The Chaperone System in Breast Cancer: Roles and Therapeutic Prospects of the Molecular Chaperones Hsp27, Hsp60, Hsp70, and Hsp90

Breast cancer (BC) is a major public health problem, with key pieces of information needed for developing preventive and curative measures still missing. For example, the participation of the chaperone system (CS) in carcinogenesis and anti-cancer responses is poorly understood, although it can be predicted to be a crucial factor in these mechanisms. The chief components of the CS are the molecular chaperones, and here we discuss four of them, Hsp27, Hsp60, Hsp70, and Hsp90, focusing on their pro-carcinogenic roles in BC and potential for developing anti-BC therapies. These chaperones can be targets of negative chaperonotherapy, namely the elimination/blocking/inhibition of the chaperone(s) functioning in favor of BC, using, for instance, Hsp inhibitors. The chaperones can also be employed in immunotherapy against BC as adjuvants, together with BC antigens. Extracellular vesicles (EVs) in BC diagnosis and management are also briefly discussed, considering their potential as easily accessible carriers of biomarkers and as shippers of anti-cancer agents amenable to manipulation and controlled delivery. The data surveyed from many laboratories reveal that, to enhance the understanding of the role of the CS in BS pathogenesis, one must consider the CS as a physiological system, encompassing diverse members throughout the body and interacting with the ubiquitin–proteasome system, the chaperone-mediated autophagy machinery, and the immune system (IS). An integrated view of the CS, including its functional partners and considering its highly dynamic nature with EVs transporting CS components to reach all the cell compartments in which they are needed, opens as yet unexplored pathways leading to carcinogenesis that are amenable to interference by anti-cancer treatments centered on CS components, such as the molecular chaperones.     

Triiodothyronine Acts as a Smart Influencer on Hsp90 via a Triiodothyronine Binding Site

Microarray-based experiments revealed that thyroid hormone triiodothyronine (T3) enhanced the binding of Cy5-labeled ATP on heat shock protein 90 (Hsp90). By molecular docking experiments with T3 on Hsp90, we identified a T3 binding site (TBS) near the ATP binding site on Hsp90. A synthetic peptide encoding HHHHHHRIKEIVKKHSQFIGYPITLFVEKE derived from the TBS on Hsp90 showed, in MST experiments, the binding of T3 at an EC₅₀ of 50 μM. The binding motif can influence the activity of Hsp90 by hindering ATP accessibility or the release of ADP.     

Hsp90 Inhibition: A Promising Therapeutic Approach for ARSACS

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease caused by mutations in the SACS gene, encoding the 520 kDa modular protein sacsin, which comprises multiple functional sequence domains that suggest a role either as a scaffold in protein folding or in proteostasis. Cells from patients with ARSACS display a distinct phenotype including altered organisation of the intermediate filament cytoskeleton and a hyperfused mitochondrial network where mitochondrial respiration is compromised. Here, we used vimentin bundling as a biomarker of sacsin function to test the therapeutic potential of Hsp90 inhibition with the C-terminal-domain-targeted compound KU-32, which has demonstrated mitochondrial activity. This study shows that ARSACS patient cells have significantly increased vimentin bundling compared to control, and this was also present in ARSACS carriers despite them being asymptomatic. We found that KU-32 treatment significantly reduced vimentin bundling in carrier and patient cells. We also found that cells from patients with ARSACS were unable to maintain mitochondrial membrane potential upon challenge with mitotoxins, and that the electron transport chain function was restored upon KU-32 treatment. Our preliminary findings presented here suggest that targeting the heat-shock response by Hsp90 inhibition alleviates vimentin bundling and may represent a promising area for the development of therapeutics for ARSACS.     

Development of Computational Approaches with a Fragment-Based Drug Design Strategy: In Silico Hsp90 Inhibitors Discovery

A semi-exhaustive approach and a heuristic search algorithm use a fragment-based drug design (FBDD) strategy for designing new inhibitors in an in silico process. A deconstruction reconstruction process uses a set of known Hsp90 ligands for generating new ones. The deconstruction process consists of cutting off a known ligand in fragments. The reconstruction process consists of coupling fragments to develop a new set of ligands. For evaluating the approaches, we compare the binding energy of the new ligands with the known ligands.     

Inhibition of Hepatitis C Replication by Targeting the Molecular Chaperone Hsp90: Synthesis and Biological Evaluation of 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole Derivatives

Cellular chaperones that belong to the heat-shock protein 90 (Hsp90) family are a prerequisite for successful viral propagation for most viruses. The hepatitis C virus (HCV) uses Hsp90 for maturation, folding, and modification of viral proteins. Based on our previous discovery that marine alkaloid analogues with a 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2-amine structure show inhibition of HCV replication and binding to Hsp90, a series of twelve novel compounds based on this scaffold was designed and synthesized. The aim was improved Hsp90 affinity and anti-HCV activity. Through structural optimization, improved binding to Hsp90 and specific HCV inhibition in genotype 1b and 2a replicon models was achieved for three compounds belonging to the newly synthesized series. Furthermore, these compounds efficiently inhibited replication of full-length HCV genotype 2a in a reporter virus RNA assay with IC₅₀ values ranging from 0.03 to 0.6 μm .     

The Activity and Stability of p56Lck and TCR Signaling Do Not Depend on the Co-Chaperone Cdc37

Lymphocyte-specific protein tyrosine kinase (Lck) is a pivotal tyrosine kinase involved in T cell receptor (TCR) signaling. Because of its importance, the activity of Lck is regulated at different levels including phosphorylation of tyrosine residues, protein–protein interactions, and localization. It has been proposed that the co-chaperone Cdc37, which assists the chaperone heat shock protein 90 (Hsp90) in the folding of client proteins, is also involved in the regulation of the activity/stability of Lck. Nevertheless, the available experimental data do not clearly support this conclusion. Thus, we assessed whether or not Cdc37 regulates Lck. We performed experiments in which the expression of Cdc37 was either augmented or suppressed in Jurkat T cells. The results of our experiments indicated that neither the overexpression nor the suppression of Cdc37 affected Lck stability and activity. Moreover, TCR signaling proceeded normally in T cells in which Cdc37 expression was either augmented or suppressed. Finally, we demonstrated that also under stress conditions Cdc37 was dispensable for the regulation of Lck activity/stability. In conclusion, our data do not support the idea that Lck is a Cdc37 client.     

The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90

The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP-driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N-terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD−MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD−MD arrangement with the accessibility of a client binding site on the NTD.     

Spatial Temporal Dynamics and Molecular Evolution of Re-Emerging Rabies Virus in Taiwan

Taiwan has been recognized by the World Organization for Animal Health as rabies-free since 1961. Surprisingly, rabies virus (RABV) was identified in a dead Formosan ferret badger in July 2013. Later, more infected ferret badgers were reported from different geographic regions of Taiwan. In order to know its evolutionary history and spatial temporal dynamics of this virus, phylogeny was reconstructed by maximum likelihood and Bayesian methods based on the full-length of glycoprotein (G), matrix protein (M), and nucleoprotein (N) genes. The evolutionary rates and phylogeographic were determined using Beast and SPREAD software. Phylogenetic trees showed a monophyletic group containing all of RABV isolates from Taiwan and it further separated into three sub-groups. The estimated nucleotide substitution rates of G, M, and N genes were between 2.49 × 10⁻⁴–4.75 × 10⁻⁴ substitutions/site/year, and the mean ratio of dN/dS was significantly low. The time of the most recent common ancestor was estimated around 75, 89, and 170 years, respectively. Phylogeographic analysis suggested the origin of the epidemic could be in Eastern Taiwan, then the Formosan ferret badger moved across the Central Range of Taiwan to western regions and separated into two branches. In this study, we illustrated the evolution history and phylogeographic of RABV in Formosan ferret badgers.     

Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N⁷/N⁹-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC₅₀ values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.     

Structural Communication between the E. coli Chaperones DnaK and Hsp90

The 70 kDa and 90 kDa heat shock proteins Hsp70 and Hsp90 are two abundant and highly conserved ATP-dependent molecular chaperones that participate in the maintenance of cellular homeostasis. In Escherichia coli, Hsp90 (Hsp90Ec) and Hsp70 (DnaK) directly interact and collaborate in protein remodeling. Previous work has produced a model of the direct interaction of both chaperones. The locations of the residues involved have been confirmed and the model has been validated. In this study, we investigate the allosteric communication between Hsp90Ec and DnaK and how the chaperones couple their conformational cycles. Using elastic network models (ENM), normal mode analysis (NMA), and a structural perturbation method (SPM) of asymmetric and symmetric DnaK-Hsp90Ec, we extract biologically relevant vibrations and identify residues involved in allosteric signaling. When one DnaK is bound, the dominant normal modes favor biological motions that orient a substrate protein bound to DnaK within the substrate/client binding site of Hsp90Ec and release the substrate from the DnaK substrate binding domain. The presence of one DnaK molecule stabilizes the entire Hsp90Ec protomer to which it is bound. Conversely, the symmetric model of DnaK binding results in steric clashes of DnaK molecules and suggests that the Hsp90Ec and DnaK chaperone cycles operate independently. Together, this data supports an asymmetric binding of DnaK to Hsp90Ec.     

Carborane-Based Analogues of 5-Lipoxygenase Inhibitors Co-inhibit Heat Shock Protein 90 in HCT116 Cells

5-Lipoxygenase converts arachidonic acid into leukotrienes, which are involved in inflammation and angiogenesis. The introduction of carboranes can improve the pharmacokinetic behavior of metabolically less stable pharmaceutics. Herein we report the syntheses of several carborane-based inhibitors of the 5-lipoxygenase pathway. The isosteric replacement of phenyl rings by carboranes leads to improved cytotoxicity toward several melanoma and colon cancer cell lines. For the colon cancer cell line HCT116, the co-inhibition of heat shock protein 90 was observed.     

