Targeting the hydrophobic region of Hsp90’s ATP binding pocket with novel 1,3,5-triazines

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in regulating the maturation and stabilization of many oncogenic proteins. In an attempt to discover a new class of Hsp90 inhibitors, a series of 1,3,5-triazine compounds were rationally designed, synthesized, and their biological activities were evaluated. Compound 3b was found to degrade Hsp90’s client proteins of Her2, Met and Akt and to induce the expression level of Hsp70. The binding mode of 3b in the ATP-binding site of Hsp90 was predicted by the molecular docking.     

New heat shock protein (Hsp90) inhibitors,designed by pharmacophore modeling and virtual screening: synthesis,biological evaluation and molecular dynamics studies

Abstract

Inhibition of heat shock protein 90 (Hsp90) is known to be a significantly effective strategy in cancer therapy. Here, pyrazolopyranopyrimidine derivatives were characterized as new Hsp90 inhibitors. The molecules’ key structure (ZINC02819805) was determined by utilizing a pharmacophore model virtual screening workflow. Structural optimization was then carried out on compound ZINC02819805, pyrazolopyranopyrimidine derivatives were designed and six chosen derivatives were synthesized. The inhibition of Hsp90 ATPase activity of synthesized compounds revealed that para methylphenyl derivative of pyrazolopyranopyrimidine (HM3) was the most potent inhibitor (IC₅₀ = 5.5?µM). The anti-proliferative activity of this compound was evaluated against a panel of cell lines including MCF-7, HeLa and HUVEC (IC₅₀ = 1.28?µM, IC₅₀ = 1.74?µM and IC₅₀ = 61.48?µM respectively) by MTT method. The western blot analysis of treated MCF-7 cells with compound HM3 showed that the expression level of Hsp70 and Her2 proteins changed. The high level of Hsp70 expression and low level of Her2 expression suggest that compound HM3 exhibits inhibitory effect on Hsp90. Finally, the key interactions between HM3 and Hsp90 protein were studied by molecular dynamics simulation and showed that compound HM3 was stable in Hsp90 active cite during 200?ns simulation. Abbreviations Hsp90 Heat shock protein 90

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ATP adenosine triphosphate

MD molecular dynamics simulation

RMSD root-mean-square deviation

RMSF root-mean-square fluctuation

Rg gyration radius

m-SABNPs boehmite nanoparticles-supported sulfamic acid

Communicated by Ramaswamy H. Sarma     

Quinazoline Based HSP90 Inhibitors: Synthesis,Modeling Study and ADME Calculations Towards Breast Cancer Targeting

A new 2-thioquinazolinones series was designed and synthesized as HSP90 inhibitors based on the structure of hit compound VII obtained by virtual screening approach. Their in vitro anti-proliferative activity was evaluated against three human cancer cell lines rich in HSP90 namely; colorectal carcinoma (HCT-116), and cervical carcinoma (Hela), breast carcinoma (MCF-7). Compounds 5a, 5d, 5e and 9h showed a significant broad spectrum anti-proliferative activity against all tested cell lines. They were characterized by potent effect against breast cancer in particular with IC₅₀ of 11.73, 8.56, 7.35 and 9.48 μM, respectively against Doxorubicin (IC₅₀ 4.17 μM). HSP90 ATPase activity inhibition assay were conducted where compound 5d exhibited the best IC₅₀ with 1.58 μM compared to Tanespimycin (IC₅₀ = 2.17 μM). Compounds 5a and 9h showed higher IC₅₀ values of 3.21 and 3.41 μM, respectively. The effects of 5a, 5d and 9h on Her2 (a client proteins of HSP90) and HSP70 were evaluated in MCF-7 cells. All tested compounds were found to reduce Her2 protein expression levels and induce Hsp70 protein expression levels significantly, emphasizing that antibreast cancer effect is a consequence of HSP90 chaperone inhibition. Cell cycle analysis of MCF-7 cells treated with 5d showed cell cycle arrest at G2/M phase 38.89% and pro-apoptotic activity as indicated by annexin V-FITC staining by 22.42%. Molecular docking studies suggested mode of interaction to HSP90 via hydrogen bonding. ADME properties prediction of the active compounds suggested that they could be used as orally absorbed anticancer drug candidates.     

