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

Structure of the 54 tested compounds.

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

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

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

SPR analysis results.

<p>Sensorgrams obtained by injecting different concentrations (from 0.020 to 1 <i>µ</i>M) of <b>1</b> (A), <b>2</b> (B), <b>3</b> (C), <b>4</b> (D), <b>5</b> (E), <b>6</b> (F) <b>7</b> (G) and radiciol (H) on immobilized Hsp90.</p

Docking calculation results.

<p>Three dimensional models (A and B) of (+)-lentiginosine (1, yellow) and (−)-lentiginosine (2, green) with HSP90. The target molecule is depicted by sky blue ribbon and the crucial amino acids by cpk (by atom type: C, purple; O, red; N, dark blue, H, white).</p

Structure of compounds 1–7.

<p>Structure of compounds 1–7.</p