Functional Analysis of Hsp70 Inhibitors

<div><p>The molecular chaperones of the Hsp70 family have been recognized as targets for anti-cancer therapy. Since several paralogs of Hsp70 proteins exist in cytosol, endoplasmic reticulum and mitochondria, we investigated which isoform needs to be down-regulated for reducing viability of cancer cells. For two recently identified small molecule inhibitors, VER-155008 and 2-phenylethynesulfonamide (PES), which are proposed to target different sites in Hsp70s, we analyzed the molecular mode of action in vitro. We found that for significant reduction of viability of cancer cells simultaneous knockdown of heat-inducible Hsp70 (HSPA1) and constitutive Hsc70 (HSPA8) is necessary. The compound VER-155008, which binds to the nucleotide binding site of Hsp70, arrests the nucleotide binding domain (NBD) in a half-open conformation and thereby acts as ATP-competitive inhibitor that prevents allosteric control between NBD and substrate binding domain (SBD). Compound PES interacts with the SBD of Hsp70 in an unspecific, detergent-like fashion, under the conditions tested. None of the two inhibitors investigated was isoform-specific.</p></div

VER-155008 inhibits Hsp70's ATPase activity in a competitive manner and slows down nucleotide association.

<p>Steady-state ATPase activity of Hsp70 (4 µM) in the absence (<b>A, C</b>) or presence (<b>B, C</b>) of 1 µM Hdj1. VER-155008 (<b>A, B</b>) and PES (<b>C, D</b>) were added as indicated. <b>D</b>, zoom of <b>C</b> for 0 to 12 µM ATP. <i>K_M</i> and <i>K_i</i> values for VER-155008 were determined by global fitting of the data. Differences in K_M and k_cat in the presence of PES as compared to the DMSO control were not statistically significant (t-test, p values of 0.27 to 0.85). In contrast, differences in apparent K_M observed in the presence of VER-155008 was statistically significant (p = 0.05 in absence of Hdj1; p = 0.005 in presence of Hdj1). Error bars represent the standard errors of at least three independent measurements. <b>E, F,</b> Association of MABA-ADP (0.5 µM) to nucleotide-free Hsp70 (0.5 µM) in the presence of VER-155008 (20 µM) and PES (160 µM). Changes in fluorescence intensity were followed in a stopped flow device for 20 (<b>E</b>) and 500 seconds (<b>F</b>). Comparison of the ADP association rates in the absence and presence of PES using the students t-test resulted in a P value of 0.41, indicating that the differences are not statistically significant.</p

Simultaneous knockdown of HSPA1 and HSPA8 are necessary to compromise viability of MDA-MB-468 breast cancer cells.

<p>MDA-MB-468 cells were transfected with siRNAs targeting the indicated heat shock proteins. For HSPA1 as well as HSPA8 and HSPA1+HSPA8 several single siRNAs or mixes were used marked with #1–3. <b>A</b>, Cell viability was measured six days after transfection by using Alamar Blue reagent. As positive control a pool of siRNAs against the mitotic kinase PLK1 was transfected. As negative controls non-targeting siRNAs (Co_4 and Co_5) were used, additionally cells were incubated only with the transfection reagent without siRNAs (TM) or left untreated (untr.). Viability is shown as % of TM. Only simultaneous transfection of HSPA1 and HSPA8 specific siRNAs induced a significant loss of viability. Additional knockdown of HSPA2 or HSPA5 had no further effect. <b>B</b> and <b>C</b>, Knockdown was confirmed by Western Blot analysis using HSPA1 and/or HSPA8 specific antibodies and infrared-labeled secondary antibodies three days after transfection of siRNAs. Knockdown efficiency was quantified by detecting signals with Odyssey infrared imaging system and is specified as % of TM.</p

VER-155008 and PES inhibit reactivation of firefly luciferase.

<p><b>A</b>, Structure of the small molecule inhibitors VER-155008 and PES. <b>B–E</b>, Refolding of thermally denatured firefly luciferase by Hsp70 (<b>B, D</b>) and Hsc70 (<b>C, E</b>) in the presence of different concentrations of VER-155008 (<b>B, C</b>) or PES (<b>D, E</b>) (80 nM luciferase, 4 µM Hsp70/Hsc70, 2 µM Hdj1, 0.4 µM Apg2). Luciferase activity is plotted as a fraction of the native luciferase control. Error bars represent standard errors of three independent experiments. ANOVA analysis of the 120-min-values indicates highly significant differences between control and inhibitor treated samples (p<0.001) except for Hsp70 inhibition by VER-155008 (p = 0.011).</p

Kinetic constants of the Hsp70-substrate and Hsp70-nucleotide interaction in the presence or absence of inhibitors.

<p>n.d., not determined.</p

Crystal structure of human Hsp70 NBD in complex with VER-155008.

<p>(PDB entry code 4IO8). <b>A</b>, Zoom into the nucleotide binding pocket with NBD in cartoon and transparent surface representation. VER-155008 interacting residues are shown as sticks with hydrogen bonds (K271, S275 and water molecules) and π-stacking R272 indicated as dotted and dashed lines, respectively. <b>B</b>, overlay of the structure of human Hsc70 NBD in complex with Bag1 and VER-155008 (green, PDB ID 3FZL; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078443#pone.0078443-Williamson1" target="_blank">[24]</a>) and our structure (gold) in cartoon representation with VER-155008 in stick representation colored according to the elements with carbon colored in green (3FZL) and gold (our structure). <b>C</b>, overlay of bovine Hsc70 NBD K71M variant in complex with ATP (blue, PDB ID 1KAX; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078443#pone.0078443-OBrien1" target="_blank">[58]</a>) and our structure (gold) in cartoon representation with VER-155008 and ATP in stick representation colored according to the elements with carbon colored in blue (1KAX) and gold (our structure).</p

Substrate binding remains unchanged in the presence of small molecules.

<p><b>A</b> and <b>B</b>, PES does not block the Hsp70-substrate interaction. Dissociation equilibrium titration of Hsp70 with d-NR-peptide. <b>A</b>, Selected fluorescence emission spectra of d-NR-peptide bound to different concentrations of Hsp70 as indicated in the presence or absence of 160 µM PES. <b>B</b>, Fluorescence intensity at 525 nm plotted against the Hsp70 concentration (one of four independent experiments shown). We determined the <i>K_d</i> values by fitting the quadratic solution of the law of mass action to the data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078443#pone-0078443-t001" target="_blank">Table 1</a>). Paired t-test analysis did not reveal any statistically significant difference between the K_d values in the absence and presence of PES (p = 0.53). <b>C</b>, Dissociation of d-NR peptide from Hsp70 in the presence of VER-155008 or PES. The fluorescence decay was fitted by a single exponential decay to obtain <i>k_off</i> values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078443#pone-0078443-t001" target="_blank">Table 1</a>). <b>D</b>, Fluorescence changes observed during the association of 1 µM d-NR-peptide to 1 µM Hsp70 in the absence and presence of inhibitors. ANOVA analysis demonstrated that neither VER-155008 nor PES caused any statistically significant difference (p = 0.32). <b>E and F</b>, ATP-stimulated dissociation of d-NR-peptide from Hsp70 in the presence of different concentrations of VER-155008 (<b>E</b>) and PES (<b>F</b>) as measured in a stopped flow apparatus. ANOVA analysis of the results show that the differences observed in the presence of VER-155008 are highly statistically significant (p<0.0001). In contrast, differences in the presence of PES are not significant (p = 0.63). The individual curves in <b>C–F</b> are y-transformed for better visibility.</p

Discovery of Cycloalkyl[<i>c</i>]thiophenes as Novel Scaffolds for Hypoxia-Inducible Factor-2α Inhibitors

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy–aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology