Reactive oxygen species mediate hepatotoxicity induced by the Hsp90 inhibitor geldanamycin and its analogs

Geldanamycin (GM), a benzoquinone ansamycin antibiotic, is a natural product inhibitor of Hsp90 with potent and broad anti-cancer properties. Because of its adverse effects on liver, its less toxic derivatives 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) are currently being evaluated for the treatment of cancer. Previously, it has been demonstrated that the redox cycling of GM by NADPH-cytochrome P450 reductase leads to the formation of the GM semiquinone and superoxide radicals, the latter being identified using spin-trapping. We hypothesized that the different hepatotoxicity induced by GM, 17-AAG and 17-DMAG reflects the redox active properties of the quinone moiety and possibly the extent of superoxide formation, which may stimulate cellular oxidative injury. Our data demonstrate that superoxide can be efficiently trapped during the reduction of GM, 17-AAG and 17-DMAG by NADPH-cytochrome P450 reductase, and that superoxide formation rate followed the order 17-DMAG > 17-AAG > GM. In the absence of superoxide scavengers, the rate of NADPH oxidation followed the order 17-DMAG > GM > 17-AAG. The half-wave one-electron reduction potentials (E_1/2) of GM, 17-AAG and 17-DMAG in DMSO have been determined to be -0.37, -0.13 and -0.015 V (vs. Ag/AgCl), respectively. If the same order of E_1/2 follows in neutral aqueous media, thermodynamic considerations imply that 17-DMAG is more readily reduced by the P450 reductase as well as by superoxide. The order of the drug cytotoxicity toward rat primary hepatocytes, as determined by their effect on cell viability and on intracellular oxidant level, was opposite to the order of E_1/2 of the respective quinone/semiquinone couples. These results suggest that hepatotoxicity exhibited by the Hsp90 inhibitors belonging to benzoquinone ansamycins could be attributed to superoxide. The apparent discrepancy between the order of toxicity and the orders of superoxide formation rate, which is correlated with E_1/2, is discussed.     

Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells

The molecular chaperone heat shock protein 90 (Hsp90) affects the function of many oncogenic signaling proteins including nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in anaplastic large cell lymphoma (ALCL). While ALK-positive ALCL cells are sensitive to the Hsp90 inhibitor and the geldanamycin (GA) analog, 17-allylamino-17-demethoxygeldanamycin (17-AAG), the proteomic effects of these drugs on ALK-positive ALCL cells are unpublished. In this study, we investigated the cellular, biologic, and proteomic changes occurring in ALK-positive ALCL cells in response to GA treatment. GA induced G2/M cell cycle arrest and caspase-3-mediated apoptosis. Furthermore, quantitative proteomic changes analyzed by cleavable isotope-coded affinity tag-LC-MS/MS (cICAT-LC-MS/MS) identified 176 differentially expressed proteins. Out of these, 49 were upregulated 1.5-fold or greater and 70 were downregulated 1.5-fold or greater in GA-treated cells. Analysis of biological functions of differentially expressed proteins revealed diverse changes, including induction of proteins involved in the 26S proteasome as well as downregulation of proteins involved in signal transduction and protein and nucleic acid metabolism. Pathway analysis revealed changes in MAPK, WNT, NF-kappaB, TGFbeta, PPAR, and integrin signaling components. Our studies reveal some of the molecular and proteomic consequences of Hsp90 inhibition in ALK-positive ALCL cells and provide novel insights into the mechanisms of its diverse cellular effects.     

Suppression of the mTOR-raptor signaling pathway by the inhibitor of heat shock protein 90 geldanamycin

Heat shock protein 90 (Hsp90) was co-immunoprecipitated with raptor, the binding partner of the mammalian target of rapamycin (mTOR) from HEK293 cells. Hsp90 was detected in the anti-raptor antibody immunoprecipitates prepared from the cell extract by immunoblot analysis using the anti-Hsp90 antibody, and the association of these two proteins was confirmed by immunoprecipitation from the cells co-expressing Hsp90 and raptor as epitope-tagged molecules. Geldanamycin, a potent inhibitor of Hsp90, disrupted the in vivo binding of Hsp90 to raptor without affecting the association of raptor and mTOR, and suppressed the phosphorylation by mTOR of the downstream translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The protein kinase activity of S6K as well as the phosphorylation of the substrate, 40S ribosomal protein S6, were lowered in the geldanamycin-treated cells. These results indicate that Hsp90 is involved in the regulation of protein translation by facilitating the phosphorylation reaction of 4E-BP1 and S6K catalyzed by the mTOR/raptor complex through the association with raptor, and that the mTOR signaling pathway is a novel target of geldanamycin.     

Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.     

Therapeutic and diagnostic implications of Hsp90 activation

The molecular chaperone heat-shock protein 90 (Hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. Hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is presently in clinical trials. Recent work has identified the role of Hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by Hsp90 inhibitors is the result of an activated, high-affinity conformation of Hsp90 in tumors. This review discusses these recent advances in the understanding of tumor Hsp90 for the treatment and diagnosis of cancer. In addition, the role of Hsp90 in non-oncological diseases will also be discussed.     

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90)

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.     

