Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.     

Discovery of NMS-E973 as novel,selective and potent inhibitor of heat shock protein 90 (Hsp90)

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.     

C-terminal modulators of heat shock protein of 90 kDa (HSP90): State of development and modes of action

Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as “C-terminal inhibitors” have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites.     

Inhibition of translation by cytotrienin A--a member of the ansamycin family

The ansamycins are a diverse and often physiologically active group of compounds that include geldanamycin and rifamycin, inhibitors of heat shock protein 90 and prokaryotic DNA-dependent RNA synthesis, respectively. Cytotrienin A is an ansamycin-type small molecule with potent antiproliferative and proapoptotic properties. Here, we report that this compound inhibits eukaryotic protein synthesis by targeting translation elongation and interfering with eukaryotic elongation factor 1A function. We also find that cytotrienin A prevents HUVEC tube formation and diminishes microvessel formation in the chorioallantoic membrane assay. These results provide a molecular understanding into cytotrienin A's previously reported properties as an anticancer apoptosis-inducing drug.     

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.     

Microwave-assisted one step synthesis of 8-arylmethyl-9H-purin-6-amines

Molecular chaperone heat shock protein 90 (Hsp90) is an important target in cancer and neurodegenerative diseases, and has rapidly become the focus of several drug discovery efforts. Among small molecule Hsp90 inhibitors with clinical applicability are derivatives of 8-arylmethyl-9H-purin-6-amine class. Here we report the use of microwave-assisted chemistry for the successful one-pot delivery of 8-arylmethyl-9H-purin-6-amines. We discuss the applicability as well as the limitations of this method towards the creation of a large chemical diversity in the 8-arylmethyl-9H-purin-6-amine series.     

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.     

The Antitumor Activity of the Novel Compound Jesridonin on Human Esophageal Carcinoma Cells

Jesridonin, a small molecule obtained through the structural modification of Oridonin, has extensive antitumor activity. In this study, we evaluated both its in vitro activity in the cancer cell line EC109 and its in vivo effect on tumor xenografts in nude mice. Apoptosis induced by Jesridonin was determined using an MTT assay, Annexin-V FITC assay and Hoechest 33258 staining. Apoptosis via mitochondrial and death receptor pathways were confirmed by detecting the regulation of MDM2, p53, and Bcl-2 family members and by activation of caspase-3/-8/-9. In addition, vena caudalis injection of Jesridonin showed significant inhibition of tumor growth in the xenograft model, and Jesridonin-induced cell apoptosis in tumor tissues was determined using TUNEL. Biochemical serum analysis of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT), total protein (TP) and albumin (ALB) indicated no obvious effects on liver function. Histopathological examination of the liver, kidney, lung, heart and spleen revealed no signs of JD-induced toxicity. Taken together, these results demonstrated that Jesridonin exhibits antitumor activity in human esophageal carcinomas EC109 cells both in vitro and in vivo and demonstrated no adverse effects on major organs in nude mice. These studies provide support for new drug development.     

Extracellular HSP90: Conquering the cell surface

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone, assisting intracellularly in the folding and conformational regulation of a multitude of client proteins that play a crucial role in growth, cell survival and developmental processes(1). Moreover HSP90 interacts with a great number of molecules that are involved in the development and/or survival of cancer cells, allowing mutant proteins to retain or gain function while permitting cancer cells to tolerate the imbalanced signaling that such oncoproteins create (2,3). Prime examples include the HER-2 receptor, c-Raf-1, Akt/PKB, CDK4, and mutant p53 (4,5). Highly specific inhibitors of HSP90 have been identified and are currently under clinical evaluation. These include geldanamycin and its derivatives 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxygeldanamycin, which inhibit cancer cell proliferation in vitro and tumor growth in vivo (6-9).     

TAp73 Protein Stability Is Controlled by Histone Deacetylase 1 via Regulation of Hsp90 Chaperone Function

Histone deacetylases (HDACs) play important roles in fundamental cellular processes, and HDAC inhibitors are emerging as promising cancer therapeutics. p73, a member of the p53 family, plays a critical role in tumor suppression and neural development. Interestingly, p73 produces two classes of proteins with opposing functions: the full-length TAp73 and the N-terminally truncated ΔNp73. In the current study, we sought to characterize the potential regulation of p73 by HDACs and found that histone deacetylase 1 (HDAC1) is a key regulator of TAp73 protein stability. Specifically, we showed that HDAC1 inhibition by HDAC inhibitors or by siRNA shortened the half-life of TAp73 protein and subsequently decreased TAp73 expression under normal and DNA damage-induced conditions. Mechanistically, we found that HDAC1 knockdown resulted in hyperacetylation and inactivation of heat shock protein 90, which disrupted the interaction between heat shock protein 90 and TAp73 and thus promoted the proteasomal degradation of TAp73. Functionally, we found that down-regulation of TAp73 was required for the enhanced cell migration mediated by HDAC1 knockdown. Together, we uncover a novel regulation of TAp73 protein stability by HDAC1-heat shock protein 90 chaperone complex, and our data suggest that TAp73 is a critical downstream mediator of HDAC1-regulated cell migration.     

