Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

Modified biphenyl Hsp90 C-terminal inhibitors for the treatment of cancer

Heat Shock Protein 90 (Hsp90) is a molecular chaperone under clinical investigation for the treatment of neurodegenerative diseases and cancer. Neuroprotective Hsp90 C-terminal inhibitors (novologues) contain a biaryl ring system, and include KU-596, which was modified and investigated for potential anti-cancer activity. Incorporation of a benzamide group onto the biaryl novologues in lieu of the acetamide yielded compounds that manifest anti-cancer activity. Further exploration of the central phenyl ring led to compounds with enhanced anti-proliferative activity. The design, synthesis, and evaluation of these new analogs against breast and prostate cancer cell lines is reported herein, where it was found that 8b and 10 manifest potent anti-proliferative activity and a robust degradation of Hsp90 client-dependent proteins.     

Novologues containing a benzamide side chain manifest anti-proliferative activity against two breast cancer cell lines

Hsp90 represents a promising target for the development of both anti-cancer and neuroprotective agents. Structure–activity relationship studies on novobiocin and novobiocin analogues, led to the development of KU-32 and recently, KU-596, as lead compounds for the potential treatment of neurodegenerative diseases. Similar to KU-32, we have demonstrated that upon replacement of the acetamide side chain present in KU-32 with a benzamide, this neuroprotective agent was transformed into a scaffold that manifests anti-proliferative activity. To assess structure–activity relationships for this new scaffold, a library of benzamide-containing novologues was prepared and evaluated against two breast cancer cell lines. Compound 14a manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation in a concentration-dependent manner.     

Plant Hsp90 family with special reference to rice

Hsp90 family represents a group of highly conserved and strongly expressed proteins present in almost all biological species. Heat shock proteins in the range of 90 kDa have been detected in a range of plant species andhsp90 genes have been cloned and characterized in selected instances. However, the expression characteristics of plant Hsp90 are poorly understood. Work on expression characteristics of rice Hsp90 is reviewed in this paper. Experimental evidence is provided for indicating that while the rice 87 kDa protein is transiently synthesized within initial 2 h of heat shock, high steady-state levels of this protein are retained even under prolonged high temperature stress conditions or recovery following 4 h heat shock. It is further shown that fifteen different wild rices accumulate differential levels of these proteins in response to heat shock treatment.     

Rapamycin conditionally inhibits Hsp90 but not Hsp70 mRNA translation in <Emphasis Type="Italic">Drosophila</Emphasis>: implications for the mechanisms of Hsp mRNA translation

Rapamycin inhibits the activity of the target of rapamycin (TOR)-dependent signaling pathway, which has been characterized as one dedicated to translational regulation through modulating cap-dependent translation, involving eIF4E binding protein (eIF4E-BP) or 4E-BP. Results show that rapamycin strongly inhibits global translation in Drosophila cells. However, Hsp70 mRNA translation is virtually unaffected by rapamycin treatment, whereas Hsp90 mRNA translation is strongly inhibited, at normal growth temperature. Intriguingly, during heat shock Hsp90 mRNA becomes significantly less sensitive to rapamycin-mediated inhibition, suggesting the pathway for Hsp90 mRNA translation is altered during heat shock. Reporter mRNAs containing the Hsp90 or Hsp70 mRNAs’ 5′ untranslated region recapitulate these rapamycin-dependent translational characteristics, indicating this region regulates rapamycin-dependent translational sensitivity as well as heat shock preferential translation. Surprisingly, rapamycin-mediated inhibition of Hsp90 mRNA translation at normal growth temperature is not caused by 4E-BP-mediated inhibition of cap-dependent translation. Indeed, no evidence for rapamycin-mediated impaired eIF4E function is observed. These results support the proposal that preferential translation of different Hsp mRNA utilizes distinct translation mechanisms, even within a single species.     

Hsp90 chaperones have an energetic hot‐spot for binding inhibitors

Although Hsp90‐family chaperones have been extensively targeted with ATP‐competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP‐binding pocket. A large‐scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen‐bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle‐specific Hsp90 paralogs. For inhibitors in the resorcinol family, the D93:inhibitor hydrogen‐bond is pH‐dependent because the associated inhibitor hydroxyl group is titratable, rationalizing a linked‐protonation event previously observed by the Matulis group. The inhibitor hydroxyl group pK_a associated with the D93 hydrogen‐bond is therefore critical for optimizing the affinity of resorcinol derivatives, and we demonstrate that spectrophotometric measurements can determine this pK_a value. Quantifying the energetic contribution of the D93 hotspot is best achieved with the mitochondrial Hsp90 paralog, yielding 3–6 kcal/mol of stabilization (35–60% of the total binding energy) for a diverse set of inhibitors. The Hsp90 Asp93?Asn substitution has long been known to abolish nucleotide binding, yet puzzlingly, native sequences of structurally similar ATPases, such as Topoisomerasese II, have an asparagine at this same crucial site. While aspartate and asparagine sidechains can both act as hydrogen bond acceptors, we show that a steric clash prevents the Hsp90 Asp93?Asn sidechain from adopting the necessary rotamer, whereas this steric restriction is absent in Topoisomerasese II.     

