Structural and energetic insight into the isoform-selective inhibitors of tumour marker Hsp90 against Grp94

The heat shock protein 90 (Hsp90) represents a new avenue for cancer therapy. A novel benzolactam inhibitor, compound 31, showed a great selectivity for Hsp90α and Hsp90β against Grp94. However, the structural basis for the great selectivity of compound 31 for Hsp90α/β versus Grp94 remains poorly understood. In this study, we carried out molecular docking, molecular dynamics simulations and binding free energy calculations (MM-GBSA) to address the isoform selective property. Molecular docking studies indicated the different binding modes of the Hsp90 and Grp94 with compound 31. The MM-GBSA calculations revealed that the hydrophobic interactions between compound 31 and proteins contributed the most to the binding affinity and the Grp94/compound 31 complex could result in a less energy-favourable complex compared with the Hsp90α/compound 31 and the Hsp90β/compound 31 complexes. This may render the weak binding of compound 31 to the Grp94. This study may be helpful for the future design of novel and selective Hsp90 inhibitors.     

Grp94, the endoplasmic reticulum Hsp90, has a similar solution conformation to cytosolic Hsp90 in the absence of nucleotide

The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide‐dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species‐specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP‐bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X‐ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair‐like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs.     

Discovery of 2-((4-resorcinolyl)-5-aryl-1,2,3-triazol-1-yl)acetates as potent Hsp90 inhibitors with selectivity over TRAP1

As the most abundant heat shock protein (HSP), Hsp90 is actively involved in tumor cell growth and various responses to anti-carcinogenic stress. Hsp90 has thus emerged as a potential drug target. A structure-based drug design approach was applied to develop novel resorcinolyltriazole derivatives as Hsp90 inhibitors. Structure-activity relationships (SARs) and molecular docking were investigated to provide a rationale for binding affinity and paralog selectivity. Click chemistry between iodoethynylresorcinol and an azido derivative was used to synthesize a new family of 2-((4-resorcinolyl)-5-aryl-1,2,3-triazol-1-yl) acetates that exhibited Hsp90 binding affinities of 40–100 nM (IC₅₀). Among the synthesized molecules, the triazole alkyl acetates displayed the highest Hsp90 binding affinities. Their potency against Hsp90 was over 100-fold stronger than against TRAP1 and 1–3-fold stronger than against Grp94. In particular, compounds 18, 19, and 30 had Hsp90 inhibitory activities of ~45 nM (IC₅₀) and they displayed over 350-fold selectivity for Hsp90 over TRAP1.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

The ER Chaperones BiP and Grp94 Regulate the Formation of Insulin-Like Growth Factor 2 (IGF2) Oligomers

While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.     

Synthesis and biological evaluation of arylated novobiocin analogs as Hsp90 inhibitors

Novobiocin analogs lacking labile glycosidic ether have been designed, synthesized and evaluated for Hsp90 inhibitory activity. Replacement of the synthetically complex noviose sugar with simple aromatic side chains produced analogs that maintain moderate cytotoxic activity against MCF7 and SkBR3 breast cancer cell-lines. Rationale for the preparation of des-noviose novobiocin analogs in addition to their synthesis and biological evaluation are presented herein.     

Towards the discovery of drug-like epigallocatechin gallate analogs as Hsp90 inhibitors

(−)-Epigallocatechin gallate (EGCG) is the major flavonoid of green tea and has been widely explored for a range of biological activities including anti-infective, anti-inflammatory, anti-cancer, and neuroprotection. Existing structure–activity data for EGCG has been largely limited to exploration of simple ethers and hydroxyl deletion. EGCG has poor drug-like properties because of multiple phenolic hydroxyl moieties and a metabolically labile ester. This work reports a substantial expansion of structure–activity understanding by exploring a range of semi-synthetic and synthetic derivatives with ester replacements and variously substituted aromatic and alicyclic groups containing more drug-like substituents. Structure–activity relationships for these molecules were obtained for Hsp90 inhibition. The results indicate that amide and sulfonamide linkers are suitable ester replacements. Hydroxylated aromatic rings and the cis-stereochemistry in EGCG are not essential for Hsp90 inhibition. Selected analogs in this series are more potent than EGCG in a luciferase refolding assay for Hsp90 activity.     

Grp94 acts as a mediator of curcumin‐induced antioxidant defence in myogenic cells

