Schematic representation of the results obtained from limited proteolysis experiments.

<p>The preferential cleavage sites detected on recombinant Hsp90α and on the Hsp90α/geraniin complex are in black. The Hsp90α <i>N</i>-terminal domain is highlighted in light grey, the middle domain is boxed and the <i>C</i>-terminal domain is highlighted in grey.</p

Structure of the 54 tested compounds.

<p>Geraniin structure (54) is evidenced.</p

Inhibition of Hsp90α activity.

<p>ATPase activity of the chaperone was evaluated in the presence of different concentrations of geraniin, 17-AAG and HA (<b>A</b>). Data are reported as the residual ATPase activity (%) compared to that observed for an untreated sample. Data are the mean of three independent experiments performed in triplicate and were analyzed by t test (HA vs testing compounds): The error bar represents the standard deviation of nine measurements, while * indicates significance at P<0.01. Aggregation kinetics of citrate synthase (CS) at 43°C determined by light scattering <b>(B</b>)<b>.</b> The spontaneous aggregation of CS at 43°C (♦) and the aggregation of CS at 43°C in the presence of 0.075 µM Hsp90 α and 0.3 µM ATP (▴), 0.075 µM Hsp90 α, 0.3 µM ATP and 0.3 µM geraniin (▪), or 0.075 µM Hsp90α, 0.3 µM ATP and 0.3 µM HA (•) are shown. Kinetics traces reported are the averages of three separated measurements; the error bar represents the standard deviation of three measurements.</p

Cell viability (%) of cancer cells and normal cells treated for 24 h with geraniin or 17-AAG.

<p>Jurkat, HeLa and PBMC cells were incubated for 24(<b>A</b>) or 17-AAG (<b>B</b>) used in different concentrations (0.5–50 µM) and (0.1–20 µM), respectively, and processed for cell proliferation determination by the MTT assay.</p

Effect of geraniin on Hsp90 client protein levels.

<p>Equal amounts (30 µg) of whole-cell lysates were separated on SDS-PAGE and client proteins were visualized by western blot analysis using specific antibodies. Actin was used as loading control. Total cellular proteins were extracted 24 h after treatment with geraniin (2.5 µM, 5 µM and 10 µM) in Jurkat cells (<b>A</b>) or geraniin (1 µM, 0.7 µM and 0.5 µM) in HeLa cells (<b>B</b>), ctrl = control cells treated with DMSO). The blots shown are representative of three different experiments with similar results.</p

SPR results.

<p>Sensorgrams obtained by injecting 25(▪), 50 nM (♦), 250 nM (▴) and 1 µM (•) of geraniin (<b>A</b>), compound <b>1</b> (<b>B</b>), 17-AAG (<b>C</b>), and HA (<b>D</b>) on immobilized Hsp90α.</p

Effects of geraniin and 17-AAG on cell cycle progression.

<p>Percentage of cell cycle stages was analyzed by flow cytometry, (<b>A</b>) PI-stained viable Jurkat cells treated with DMSO, 0.7 µM geraniin or 10 µM 17-AAG for 24 h. (<b>B</b>) PI-stained viable HeLa cells treated with DMSO, 5 µM geraniin or 0.2 µM 17-AAG for 24 h, (<b>C</b>) The percentage of hypodiploid cells as treated in <b>A</b> and <b>B</b>. Results are expressed as means ± SD of three experiments performed in duplicate (***<i>P</i><0.001).</p

Thermodynamic constants measured by SPR for the interaction between tested compounds and immobilized HSP90α.

<p>Thermodynamic constants measured by SPR for the interaction between tested compounds and immobilized HSP90α.</p

Sesterterpenes as Tubulin Tyrosine Ligase Inhibitors. First Insight of Structure−Activity Relationships and Discovery of New Lead

Twenty-four new sesterterpenes, compounds 1−24, were isolated from the aerial parts of Salvia dominica. Their structures were elucidated by 1D and 2D NMR experiments as well as ESIMS analysis and chemical methods. The evaluation of the biological activity of Salvia dominica sesterterpenes by means of a panel of chemical and biological approaches, including chemical proteomics, surface plasmon resonance (SPR) measurements, and biochemical assays were realized. Obtained results showed that 18 out of the 24 sesterterpene lactones isolated from Salvia dominica interact with tubulin−tyrosine ligase (TTL) an enzyme involved in the tyrosination cycle of the C-terminal of tubulin, and inhibit TTL activity in cancer cells. Besides, results of our studies provided an activity/structure relationship that can be used to design effective TTL inhibitors

Bioactive Limonoids from the Leaves of <i>Azaridachta indica</i> (Neem)

Eight new limonoids (1–8) and one new phenol glycoside (9), along with six known compounds, were isolated from the leaves of Azaridachta indica. The structures of 1–9 were elucidated on the basis of spectroscopic data analysis. Compounds isolated were assayed for their cytotoxicity against different cancer cell lines. Moreover, their ability to interact with the molecular chaperone Hsp90, affecting its biological activity, was tested