Molecular-level interplay between intrinsically disordered clients and Hsp90

Proteostasis is maintained by a network of molecular chaperones, a prominent member of which is the 90-kilodalton heat shock protein Hsp90. The chaperone function of Hsp90 has been extensively reviewed previously, emphasizing its ATPase activity and remodeling of folded client proteins. Experimental evidence implicating Hsp90 in neurodegenerative diseases has bolstered interest in the noncanonical chaperoning of intrinsically disordered protein (IDPs), however the interplay between Hsp90 and its disordered clients remains poorly understood. In this review we describe recent advances that have contributed to our understanding of the intricate mechanisms characterizing Hsp90-mediated chaperoning of the IDPs tau and α-synuclein and survey emerging insights into the modulation of the chaperone-client interplay in the context of neurodegeneration.     

Synthesis and evaluation of a novel class Hsp90 inhibitors containing 1-phenylpiperazine scaffold

Previously, we identified 1-(2-(4-bromophenoxy)ethoxy)-3-(4-(2-methoxyphenyl)piperazin-1-yl)propan-2-ol (1) as a novel Hsp90 inhibitor with moderate activity through virtual screening. In this study, we report the optimization process of 1. A series of analogues containing the 1-phenylpiperazine core scaffold were synthesized and evaluated. The structure–activity relationships (SAR) for these compounds was also discussed for further molecular design. This effort afforded the most active inhibitor 13f with improved activity in not only target-based level, but also cell-based level compared with the original hit 1.     

The role of Hsp90 in cell response to hyperthermia

(1) Preincubation of SKOV3 human ovarian carcinoma cells with a non-toxic dose of Geldanamycin resulted in exacerbation of hyperthermia-induced cytotoxicity and re-distribution of dying cells toward necrosis. (2) Exposure of primary human ovarian carcinoma cells to mild hyperthermia (42 °C for 2 h) led, after a recovery period of 16 h, to several-fold increase in the levels of Hsp90 and ErbB2, to a moderate decrease in the levels of phospho-Erk1/2, whereas the level of β-catenin appeared to be unchanged. (3) The inhibitors of Hsp90 (Geldanamycin and Novobiocin) significantly affected the cell signaling of heat-pretreated cultures. (4) The results suggest that Hsp90 plays a pivotal role in cell response to hyperthermia. (5) A combination of Hsp90 inhibitors with hyperthermia may considerably increase the efficacy of thermotherapy.     

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1^G93A transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1^G93A by gene transfer or in presymptomatic SOD1^G93A transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.     

热激蛋白90抑制剂

分子伴侣热激蛋白90(heat-shock protein 90,Hsp90)在生物体内具有重要的生理功能,它在许多肿瘤细胞中表达增加。临床研究发现Hsp90抑制剂单一用药或者联合用药都具有较好的抗肿瘤效果,因此目前Hsp90被认为是癌症治疗一个非常有潜力的靶标。本文总结了Hsp90的结构功能、Hsp90抑制剂的作用机理以及Hsp90抑制剂的临床应用前景,希望为设计和开发新的Hsp90抑制剂提供一定的参考。     

Synthesis and evaluation of 4-(2-hydroxypropyl)piperazin-1-yl) derivatives as Hsp90 inhibitors

We previously reported 4-(3-((6-bromonaphthalen-2-yl)oxy)-2-hydroxypropyl)-N,N-dimethylpiperazine-1-sulfonamide (1) as a novel heat shock protein 90 inhibitor with moderate activity. In our ongoing efforts for the discovery of Hsp90 modulators we undertake structural investigations on 1. Series of the titled compound were designed, synthesized and evaluated. We have found that compounds with a hydroxyl group at C-4 of the aryl ring on the piperazine moiety possess Hsp90 inhibition properties. Compound 6f with improved activity could be further developed and optimized as Hsp90 inhibitor.     