Curcumin is a non‐toxic polyphenol with pleiotropic activities and limited bioavailability. We investigated whether a brief exposure to low doses of curcumin would induce in the myogenic C2C12 cell line an endoplasmic reticulum (ER) stress response and protect against oxidative stress. A 3‐hr curcumin administration (5–10 μM) increased protein levels of the ER chaperone Grp94, without affecting those of Grp78, calreticulin and haeme‐oxygenase‐1 (HO‐1). Exposure of cells to hydrogen peroxide 24 hrs after the curcumin treatment decreased caspase‐12 activation, total protein oxidation and translocation of NF‐κB to the nucleus, compared with untreated cells. Grp94 overexpression, achieved by means of either stable or transient trasfection, induced comparable cytoprotective effects to hydrogen peroxide. The delayed cytoprotection induced by curcumin acted through Grp94, because the curcumin‐induced increase in Grp94 expression was hampered by either stable or transient transfection with antisense cDNA; in these latter cells, the extent of total protein oxidation, as well as the translocation of NF‐κB to the nucleus, and the percentage of apoptotic cells were comparable to those observed in both curcumin‐untreated wild‐type and empty vector transfected cells. Defining the mechanism(s) by which Grp94 exerts its antioxidant defence, the determination of cytosolic calcium levels in C2C12 cells by fura‐2 showed a significantly reduced amount of releasable calcium from intracellular stores, both in conditions of Grp94 overexpression and after curcumin pre‐treatment. Therefore, a brief exposure to curcumin induces a delayed cytoprotection against oxidative stress in myogenic cells by increasing Grp94 protein level, which acts as a regulator of calcium homeostasis.     

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.     

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.     

Genetically Engineered Hepatitis C Virus-like Particles (HCV-LPs) Tagged with SP94 Peptide to Acquire Selectivity to Liver Cancer Cells via Grp78

Targeted cancer therapy is a challenging area that includes multiple chemical and biological vehicles. Virus-like particles (VLPs) combine safety and efficacy in their roles as potential vaccines and drug delivery vehicles. In this study, we propose a novel drug delivery system based on HCV-LPs engineered with SP94 and RGD peptides mediated by a specific molecular chaperone (Grp78) associated with cancer drug resistance. The PCR primers were designed for engineering two constructs, SP94-EGFP-CORE-HIS and RGD-EGFP-CORE-HIS, by sequential PCR reactions. The two fragments were cloned into pFastBac Dual under the polyhedrin promoter and then used to produce two recombinant baculoviruses (AcSP94 and AcRGD). The VLP’s expression was optimized by recombinant virus infection with different MOIs, ranging from 1 to 20 MOI. Recombinant VLP2 were purified by Ni-NTA and their sizes and shapes were confirmed with TEM. They were incubated with different types of cells prior to examination using the fluorescence microscope to test the binding specificity. The effect of the overexpression of the Grp78 on the binding affinity of the engineered VLPs was tested in HepG2 and HeLa cells. The protocol optimization revealed that MOI 10 produced the highest fluorescence intensities after 72 h for the two recombinant proteins (SP94-core and RGD-core). Moreover, the binding assay tested on different types of mammalian cells (HeLa, HEK-293T, and HepG2 cells) showed green fluorescence on the periphery of all tested cell lines when using the RGD-core protein; while, the SP94-core protein showed green fluorescence only with the liver cancer cells, HepG2 and HuH7. Overexpression of Grp78 in HepG2 and HeLa cells enhanced the binding efficiency of the engineered VLPs. We confirmed that the SP94 peptide can be specifically used to target liver cancer cells, while the RGD peptide is sufficiently functional for most types of cancer cells. The overexpression of the Grp78 improved the binding capacity of both SP94 and RGD peptides. It is worth noting that the SP94 peptide can function properly as a recombinant peptide, and not only as a chemically conjugated peptide, as heretofore commonly used.     

The expression of glucose regulated protein—94 in colorectal carcinoma cells treated by sodium butyrate

The expression of glucose regulated protein 94 (GPR94) during the treatment of human colorectal carcinoma cell lineClone A cells with codium butyrate was studied.Dodium butyrate (SB) can cause functional and morphological effects on Clone A cells including growth arrest at G0/G1 stage and cell differentiation as observed by morphological changes,MTT and flow cytometry assays,as well as reduced Grp94 gene expression as shown by Northern blot and Western blot assays.The possible mechanism of the correlation between Grp94 gene expression and tumor growth inhibition and cell differentiation is briefly discussed.     

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 biological evaluation of coumarin replacements of novobiocin as Hsp90 inhibitors

Since Hsp90 modulates all six hallmarks of cancer simultaneously, it has become an attractive target for the development of cancer chemotherapeutics. In an effort to develop more efficacious compounds for Hsp90 inhibition, novobiocin analogues were prepared by replacing the central coumarin core with naphthalene, quinolinone, and quinoline surrogates. These modifications allowed for modification of the 2-position, which was previously unexplored. Biological evaluation of these compounds suggests a hydrophobic pocket about the 2-position of novobiocin. Anti-proliferative activities of these analogues against multiple cancer cell lines identified 2-alkoxyquinoline derivatives to exhibit improved activity.     

GRP94的功能及其与肿瘤的相关性

葡萄糖调节蛋白94（gloucose requlated protein 94,GRP94）是HSP90家族的成员,主要定位于内质网中.GRP94作为分子伴侣参与蛋白质的折叠、转运和分泌外,还参与了细胞凋亡以及抗原的提呈.GRP94与肿瘤的发生以及进展密切相关,肿瘤的分化程度越低,GRP94的表达也越高.GRP94又可将肿瘤特异性抗原提呈给专业提呈细胞启动特异性的免疫反应.GRP94在肿瘤的发生及转归中发挥了重要的作用.     

Synthesis and biological evaluation of 2,4-diaminoquinazoline derivatives as novel heat shock protein 90 inhibitors

Novel 2,4-diaminoquinazoline derivatives originating from a virtual screening approach were designed, synthesized and their biological activities as heat shock protein 90 (Hsp90) inhibitors were evaluated. The prepared compounds exhibited significant anti-proliferative activities against DU-145, HT-29, HCT-116, A375P and MCF-7 cancer cell lines. The selected compounds were tested against Her2, a client protein of Hsp90, and showed significant reduction in Her2 protein expression. Compound 6b was found the most potent, reduced Her2 protein expression levels and induced Hsp70 protein expression levels significantly.     

An investigation into the potential use of serum Hsp70 as a novel tumour biomarker for Hsp90 inhibitors

Hsp90 inhibitors are under investigation in multiple human clinical trials for the treatment of cancers, including myeloma, breast cancer, prostate, lung, melanoma, gastrointestinal stromal tumour and acute myeloid leukaemia. The pharmacodynamic activity of Hsp90 inhibitors in the clinic is currently assessed by Hsp70 induction in peripheral blood mononuclear cells using Western blot analysis, a method that is laborious, semiquantitative and difficult to implement in the clinic. Since Hsp70 was reported to be secreted by tumour cells and elevated in sera of cancer patients, serum Hsp70 has been evaluated as a potentially more robust, easily and reproducibly measured biomarker of Hsp90 inhibition as an alternative to cytosolic Hsp70. A highly sensitive and specific electrochemiluminescent ELISA was developed to measure serum Hsp70 and employed to evaluate Hsp70 levels in both ex vivo and xenograft samples. In ex vivo studies, maximal secretion of Hsp70 by tumour cells was observed between 48 and 72?h after exposure to Hsp90 inhibitors. In in vivo studies a 3–4-fold increase in serum Hsp70 was observed following treatment with BIIB021 in tumour-bearing mice. Strikingly, secreted Hsp70 was detectable in mice transplanted with human tumours but not in naive mice indicating a direct origination from the transplanted tumours. Analysis of clinical samples revealed low baseline levels (2–15?ng ml^?1) of Hsp70 in the serum of cancer patients and normal donors. Together these findings in laboratory studies and archived cancer patient sera suggest that serum Hsp70 could be a novel biomarker to assess reliably the pharmacological effects of Hsp90 inhibitors in clinical trials, especially under conditions where collection of tumour biopsies is not feasible.     

Synthesis and evaluation of biotinylated sansalvamide A analogs and their modulation of Hsp90

Described are the syntheses of three sansalvamide A derivatives that contain biotinylated tags at individual positions around the macrocycle. The tagged derivatives indicated in protein pull-down assays that they bind to Hsp90 at the same binding site (N-Middle domain) as the San A-amide peptide. Further, these compounds inhibit binding between Hsp90 and multiple C-terminal client proteins. This interaction is unique to the San A analogs indicating they can be tuned for selectivity against Hsp90 client/co-chaperone proteins.     

Chaperone gp96-independent inhibition of endotoxin response by chaperone-based peptide inhibitors

HSP90 chaperones a large number of proteins, and it plays essential roles in multiple signaling pathways to maintain protein homeostasis in the cytosol. In addition, HSP90 has been implicated in mediating recognition of lipopolysaccharide (LPS). However, no pharmacologic agents have been developed to interrogate this pathway. Herein we demonstrate that a peptide-based inhibitor that was previously reported to inhibit the master Toll-like receptor-chaperone gp96, an endoplasmic reticulum paralog of HSP90, in fact blocks HSP90-LPS interaction. It inhibited the binding of LPS to the cell surface of both wild type and gp96-null cells and thereby abrogated the cellular response to LPS but not to other Toll-like receptor ligands. We also generated a series of peptide derivatives (named peptide inhibitors of endotoxin responsiveness (PIERs)) from the N-terminal helix structure of HSP90 and demonstrated their effectiveness in blocking LPS activity. PIER inhibition of LPS signaling was partially reversed by CD14 expression. Moreover, we found that a cell-permeable PIER abrogated HSP90 function and caused degradation of multiple known HSP90 client proteins in cancer cells. Thus, targeting HSP90 is a promising modality for treatment of both LPS-mediated pathology and cancer.     

Synthesis and in vitro antiproliferative activity of C5-benzyl substituted 2-amino-pyrrolo[2,3-d]pyrimidines as potent Hsp90 inhibitors

A novel series of heat shock protein 90 (Hsp90) inhibitors was identified by X-ray crystal analysis of complex structures at solvent-exposed exit pocket C. The 2-amino-pyrrolo[2,3-d]pyrimidine derivatives, 7-deazapurines substituted with a benzyl moiety at C5, showed potent Hsp90 inhibition and broad-spectrum antiproliferative activity against NCI-60 cancer cell lines. The most potent compound, 6a, inhibited Hsp90 with an IC₅₀ of 36 nM and showed a submicromolar mean GI₅₀ value against NCI-60 cell lines. The interaction of 6a at the ATP-binding pocket of Hsp90 was confirmed by X-ray crystallography and Western blot analysis.