HSP70 Inhibitor VER155008 Research Articles

HSP70 contributes to pathogenesis of fulminant hepatitis induced by coronavirus

Fulminant viral hepatitis (FH) represents a significant clinical challenge, with its pathogenesis not yet fully elucidated. Heat shock protein (HSP)70, a molecular chaperone protein with a broad range of cytoprotective functions, is upregulated in response to stress. However, the role of HSP70 in FH remains to be investigated. Notably, HSP70 expression is upregulated in the livers of coronavirus-infected mice and patients. Therefore, we investigated the mechanistic role of HSP70 in coronavirus-associated FH pathogenesis. FH was induced in HSP70-deficient (HSP70 KO) mice or in WT mice treated with the HSP70 inhibitor VER155008 when infected with the mouse hepatitis virus strain A59 (MHV-A59). MHV-A59-infected HSP70 KO mice exhibited significantly reduced liver damage and mortality. This effect was attributed to decreased infiltration of monocyte-macrophages and neutrophils in the liver of HSP70 KO mice, resulting in lower levels of inflammatory cytokines such as IL-1β, TNFα, and IL-6, and a reduced viral load. Moreover, treatment with the HSP70 inhibitor VER155008 protected mice from MHV-A59-induced liver damage and FH mortality. In summary, HSP70 promotes coronavirus-induced FH pathogenesis by enhancing the infiltration of monocyte-macrophages and neutrophils and promoting the secretion of inflammatory cytokines. Therefore, HSP70 is a potential therapeutic target in viral FH intervention.

Small-molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in CML cells overcomes BCR-ABL-independent TKI resistance.

Herein, we screened a novel inhibitor of the Hsp70-Bim protein-protein interaction (PPI), S1g-2, from a Bcl-2 inhibitor library; this compound specifically disrupted the Hsp70-Bim PPI by direct binding to an unknown site adjacent to that of an allosteric Hsp70 inhibitor MKT-077, showing binding affinity in sub-μM concentration range. S1g-2 exhibited overall 5-10-fold higher apoptosis-inducing activity in CML cells, primary CML blasts, and BCR-ABL-transformed BaF3 cells than other cancer cells, normal lymphocytes, and BaF3 cells, illustrating Hsp70-Bim PPI driven by BCR-ABL protects CML through oncoclient proteins that enriched in three pathways: eIF2 signaling, the regulation of eIF4E and p70S6K signaling, and the mTOR signaling pathways. Moreover, S1g-2 progressively enhanced lethality along with the increase in BCR-ABL-independent TKI resistance in the K562 cell lines and is more effective in primary samples from BCR-ABL-independent TKI-resistant patients than those from TKI-sensitive patients. By comparing the underlying mechanisms of S1g-2, MKT-077, and an ATP-competitive Hsp70 inhibitor VER-155008, the Hsp70-Bim PPI was identified to be a CML-specific target to protect from TKIs through the above three oncogenic signaling pathways. The in vivo activity against CML and low toxicity endows S1g-2 a first-in-class promising drug candidate for both TKI-sensitive and resistant CML.

Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90β and the androgen receptor, induces apoptosis, and attenuates prostate cancer cell growth.

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90β was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.

Heat shock protein 70 dampens the inflammatory response of human PDL cells to mechanical loading in vitro.

Previously, we demonstrated an inflammatory response of human PDL (hPDL) cells to mechanical loading. The cellular reaction was dampened by heat pre-treatment suggesting a protective role for heat shock proteins (HSP) during stress-induced ischemia. Here we explored if HSP70, which has already been documented in the pressure zone of tooth movement, might be regulatorily involved in the attenuation of the inflammatory response. Fifth passage hPDL cells were mechanically loaded in the presence of the HSP70 inhibitor VER155008. Cell morphology, HSP70 expression, viability, IL-6 and IL-8 expression were determined by means of microscopy, realtime-PCR and ELISA. The conditioned medium of mechanically loaded and pre-treated hPDL cells was used to culture monocytes to identify a potential impact on adhesion and osteoclastic differentiation capacity. Mechanical cell stress resulted in a significant increase of pro-inflammatory parameters. HSP70 inhibition led to a further enhancement of cytokine expression. The conditioned medium of mechanically loaded hPDL cells significantly increased monocyte adhesion and differentiation along the osteoclastic pathway. VER155008 pronounced this effect significantly. The results indicate a regulatory role for HSP70 in the control of the inflammatory hPDL cell response to mechanical loading and identify HSP70 as a target in the attempt to attenuate tissue damage during orthodontic tooth movement. Furthermore, the present findings point to the risk of increased periodontal destruction when medication targeting HSP70 is applied for severe medical conditions during orthodontic tooth movement.

Induction of the heat shock response in Arabidopsis by heat shock protein 70 inhibitor VER-155008.

The heat shock protein 90 (HSP90) inhibitor, geldanamycin, is a chemical inducer of the heat shock response (HSR) in Arabidopsis. Geldanamycin is thought to activate the heat shock signal by dissociating the HSP90-heat shock factor (HSF) complex. Recent studies have indicated that plant HSP70 is also associated with HSF, suggesting that inhibition of HSP70 may induce the HSR. However, no studies have been conducted to test this hypothesis. Here, we found that a specific HSP70 inhibitor VER-155008 activated the promoter of a small HSP gene (At1 g53540, HSP17.6C-CI) of Arabidopsis, which was shown to be activated by geldanamycin and other HSP90 inhibitors. The production of HSP17.6C-CI, HSP70 and HSP90.1 proteins in Arabidopsis was enhanced by the addition of VER-155008. The reduction of chlorophyll contents by heat shock was ameliorated by VER-155008. Chaperone analyses indicated that VER-155008 inhibited the chaperone activities of wheat germ extract and human HSP70/HSP40, respectively. These results suggest that the inhibition of HSP70 by VER-155008 enhanced the heat tolerance of Arabidopsis by inducing the HSR in the plant.

Role of HSP70 protein in human periodontal ligament cell function and physiology

ObjectiveHeat pre-treatment of mechanically loaded human periodontal ligament cells (hPDL) dampens the inflammatory cellular response, as evidenced by a reduced expression of pro-inflammatory cytokines, inhibition of monocyte adhesion and osteoclastic differentiation. These findings imply heat shock proteins (HSP) as cell protective molecules acting in the PDL that are up-regulated upon ischemia caused by mechanical loading. HSP70 and its inhibition by VER155008 as the active agent in several pharmaceuticals are established targets and strategies, respectively, in the treatment of neoproliferative diseases. However, the effect of both players on periodontal remodeling in unknown. Therefore, we analyzed the role of HSP70 and its frequently used inhibitor VER155008 in the regulation of physiological hPDL cell functions and immune cell interaction. Materials and MethodsFifth passage hPDL cells were cultured in the presence of 25μm HSP70 inactivating agent VER155008. At harvest, HSP70 expression, cell proliferation, and parameters of cell interaction, colony formation and wound healing were analyzed by means of real-time PCR, immunohistochemistry, Western blot, biochemical MTS assay, microscopy, and functional assays for monocyte adhesion and differentiation. ResultsBasal HSP70 expression and hPDL cell morphology were not affected by HSP70 inhibitor VER155008. In contrast, cell proliferation, tissue defect healing, and colony formation were reduced significantly following HSP70 inhibition, whereas apoptosis and necrosis, monocyte adhesion and osteoclastic differentiation were markedly increased. ConclusionsThe present data indicate a regulatory role for HSP70 protein in hPDL cell biology. Clinical relevanceThese findings identify HSP70 as a promising target in the attempt to modify periodontal remodeling and point to potential periodontal side effects of HSP70 pharmaceutical usage.

Hsp90 Inhibition Reduces TLR5 Surface Expression and NF-κB Activation in Human Myeloid Leukemia THP-1 Cells.

Tumors highly express active heat shock protein 90 (Hsp90), which is involved in tumor survival and progression. Enhanced Toll-like receptor (TLR) 5 expression and signaling were reported to be associated with acute myeloid leukemia. In the present study, we investigated the possible modulatory effects of Hsp90 inhibitors on TLR5 expression and signaling in the human myeloid leukemia cell line THP-1. Cells were pretreated with various concentrations of the Hsp90 inhibitor geldanamycin (GA) or the Hsp70 inhibitor VER155008, followed by stimulation with bacterial flagellin. Flagellin-induced nuclear factor-κB (NF-κB) activation was significantly reduced by treatment with GA or VER155008. To elucidate the underlying mechanism of this effect, mRNA and cell surface expression of TLR5 was examined. TLR5 mRNA expression was enhanced by both GA and VER155008, whereas cell surface expression of TLR5 was reduced by three different Hsp90 inhibitors, including GA, 17-(allylamino)-17-demethoxygeldanamycin, and radicicol, and an Hsp70 inhibitor. The inhibitory effect of Hsp90 inhibitors was much higher than that of Hsp70 inhibitor. Our results suggest that Hsp90 inhibitors suppress TLR5 surface expression and activation of NF-κB in THP-1 cells in response to TLR5 ligand, and these inhibitory effects may be associated with the possible mechanisms by which Hsp90 inhibitors suppress myeloid leukemia.

Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors

A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein–protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies.

Lipopolysaccharide and heat stress impair the estradiol biosynthesis in granulosa cells via increase of HSP70 and inhibition of smad3 phosphorylation and nuclear translocation

LPS and heat stress have been shown to exert various toxic effects in animals, as they induce estradiol biosynthesis dysfunction in granulosa cells (GCs) and result in low reproductive performance. However, there is limited information regarding their detailed mechanisms. In the present study, primary cultured porcine GCs were treated with LPS (1000ng/mL for 48h), or heat stress (41°C for 3h), in vitro, with or without the HSP70 inhibitor VER155008 (10μM), to investigate their potential mechanisms. To mimic the spike in HSP70 from LPS and heat stress, treatments with only the HSP70 activator STA-4783 (10μM for 3h or 48h) were also performed. We found that LPS and heat stress treatments could significantly reduce the expressions of FSHR and CYP19A1; associated with a reduction in estradiol concentrations; and increased in HSP70 expression both at mRNA and protein levels. While, VER155008 attenuation of LPS and heat stress induced HSP70 upregulation can restore the expressions of FSHR and CYP19A1. Furthermore, STA-4783 treatment alone can mimic the effects of LPS and heat stress treatments. Following immunofluorescence staining and western blot analysis showed that Smad3 phosphorylation and nuclear translocation were also inhibited by LPS, heat stress and STA-4783 treatments. We also examined the interactions between HSP70 and Smad3 by yeast two-hybrid screening, the results revealed that HSP70 indirectly interacted with Smad3. Thus, our results suggested that LPS and heat stress could impair estradiol biosynthesis in GCs via increased HSP70 and indirect inhibition of Smad3 phosphorylation and nuclear translocation.

Incomplete radiofrequency ablation accelerates proliferation and angiogenesis of residual lung carcinomas via HSP70/HIF-1α

Radiofrequency ablation (RFA) therapy has been proved effective and feasible for lung cancer. However, the molecular mechanisms of local lung cancer recurrence following RFA are poorly understood. The present study aimed to evaluate the ability of HSP70/HIF-1α to affect the proliferation and angiogenesis of non-small cell lung cancers (NSCLCs) following insufficient RFA to uncover the molecular mechanisms of local recurrence. In vitro heat treatment was used to establish sublines of NCI-H1650 cells. The NCI-H1650 subline that was established by heat treatment at 54°C had a relatively higher viability and significantly elevated heat tolerance (compared to the parental strain). After treatment with the HSP70 inhibitor VER-155008, the HIF-1α inhibitor YC-1 and PI3K/Akt inhibitor wortmannin, the viability and proliferation rate of the cells was measured. At the same time, HSP70, HIF-1α and Akt were detected by real-time PCR and western blotting. In vivo xenograft tumors were created by subcutaneously inoculating nude mice with NCI-H1650 cells. HSP70, HIF-1α and Akt were detected by western blotting, and CD34 expression was detected by immunohistochemistry before and after RFA or treatment with the VER-155008, YC-1 or wortmannin inhibitors. The heat-adapted NCI-H1650 subline established in vitro had a higher viability and proliferative activity compared to parental cells. Inhibiting HSP70/HIF-1α abolished this difference. Blocking the PI3K/Akt signaling pathway decreased HSP70/HIF-1α expression levels. In vivo, we found that incomplete RFA treatment promoted HSP70/HIF-1α and CD34 expression. Additionally, the combination of RFA and treatment targeting HSP70/HIF-1α resulted in a synergistic reduction in tumor growth compared to incomplete RFA alone. The PI3K/Akt signaling pathway is also involved in regulating HSP70/HIF-1α expression during this process. We conclude that the accelerated proliferation and angiogenesis potential of residual lung carcinomas following RFA treatment was induced by HSP70/HIF-1α, expression of which is regulated by the PI3K/Akt signaling pathway.

Role of HSP70 in motoneuron survival after excitotoxic stress in a rat spinal cord injury model in vitro.

The outcome for gait recovery from paralysis due to spinal lesion remains uncertain even when damage is limited. One critical factor is the survival of motoneurons, which are very vulnerable cells. To clarify the early pathophysiological mechanisms of spinal damage, an in vitro injury model of the rat spinal cord caused by moderate excitotoxicity was used. With this preparation we investigated whether motoneuron survival was dependent on the expression of the neuroprotective protein HSP70. In the present study excitotoxicity evoked by kainate induced delayed (24 h) loss (35%) of motoneurons, which became pyknotic with translocation of the cell death biomarker apoptosis-inducing factor (AIF) to the nucleus. This process was concomitant with suppression of locomotor network electrical activity. Surviving cells showed strong expression of HSP70 without nuclear AIF. The HSP70 inhibitor VER155008 per se induced neurotoxicity similar to that of kainate, while the HSP90 inhibitor geldanamycin did not damage spinal tissue. Electrophysiological recording following kainate or VER155008 indicated depression of motoneuron field potentials, with decreased excitability and impaired synaptic transmission. When these two drugs were applied together, more intense neurotoxicity emerged. Our data indicate that HSP70 was one important contributor to motoneuron survival and suggest that enhancing HSP70 activity is a potential future strategy for neuroprotecting these cells.

Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer

Heat shock protein 90 (HSP90) plays a critical role in the survival of cancer cells including muscle invasive bladder cancer (MIBC). The addiction of tumor cells to HSP90 has promoted the development of numerous HSP90 inhibitors and their use in clinical trials. This study evaluated the role of inhibiting HSP90 using STA9090 (STA) alone or in combination with the HSP70 inhibitor VER155008 (VER) in several human MIBC cell lines. While both STA and VER inhibited MIBC cell growth and migration and promoted apoptosis, combination therapy was more effective. Therefore, the signaling pathways involved in MIBC were systematically interrogated following STA and/or VER treatments. STA and not VER reduced the expression of proteins in the p53/Rb, PI3K and SWI/SWF pathways. Interestingly, STA was not as effective as VER or combination therapy in degrading proteins involved in the histone modification pathway such as KDM6A (demethylase) and EP300 (acetyltransferase) as predicted by The Cancer Genome Atlas (TCGA) data. This data suggests that dual HSP90 and HSP70 inhibition can simultaneously disrupt the key signaling pathways in MIBC.

The hsp70 inhibitor VER155008 induces paraptosis requiring de novo protein synthesis in anaplastic thyroid carcinoma cells

In this study, we evaluated the effect of the hsp70 inhibitor VER155008 on survival of anaplastic thyroid carcinoma (ATC) cells. In ATC cells, VER155008 increased the percentages of dead cells and vacuolated cells. VER155008 did not lead to the cleavage of caspase-3 protein regardless of pretreatment with z-VAD-fmk. VER155008 increased LC3-II protein levels but the protein levels were not changed by autophagy inhibitors. VER155008 caused the dilatation of endoplasmic reticulum (ER), and the increased mRNA levels of Bip and CHOP, suggesting paraptosis. VER155008-induced paraptosis was attenuated by pretreatment with cycloheximide. In conclusion, VER155008 induces paraptosis characterized by cytoplasmic vacuolation, independence of caspase, dilatation of ER and induction of ER stress markers in ATC cells. Moreover, VER155008-induced paraptosis requires de novo protein synthesis in ATC cells.

Increased antileukemic effects in human acute myeloid leukemia by combining HSP70 and HSP90 inhibitors

Background: Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSP90, and more recently HSP70, have emerged as possible therapeutic targets in human malignancies, including acute myeloid leukemia (AML).Design and methods: The authors investigated the effects of the HSP70 inhibitor VER-155008 tested alone or in combination with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) on proliferation, viability, constitutive cytokine release and intracellular HSP levels of primary human AML cells.Results: VER-155008 caused a dose-dependent inhibition of cytokine-dependent AML cell proliferation both in suspension cultures and in a colony formation assay, and the drug also had a proapoptotic effect. HSP70 and HSP90 inhibition had additive antiproliferative and proapoptotic effects. VER-155008 caused a strong inhibition of the constitutive AML cell release of several growth factors/regulators of hematopoiesis (i.e., TNF-α, VEGF, IL-3, IL-1β, IL-1 receptor antagonist), but had relatively weak effects on the constitutive chemokine release. HSP70 inhibition did not induce any compensatory increase of other HSPs.Conclusion: HSP70 inhibition has antileukemic effects when tested alone, and the combination of HSP70 and HSP90 inhibition seems to have additive antileukemic effects for primary human AML cells in vitro.