Down-regulation of MSH2 expression by an Hsp90 inhibitor enhances pemetrexed-induced cytotoxicity in human non-small-cell lung cancer cells

Elevated heat shock protein 90 (Hsp90) expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against NSCLC. However, the efficacy of the combination of pemtrexed and Hsp90 inhibitor to prolong the survival of patients with NSCLC still remains unclear. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and defects or polymorphisms of MSH2 have been found in lung cancer. In this study, we evaluated the effects of pemetrexed on NSCLC cell lines (H520 and H1703) and found that treatment with this drug at 20–50 µM increased the MSH2 mRNA and protein levels in a MKK3/6–p38 MAPK signal activation-dependent manner. Furthermore, the knockdown of MSH2 expression by transfection with small interfering RNA of MSH2 or the blockage of p38 MAPK activation by SB202190 enhanced the cytotoxicity of pemetrexed. Combining the drug treatment with an Hsp90 inhibitor resulted in an enhanced pemetrexed-induced cytotoxic effect, accompanied with the reduction of MSH2 protein and mRNA levels. The expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored the MSH2 protein levels and cell survival in NSCLC cells co-treated with pemetrexed and Hsp90 inhibitor. In this study, we have demonstrated that down-regulation of the MKK3/6–p38 MAPK signal with the subsequent reduction of MSH2 enhanced the cytotoxic effect of pemetrexed in H520 and H1703 cells. The results suggest a potential future benefit of combining pemetrexed and the Hsp90 inhibitor to treat lung cancer.     

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.     

Identification,Design and Bio-Evaluation of Novel Hsp90 Inhibitors by Ligand-Based Virtual Screening

Heat shock protein 90 (Hsp90), whose inhibitors have shown promising activity in clinical trials, is an attractive anticancer target. In this work, we first explored the significant pharmacophore features needed for Hsp90 inhibitors by generating a 3D-QSAR pharmacophore model. It was then used to virtually screen the SPECS databases, identifying 17 hits. Compound S1 and S13 exhibited the most potent inhibitory activity against Hsp90, with IC₅₀ value 1.61±0.28 μM and 2.83±0.67 μM, respectively. Binding patterns analysis of the two compounds with Hsp90 revealed reasonable interaction modes. Further evaluation showed that the compounds exhibited good anti-proliferative effects against a series of cancer cell lines with high expression level of Hsp90. Meanwhile, S13 induced cell apoptosis in a dose-dependent manner in different cell lines. Based on the consideration of binding affinities, physicochemical properties and toxicities, 24 derivatives of S13 were designed, leading to the more promising compound S40, which deserves further optimization.     

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.     

Discovery of novel heat shock protein (Hsp90) inhibitors based on luminespib with potent antitumor activity

A series of isosteric surrogates of the 4-phenyl group in luminespib were investigated as new scaffolds of the Hsp90 inhibitor for the discovery of novel antitumor agents. Among the synthesized surrogates of isoxazole and pyrazole, compounds 4a, 5e and 12b exhibited potent Hsp90 inhibition in ATPase activity and Her2 degradation assays and significant antitumor activity in A2780 and HCT116 cell lines. Animal studies indicated that compared to luminespib, their activities were superior in A2780 or NCI-H1975 tumor xenograft models. A molecular modeling study demonstrated that compound 4a could fit nicely into the N-terminal ATP binding pocket.     

Down regulation effect of Rosmarinus officinalis polyphenols on cellular stress proteins in rat pheochromocytoma PC12 cells

Polyphenols are known to exhibit wide spectrum of benefit for brain health and to protect from several neurodegenerative diseases. The present study was sought to determine the neuroprotective effects of Rosmarinus officinalis' polyphenols (luteolin, carnosic acid, and rosmarinic acid) through the investigation of stress-related proteins. We carried out measurement of the expression of heat-shock protein (Hsp) 47 promoter in heat stressed Chinese hamster ovary transfected cells. We performed proteomic analysis and confirmed gene expression by real time PCR in PC12 cells. Results showed that these compounds modulated significant and different effects on the expression of 4 stress-related proteins: heat shock protein 90 α (Hsp90), Transitional endoplasmic reticulum ATPase (VCP/p97), Nucleoside diphosphate kinase (NDK), and Hypoxia up-regulated protein 1 (HYOU1)) at translational and post translational levels in PC12 cells and they downregulated the expression of Hsp47 activity in Chinese hamster transformed cells. These findings suggest that luteolin, carnosic acid, and rosmarinic acid may modulate the neuroprotective defense system against cellular stress insults and increase neuro-thermotolerance.     