Hsp90 Maintains the Stability and Function of the Tau Phosphorylating Kinase GSK3β

Hyperphosphorylation of tau leading to aggregated tau and tangle formation is a common pathological feature of tauopathies, including Alzheimer’s disease. Abnormal phosphorylation of tau by kinases, in particular GSK3β, has been proposed as a pathogenic mechanism in these diseases. In this study we demonstrate that the heat shock protein 90 (Hsp90) maintains the stability and function of the GSK3β. By using both rat primary cortical neurons and COS-7 cells, we show that Hsp90 inhibitors lead to a reduction of the protein level of GSK3β, and that this effect is associated with both a decrease in tau phosphorylation at putative GSK3β sites and an induction in heat shock protein 70 (Hsp70) levels. We further show that Hsp90 associates with the GSK3β regulating its stability and function and preventing its degradation by the proteasome.     

Structure‐Based and in silico Design of Hsp90 Inhibitors

The molecular chaperone Hsp90 is responsible for activation and stabilization of several oncoproteins in cancer cells, and has emerged as an important target in cancer treatment because of this pivotal role. In recent years, interests have arisen around structure‐based design of small molecules aimed at inhibiting the chaperone activity of Hsp90. In this review, we illustrate the recent advances in structure‐based and in silico strategies aimed at discovering and optimizing Hsp90 inhibitors.     

Discovery and biological activity of 6BrCaQ as an inhibitor of the Hsp90 protein folding machinery

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy, due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and viability. Here, a novel series of Hsp90 inhibitors containing a quinolein‐2‐one scaffold was synthesized and evaluated in cell proliferation assays. Results from these structure–activity relationships studies enabled identification of the simplified 3‐aminoquinolein‐2‐one analogue 2 b (6BrCaQ), which manifests micromolar activity against a panel of cancer cell lines. The molecular signature of Hsp90 inhibition was assessed by depletion of standard known Hsp90 client proteins. Finally, processing and activation of caspases 7, 8, and 9, and the subsequent cleavage of PARP by 6BrCaQ, suggest stimulation of apoptosis through both extrinsic and intrinsic pathways.     

TAS-116, a Well-Tolerated Hsp90 Inhibitor,Prevents the Activation of the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells

Chronic inflammation has been associated with several chronic diseases, such as age-related macular degeneration (AMD). The NLRP3 inflammasome is a central proinflammatory signaling complex that triggers caspase-1 activation leading to the maturation of IL-1β. We have previously shown that the inhibition of the chaperone protein, Hsp90, prevents NLRP3 activation in human retinal pigment epithelial (RPE) cells; these are cells which play a central role in the pathogenesis of AMD. In that study, we used a well-known Hsp90 inhibitor geldanamycin, but it cannot be used as a therapy due to its adverse effects, including ocular toxicity. Here, we have tested the effects of a novel Hsp90 inhibitor, TAS-116, on NLRP3 activation using geldanamycin as a reference compound. Using our existing protocol, inflammasome activation was induced in IL-1α-primed ARPE-19 cells with the proteasome and autophagy inhibitors MG-132 and bafilomycin A1, respectively. Intracellular caspase-1 activity was determined using a commercial caspase-1 activity kit and the FLICA assay. The levels of IL-1β were measured from cell culture medium samples by ELISA. Cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) measurements. Our findings show that TAS-116 could prevent the activation of caspase-1, subsequently reducing the release of mature IL-1β. TAS-116 has a better in vitro therapeutic index than geldanamycin. In summary, TAS-116 appears to be a well-tolerated Hsp90 inhibitor, with the capability to prevent the activation of the NLRP3 inflammasome in human RPE cells.     

抗狂犬病病毒抗体的制备及酶联免疫法的建立

目的制备具有中和活性的抗狂犬病病毒(Rabiesvirus,RV)抗体,并建立检测RV抗原的ELISA法。方法以RV全病毒免疫2只新西兰家兔,制备抗RV多克隆抗体。以RV全病毒免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0融合,建立稳定分泌抗RV单克隆抗体的杂交瘤细胞株。以小鼠中和试验(MNT)检测抗体的中和活性。以ELISA双抗体夹心法和ELISA竞争法检测RV抗原。结果2只家兔的多克隆抗体ELISA效价分别为1∶6.0×103和1∶1.2×104,纯化的兔抗RVIgG中和活性分别为46.3和29.2IU/ml。获得了9株稳定分泌抗RV的单克隆抗体杂交瘤细胞株,属于IgG1或IgG2b亚型,腹水的抗体ELISA效价为1∶1.0×105~1∶1.0×107。单克隆抗体3E5、4C2和4F8具有中和活性,Westernblot分析提示,单克隆抗体4C2是针对RV糖蛋白线性表位的抗体。以单克隆抗体4C2作为捕捉抗体,兔多克隆抗体作为检测抗体,建立了ELISA双抗体夹心法,检测RV抗原。同时建立了另一种ELISA竞争法,加入固定工作浓度的单克隆抗体4C2,与RV病毒液孵育,以ELISA间接法检测RV病毒抗原。结论所获得的狂犬病毒多克隆和单克隆抗体具有中和活性,可在ELISA中用于检测RV抗原。