A purine-type heat shock protein 90 inhibitor promotes the heat shock response in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Geldanamycin is a macrocyclic heat shock protein 90 (HSP90) inhibitor that suppresses cancer cell proliferation. Since geldanamycin also promotes the heat shock response (HSR) in cells, this compound is used as a chemical inducer of the HSR in Arabidopsis. Although many types of HSP90 inhibitors that are different from the macrocyclic types have been developed in pharmaceutical research, non-macrocyclic HSP90 inhibitors have not been investigated in terms of whether they can induce the HSR in plants. Here, we determined the HSR-inducing activities in Arabidopsis of 10 non-macrocyclic HSP90 inhibitors including 2 benzamide derivatives, 3 purine derivatives, and 5 resorcinol derivatives. Among the tested inhibitors, PU-H71, which is a purine derivative, showed the highest HSR-inducing activity. The activity of PU-H71 was significantly higher than that of geldanamycin. The application of PU-H71 induced the HSR in all Arabidopsis seedlings. The HSP17.6C-CI and HSP70 proteins accumulated after the treatment with PU-H71. The seedlings treated with PU-H71 maintained more chlorophyll than the control seedlings after the heat stress. These results suggest that the purine-derivative HSP90 inhibitor PU-H71 enhanced the heat tolerance of Arabidopsis by promoting the HSR in the plant.     

Anti-herpes simplex virus efficacies of 2-aminobenzamide derivatives as novel HSP90 inhibitors

After the widespread use of the acyclic purine nucleoside analogues for therapy of herpes simplex virus (HSV) infection since the 1980s, new antiviral strategies are urgently needed to counter the emergence of drug-resistant clinical isolates. In this report, we define the anti-HSV efficacies of three optimized 2-aminobenzamide derivatives in vitro and in vivo. The synthetic analogues SNX-25a, SNX-2112 and SNX-7081, which selectively bind to the N-terminal ATP pocket of heat shock protein 90 (HSP90), exhibited significant anti-HSV-1 and HSV-2 activities at non-cytotoxic concentrations in Vero cells, with EC(50) values close to that of acyclovir (ACV). The in vivo antiviral potentials were then confirmed using a herpes simplex keratitis (HSK) rabbit model, where eye gels containing 0.1% or 0.025% SNX-25a displayed the highest efficacies against HSV-1 infection, which were better than that obtained with 0.1% ACV. SNX-2112 and SNX-7081 gels were also effective against HSV-1 with different magnitude of activities. Our results for the first time confirmed the anti-HSV efficacies of these 2-aminobenzamide derivatives and suggest that with alternative mechanisms of action these novel HSP90 inhibitors, especially SNX-25a, could be potent as new anti-HSV clinical trial candidates.     

Immune mechanism of the antitumor effects generated by bortezomib

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8(+) T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8(+) T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8(+) T cells. CD11c(+) DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c(+) DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.     

Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network

Molecular chaperones, especially members of the heat shock protein 90 (Hsp90) family, are thought to promote tumor cell survival, but this function is not well understood. Here, we show that mitochondria of tumor cells, but not most normal tissues, contain Hsp90 and its related molecule, TRAP-1. These chaperones interact with Cyclophilin D, an immunophilin that induces mitochondrial cell death, and antagonize its function via protein folding/refolding mechanisms. Disabling this pathway using novel Hsp90 ATPase antagonists directed to mitochondria causes sudden collapse of mitochondrial function and selective tumor cell death. Therefore, Hsp90-directed chaperones are regulators of mitochondrial integrity, and their organelle-specific antagonists may provide a previously undescribed class of potent anticancer agents.     

Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.     

Low Dose Geldanamycin Inhibits Hepatocyte Growth Factor- and Hypoxia-Stimulated Invasion of Cancer Cells

Hepatocyte growth factor (HGF) receptor Met and hypoxia-inducible factor-1 (HIF-1) signaling pathways are commonly activated in aggressive tumors and promote progression. Since both Met and HIF-1α proteins are heat shock protein (Hsp) 90 clients, Hsp90 inhibitors might be expected to positively impact tumor progression. Here, we systematically evaluated the inhibitory effects of the prototypical Hsp90 inhibitor geldanamycin (GA) on cellular processes involved in invasion and angiogenesis in T24 bladder cancer cells stimulated with HGF and chemical hypoxia. First, we demonstrated the positive feedback loop between Met and HIF-1 pathways, which serves to sustain and amplifies their signaling in T24 cells. GA down-regulated Met by inhibiting new protein maturation, thereby dampening HGF signaling. HGF and chemical hypoxia with CoCl2 cooperatively promoted in vitro invasion and vascular endothelial growth factor (VEGF) secretion, while CoCl2 but not HGF activated urokinase-type plasminogen activator and matrix metalloproteinase 2, both of which promote invasion and angiogenesis. Low dose GA (100 nmol/L) inhibited these processes by suppressing both HGF and HIF-1 pathways. Notably, brief GA pretreatment inhibited in vitro invasion and VEGF secretion induced by HGF as effectively as did continuous treatment. Moreover, we found that GA inhibited activation of focal adhesion kinase, focal adhesion assembly, and actin reorganization induced by HGF and integrin engagement by extracellular matrix. Thus, GA widely suppresses extrinsic stimuli-induced signaling that contribute to tumor invasion and angiogenesis in this bladder carcinoma model, suggesting the utility of Hsp90 inhibitors in preventing tumor progression and metastasis.