of tetranortriterpenes. A high-affinity interaction with Hsp90 inducible form was uncovered for eight of these compounds, five of which are described here for the first time. By monitoring the ATPase activity and the citrate synthase thermal induced aggregation, compound 1 (cedrelosin A), 3 (7α-limonylacetate), and 5 (cedrelosin B), containing a limonol moiety, were found to be the most effective in compromising the Hsp90α chaperone activity. Consistent with these findings, the three compounds caused a depletion of c-Raf and pAkt Hsp90 client proteins in HeLa and MCF/7 cell lines. Induced fit docking protocol and molecular dynamics were used to rationalize the structural basis of the biological activity of the limonol derivatives. Taken together, these results point to limonol-derivatives as promising scaffolds for the design of novel Hsp90α inhibitors.

Cyclic peptoids have recently emerged as important examples of peptidomimetics for their interesting complexing properties and innate ability to permeate biological barriers. In the present contribution, experimental and theoretical data evidence the intricate conformational and stereochemical properties of five novel hexameric peptoids decorated with N-isopropyl, N-isobutyl, and N-benzyl substituents. Complexation studies by NMR, in the presence of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), theoretical calculations, and single-crystal X-ray analyses indicate that the conformationally stable host/guest metal adducts display architectural ordering comparable to that of the enniatins and beauvericin mycotoxins. Similarly to the natural depsipeptides, the synthetic oligolactam analogues show a correlation between ion transport abilities in artificial liposomes and cytotoxic activity on human cancer cell lines. The reported results demonstrate that the versatile cyclic peptoid scaffold, for its remarkable conformational and complexing properties, can morphologically mimic related natural products and elicit powerful biological activities.

Plant abietane diterpenoids (e.g. aethiopinone, 1- oxoaethiopinone, salvipisone and ferruginol), synthesized in the roots of several Salvia spp, have antibacterial, antifungal, sedative and anti-proliferative properties. Recently we have reported that content of these compounds in S. sclarea hairy roots is strongly depending on transcriptional regulation of genes belonging to the plastidial MEP-dependent terpenoid pathway, from which they mostly derive. To boost the synthesis of this interesting class of compounds, heterologous AtWRKY18, AtWRKY40, and AtMYC2 TFs were overexpressed in S. sclarea hairy roots and proved to regulate in a coordinated manner the expression of several genes encoding enzymes of the MEP-dependent pathway, especially DXS, DXR, GGPPS and CPPS. The content of total abietane diterpenes was enhanced in all overexpressing lines, although in a variable manner due to a negative pleiotropic effect on HR growth. Interestingly, in the best performing HR lines overexpressing the AtWRKY40 TF induced a significant 4-fold increase in the final yield of aethiopinone, for which we have reported an interesting anti-proliferative activity against resistant melanoma cells. The present results are also informative and instrumental to enhance the synthesis of abietane diterpenes derived from the plastidial MEP-derived terpenoid pathway in other Salvia species.

Transcriptional activation of genes belonging to the plastidial MEP-derived isoprenoid pathway by elicitation with methyl jasmonate and coronatine enhanced the content of bioactive abietane diterpenes in Salvia sclarea hairy roots. We have shown that aethiopinone, an abietane diterpene synthesized in Salvia sclarea roots is cytotoxic and induces apoptosis in human melanoma cells. To develop a production platform for this compound and other abietane diterpenes, hairy root technology was combined with the elicitation of methyl jasmonate (MeJA) or the phytotoxin coronatine (Cor). Both MeJA and Cor induced a significant accumulation of aethiopinone, but prolonged exposure to MeJA irremediably caused inhibition of hairy root growth, which was unaffected by Cor treatment. Considering together the fold increase in aethiopinone content and the final hairy root biomass, the best combination was a Cor treatment for 28 days, which allowed to obtain up to 105.34 ± 2.30 mg L of this compound to be obtained, corresponding to a 24-fold increase above the basal content in untreated hairy roots. MeJA or Cor elicitation also enhanced the synthesis of other bioactive abietane-quinone diterpenes. The elicitor-dependent steering effect was due to a coordinated transcriptional activation of several biosynthetic genes belonging to the plastidial MEP-derived isoprenoid pathway. High correlations between aethiopinone content and MeJA or Cor-elicited level of gene transcripts were found for DXS2 (r = 0.99), DXR (r = 0.99), and GGPPS (r = 0.98), encoding enzymes acting upstream of GGPP, the common precursor of diterpenes and other plastidial-derived terpenes, as well as CPPS (r = 0.99), encoding the enzyme involved in the first cyclization steps leading to copalyl-diphosphate, the precursor of abietane-like diterpenes. These results point to these genes as possible targets of metabolic engineering approaches to establish a more efficient production platform for such promising anti-proliferative plant-derived compounds.