4-Amino derivatives of the Hsp90 inhibitor CCT018159

Novel piperazinyl, morpholino and piperidyl derivatives of the pyrazole-based Hsp90 inhibitor CCT018159 are described. Structure-activity relationships have been elucidated by X-ray co-crystal analysis of the new compounds bound to the N-terminal domain of human Hsp90. Key features of the binding mode are essentially identical to the recently reported potent analogue VER-49009. The most potent of the new compounds has a methylsulfonylbenzyl substituent appended to the piperazine nitrogen, possesses an IC50 of less than 600 nM binding against the enzyme and demonstrates low micromolar inhibition of tumour cell proliferation.     

Synthesis of Hsp90 inhibitor dimers as potential antitumor agents

Structure-based drug design was used to systematically synthesize PU3-dimers. The cytotoxicity of PU3 dimers 6 against breast cancer cell lines was evaluated, and their potency increased as the length of the bridging linker increased. Among the compounds tested, 6e with a C-20 linker was the most potent and exhibited a 20- to 30-fold increase in activity compared with that of the parent compound 5. Western blot analyses of the cell lysates treated with 6c revealed that 6c resulted in the concentration-dependent degradation of the Hsp90 client protein Her2, which is consistent with other Hsp90 inhibitors.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.     

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.     

Changes in heat-shock protein synthesis and thermotolerance of Arabodopsis thaliana seedlings resulting from Hsp90 inhibition by geldanamycin

The influence of geldanamycin (GA), a specific inhibitor of heat-shock protein Hsp90, on the synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA under normal conditions induced synthesis of these stress proteins. Treatment of seeds with the Hsp90 inhibitor resulted in elevated constitutive levels of Hsp70 and Hsp90 in seedlings, as well as increased induction of their synthesis under heat shock. The GA effect increased with its concentration. Hsp up-regulation promoted thermotolerance of seedlings. The findings suggest autoregulation of heatshock protein synthesis and regulation of plant tolerance by Hsp90.     

Anti-NSCLC activity in vitro of Hsp90N inhibitor KW-2478 and complex crystal structure determination of Hsp90N-KW-2478

KW-2478 is a promising anti-cancer lead compound targeting to the molecular chaperone heat shock protein 90 ^N (Hsp90^N). Absence of complex crystal structure of Hsp90^N-KW-2478, however, hampered further structure optimization of KW-2478 and understanding on the molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90^N-KW-2478 was determined by X-ray diffraction (XRD, resolution limit: 1.59 Å; PDB ID: 6LT8) and their molecular interaction was analyzed in detail, which suggested that KW-2478 perfectly bound in the N-terminal ATP-binding pocket of Hsp90 to disable its molecular chaperone function, therefore suppressed or killed cancer cells. The results from thermal shift assay (TSA, ΔTm, 18.82 ± 0.51 °C) and isothermal titration calorimetry (ITC, K_d, 7.30 ± 2.20 nM) suggested that there is an intense binding force and favorable thermodynamic changes during the process of KW-2478 binding with Hsp90^N. Additionally, KW-2478 exhibited favorable anti-NSCLC activity in vitro, as it inhibited cell proliferation (IC₅₀, 8.16 μM for A549; 14.29 μM for H1975) and migration, induced cell cycle arrest and promoted apoptosis. Thirty-six novel KW-2478 derivatives were designed, based on the complex crystal structure and molecular interaction analysis of Hsp90^N-KW-2478 complex. Among them, twenty-two derivatives exhibited increased binding force with Hsp90^N evaluated by molecular docking assay. The results would provide new guidance for anti-NSCLC new drug development based on the lead compound KW-2478.     

Efficient synthesis of Hsp90 inhibitor dimers as potential antitumor agents

The PU-H58-dimers 13a–15b were efficiently synthesized and their biological properties were evaluated. The copper-catalyzed alkyne azide coupling was effective in simultaneously linking three components via a triazole formation to afford the target dimers. These synthesized dimers exhibited binding affinity to the N-terminal domain of Hsp90, cytotoxicity, and client degradation activity although these activities were comparative or weak comparable with that of the parent compound.     

Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90beta,a property that can be exploited in screens for new Hsp90 chaperone inhibitors

Heat shock protein 90 (Hsp90) is essential for activation of many of the most important regulatory proteins of eukaryotic cells. It is an extremely conserved protein, such that heterologous expressions of either human Hsp90beta or Caenorhabditis elegans Hsp90 will provide the essential Hsp90 function in yeast. The ability of these metazoan Hsp90s to provide this Hsp90 function to yeast cells requires Sti, a Hsp90 system cochaperone. Yeast that is expressing human Hsp90beta in place of the normal native yeast Hsp90 is selectively hypersensitised to Hsp90 inhibitor drugs. Hsp90 drugs are promising anticancer agents, their administration simultaneously destabilizing a number of the proteins critical to multistep carcinogenesis. Though one of these drugs (17-allylaminogeldanamycin, 17-AAG) is now progressing to Phase 2 clinical trials, there is a pressing need to identify selective Hsp90 inhibitors that are more soluble than 17-AAG. High-throughput screening for chemical agents that exert greater inhibitory effects against yeast expressing the human Hsp90beta relative to yeast expressing its native Hsp90 should therefore facilitate the search for new Hsp90 inhibitors.