Click chemistry to probe Hsp90: Synthesis and evaluation of a series of triazole-containing novobiocin analogues

A series of triazole-containing novobiocin analogues has been designed, synthesized and their inhibitory activity determined. These compounds contain a triazole ring in lieu of the amide moiety present in the natural product. The anti-proliferative effects of these compounds were evaluated against two breast cancer cell lines (SKBr-3 and MCF-7), and manifested activities similar to their amide-containing counterparts. In addition, Hsp90-dependent client protein degradation was observed via Western blot analyses, supporting a common mode of Hsp90 inhibition for both structural classes.     

Protective effects of Araloside C against myocardial ischaemia/reperfusion injury: potential involvement of heat shock protein 90

The present study was designed to investigate whether Araloside C, one of the major triterpenoid compounds isolated from Aralia elata known to be cardioprotective, can improve heart function following ischaemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. We observed that Araloside C concentration‐dependently improved cardiac function and depressed oxidative stress induced by I/R. Similar protection was confirmed in isolated cardiomyocytes characterized by maintaining Ca²⁺ transients and cell shortening against I/R. Moreover, the potential targets of Araloside C were predicted using the DDI‐CPI server and Discovery Studio software. Molecular docking analysis revealed that Araloside C could be stably docked into the ATP/ADP‐binding domain of the heat shock protein 90 (Hsp90) protein via the formation of hydrogen bonds. The binding affinity of Hsp90 to Araloside C was detected using nanopore optical interferometry and yielded KD values of 29 μM. Araloside C also up‐regulated the expression levels of Hsp90 and improved cell viability in hypoxia/reoxygenation‐treated H9c2 cardiomyocytes, whereas the addition of 17‐AAG, a pharmacologic inhibitor of Hsp90, attenuated Araloside C‐induced cardioprotective effect. These findings reveal that Araloside C can efficiently attenuate myocardial I/R injury by reducing I/R‐induced oxidative stress and [Ca²⁺]_i overload, which was possibly related to its binding to the Hsp90 protein.     

Discovery of hybrid Hsp90 inhibitors and their anti-neoplastic effects against gefitinib-resistant non-small cell lung cancer (NSCLC)

Heat shock protein 90 (Hsp90) represents an attractive cancer therapeutic target due to its role in the stabilization and maturation of many oncogenic proteins. We have designed a series of hybrid Hsp90 inhibitors by connecting the resorcinol ring of VER-49009 (2) and the trimethoxyphenyl ring of PU3 (3) using structure-based approach. Subsequent testing established that compound 1f inhibited gefitinib-resistant H1975 cell proliferation, brought about the degradation of Hsp90 client proteins including EGFR, Met, Her2 and Akt and induced the expression of Hsp70. The design, synthesis, and evaluation of 1f are described herein.     

Association between HSP90 and Her2 in Gastric and Gastroesophageal Carcinomas

Background

Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas.

Material and Methods

Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters (UICC pTNM category, tumor grading) and survival.

Results

Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis (p=0.02; p=0.004; p=0.802). Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases.

Conclusion

Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment.     

Cold stress induces antioxidants and Hsps in chicken immune organs

The aim of this study was to investigate the effects of cold stress on oxidative indexes, immune function, and the expression levels of heat shock protein (Hsp90, Hsp70, Hsp60, Hsp40, and Hsp27) in immune organs of chickens. Two hundred forty 15-day-old male chickens were randomly divided into 12 groups and kept under the temperature of (12 ± 1) °C for acute and chronic cold stress. There were one control group and five treatment groups for acute cold stress and three control groups and three treatment groups for chronic cold stress. The results showed that cold stress influence the activities of antioxidant enzymes in the immune organs. The activities of SOD and GSH-Px were first increased then decreased, and activity of total antioxidation capacity (T-AOC) was significantly decreased (P < 0.05) at the acute cold stress in chicks; however, T-AOC activities were significantly increased (P < 0.05) at the chronic cold stress in these tissues. Cold stress induced higher level of malondialdehyde (MDA) in chicken immune organs. In addition, the cytokine contents were increased in cold stress groups. As one protective factor, the expression levels of Hsps were increased significantly (P < 0.05) in both cold stress groups. These results suggested that cold stress induced the oxidative stress in the three tissues and influenced immune function of chicks. Higher expression of Hsps (Hsp90, Hsp70, Hsp60, Hsp40, and Hsp27) may play a role in protecting immune organs against cold stress.     

Discovery of 5-substituted 2-amino-4-chloro-8-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7,8-dihydropteridin-6(5H)-ones as potent and selective Hsp90 inhibitors

A new series of compounds, 5-substituted 2-amino-4-chloro-8-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7,8-dihydropteridin-6(5H)-ones, have been designed and identified as potent and selective inhibitors of Hsp90. These compounds demonstrated nanomolar potency toward both Hsp90-regulated Her2 degradation and the growth of a panel of human tumor cell lines in cell-based assays. High selectivity of these compounds toward Hsp90 was evident given that they did not inhibit a panel of 34 kinases at 10 μM. The structure–activity relationship (SAR) of this series is reported here.     

Hsp90 Inhibitors Are Efficacious against Kaposi Sarcoma by Enhancing the Degradation of the Essential Viral Gene LANA,of the Viral Co-Receptor EphA2 as well as Other Client Proteins

Heat-shock protein 90 (Hsp90) inhibitors exhibit activity against human cancers. We evaluated a series of new, oral bioavailable, chemically diverse Hsp90 inhibitors (PU-H71, AUY922, BIIB021, NVP-BEP800) against Kaposi sarcoma (KS). All Hsp90 inhibitors exhibited nanomolar EC₅₀ in culture and AUY922 reduced tumor burden in a xenograft model of KS. KS is associated with KS-associated herpesvirus (KSHV). We identified the viral latency associated nuclear antigen (LANA) as a novel client protein of Hsp90 and demonstrate that the Hsp90 inhibitors diminish the level of LANA through proteasomal degradation. These Hsp90 inhibitors also downregulated EphA2 and ephrin-B2 protein levels. LANA is essential for viral maintenance and EphA2 has recently been shown to facilitate KSHV infection; which in turn feeds latent persistence. Further, both molecules are required for KS tumor formation and both were downregulated in response to Hsp90 inhibitors. This provides a rationale for clinical testing of Hsp90 inhibitors in KSHV-associated cancers and in the eradication of latent KSHV reservoirs.     

Methylmercury Alters the Activities of Hsp90 Client Proteins,Prostaglandin E Synthase/p23 (PGES/23) and nNOS

Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E₂ (PGE₂) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O₂ ⁻ and H₂O₂ levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity.     

Effects of 50 Hz sinusoidal magnetic fields on Hsp27, Hsp70, Hsp90 expression in porcine aortic endothelial cells (PAEC)

The aim of the present study was to investigate the influence of 50 Hz sinusoidal magnetic field on Hsp27, Hsp70, and Hsp90 expression in a model of primary culture of porcine aortic endothelial cells (PAEC). We took into consideration the Hsp profile in terms of mRNA expression, protein expression and protein localization inside the cells. The choice of the cell system was motivated by the involvement of the endothelial cells in the onset of many diseases; moreover, only few reports describe the effects of extremely low frequency magnetic fields (ELF-MFs) on such cells. ELF-MF exposure induced an increase in the mRNA levels of the three proteins, which was statistically significant for Hsp70. On the contrary, we did not observe any influence on Hsp27, Hsp70, and Hsp90 protein levels. Analysis in situ by immunofluorescence revealed that ELF-MF exposure affected the cellular distribution of Hsp27; in particular a partial relocalization in the nucleus was observed.