Akt shows variable sensitivity to an Hsp90 inhibitor depending on cell context

Hsp90 inhibitors are currently in clinical trials for cancer therapy based on their ability to promote proteasomal degradation of oncogenic protein kinases and nuclear receptors. Results from recent studies suggest that cancer cells are more sensitive to these inhibitors than cells from healthy tissues. We analyzed an immortalized cell line Ba/F3 for sensitivity to the Hsp90 inhibitor geldanamycin in the absence and presence of the oncogenic tyrosine fusion kinase NPM-ALK expressed from a retroviral vector. Our results showed that NPM-ALK expression makes Akt and Cdk4 more resistant to degradation in the presence of geldanamycin, and there was a slightly reduced amount of apoptosis. The mechanism underlying the effect of NPM-ALK on Akt stability was probed by comparison of the turnover of the kinase after translation inhibition and geldanamycin treatment. We observed that Akt was degraded more rapidly in the presence of GA than upon translation inhibition without NPM-ALK expression. This suggests that NPM-ALK protects the mature kinase. Furthermore, Akt failed to bind to the Cdc37 chaperone in cells expressing NPM-ALK, which also correlates with increased Akt stability.     

Decomposition analysis of free energy profile for Hsp90-ADP association

The functional cycle of heat shock protein 90 (Hsp90) is driven and inhibited by the association/dissociation of ligand molecules. In order to understand the molecular mechanism of the association of N-terminal domain of Hsp90 (N-Hsp90) and its ligand molecule, it is necessary to investigate which part in the target system promotes or inhibits the association of N-Hsp90 and its ligand molecule. We apply the decomposition analysis for the association free energy of N-Hsp90 and ADP. The mean force calculated by thermodynamic integration method combined with molecular dynamic simulations is divided into the contributions from molecules in the target system. Van der Waals interaction of the solvent water molecules strongly stabilises the association. Three lysine residues on the surface of the N-Hsp90 pull ADP toward the binding pocket of N-Hsp90. This study elucidates the association process of ADP from the bulk region to the binding pocket of the N-terminal domain Hsp90. This approach is applicable to elucidate the association process of biomolecules.     

Circulating heat shock protein 90 (Hsp90) and autoantibodies to Hsp90 are increased in patients with atopic dermatitis

Atopic dermatitis (AD) is one of the most common chronic inflammatory dermatoses characterized by persistent itching and recurrent eczematous lesions. While the primary events and key drivers of AD are topics of ongoing debate, cutaneous inflammation due to inappropriate IgE (auto)antibody–related immune reactions is frequently considered. Highly conserved and immunogenic heat shock protein 90 (Hsp90), a key intra- and extracellular chaperone, can activate the immune response driving the generation of circulating anti-Hsp90 autoantibodies that are found to be elevated in several autoimmune disorders. Here, for the first time, we observed that serum levels of Hsp90 and anti-Hsp90 IgE autoantibodies are significantly elevated (p < 0.0001) in AD patients (n = 29) when compared to age- and gender-matched healthy controls (n = 70). We revealed a positive correlation (0.378, p = 0.042) between serum levels of Hsp90 and the severity of AD assessed by Scoring Atopic Dermatitis (SCORAD). In addition, seropositivity for anti-Hsp90 IgE has been found in 48.27% of AD patients and in 2.85% of healthy controls. Although further studies on a larger group of patients are needed to confirm presented data, our results suggest that extracellular Hsp90 and autoantibodies to Hsp90 deserve attention in the study of the mechanisms that promote the development and/or maintenance of atopic dermatitis.     

Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype

Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC₅₀ value (30–100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs.     

Independent ATPase activity of Hsp90 subunits creates a flexible assembly platform

The ATPase activity of the molecular chaperone Hsp90 is essential for its function in the assembly of client proteins. To understand the mechanism of human Hsp90, we have carried out a detailed kinetic analysis of ATP binding, hydrolysis and product release. ATP binds rapidly in a two-step process involving the formation of a diffusion-collision complex followed by a conformational change. The rate-determining step was shown to be ATP hydrolysis and not subsequent ADP dissociation. There was no evidence from any of the biophysical measurements for cooperativity in either nucleotide binding or hydrolysis for the dimeric protein. A monomeric fragment, lacking the C-terminal dimerisation domain, showed no dependence on protein concentration and, therefore, subunit association for activity. The thermodynamic linkage between client protein binding and nucleotide affinity revealed ATP bound Hsp90 has a higher affinity for client proteins than the ADP bound form. The kinetics are consistent with independent Michaelis-Menten catalysis in each subunit of the Hsp90 dimer. We propose that Hsp90 functions in an open-ring configuration for client protein activation.     

A systematic protocol for the characterization of Hsp90 modulators

Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA.