A small molecule that preferentially binds the closed conformation of Hsp90

Described is the synthesis of three different fluorescein-tagged derivatives of a macrocycle, and their binding affinity to heat shock protein 90 (Hsp90). Using fluorescence polarization anisotropy, we report the binding affinity of these fluorescein-labeled compounds to Hsp90 in its open state and ATP-dependent closed state. We show that the compounds demonstrate a conformation-dependent preference for binding to the closed state.     

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 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.     

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.     

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.     

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.     

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.     

Design,synthesis, anticancer evaluation and docking studies of new pyrimidine derivatives as potent thymidylate synthase inhibitors

Cancer is a perplexing and challenging problem for researchers. In this study, a series of 6-aryl-5-cyano-pyrimidine derivatives were designed, synthesized and evaluated for their anticancer activity against HePG-2, MCF-7 and HCT-116 cell lines. Compounds 2, 3d, 4a-c, 5, 8 and 12 displayed high anticancer activity, comparable to that of 5-fluorouracil. Additionally, those compounds with effective anticancer activity were further assessed for their ability to inhibit thymidylate synthase (TS) enzyme. All the tested compounds demonstrated a marked TS inhibitory activity (33.66–74.98%), with IC₅₀ ranging from 3.89 to 15.74 nM. Moreover, apoptosis studies were conducted on the most potent compound 8, to evaluate its proapoptotic potential. Interestingly, compound 8 induced the level of active caspase 3, and elevated the Bax/Bcl2 ratio 44 folds in comparison to the control. Finally, a molecular docking study was conducted to detect the probable interaction between the active compounds and the thymidylate synthase active site.     

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.     

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.     

Functions of the Hsp90 chaperone system: lifting client proteins to new heights

The molecular chaperone Hsp90 is an essential protein in eukaryotic organisms and is highly conserved throughout all kingdoms of life. It serves as a platform for the folding and maturation of many client proteins including protein kinases and steroid hormone receptors. To fulfill this task Hsp90 performs conformational changes driven by the hydrolysis of ATP. Further, it can resort to a broad set of co-chaperones, which fit the Hsp90 machinery to the needs of specific client proteins. During the last years the number of identified co-chaperones has been consistently rising, implying that the client spectrum of Hsp90 may be much more diverse and larger than currently known. Many cofactors contain a TPR-domain for interactions at the C-terminus of Hsp90 and in many cases their functions and client sets remain to be uncovered. Hsp90 is also a putative target to interfere with cancerous and infectious diseases. Thus the knowledge on more of its cellular functions would provide also more therapeutic options for the future. In this review we compile the current knowledge on the Hsp90 ATPase mechanism, cofactor regulation and prospects of Hsp90 inhibition.     

Synthesis and biological evaluation of new pyrazolone Schiff bases as monoamine oxidase and cholinesterase inhibitors

In the current work, Schiff base derivatives of antipyrine were synthesized. The chemical characterization of the compounds was confirmed using IR, ¹H NMR, ¹³C NMR and mass spectroscopies. The inhibitory potency of synthesized compounds was investigated towards acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and monoamine oxidases A and B (MAO-A and MAO-B) enzymes. Some of the compounds displayed significant inhibitory activity against AChE and MAO-B enzymes, respectively. According to AChE enzyme inhibition assay, compounds 3e and 3g were found as the most potent derivatives with IC₅₀ values of 0.285 µM and 0.057 µM, respectively. Also, compounds 3a (IC₅₀ = 0.114 µM), 3h (IC₅₀ = 0.049 µM), and 3i (IC₅₀ = 0.054 µM) were the most active derivatives against MAO-B enzyme activity. So as to understand inhibition type, enzyme kinetics studies were carried out. Furthermore, molecular docking studies were performed to define and evaluate the interaction mechanism between compounds 3g and 3h and related enzymes. ADME (Absorption, Distribution, Metabolism, and Excretion) and BBB (Blood, Brain, Barier) permeability predictions were applied to estimate pharmacokinetic profiles of synthesized compounds.     

Potential C-terminal-domain inhibitors of heat shock protein 90 derived from a C-terminal peptide helix

Hsp90 is a molecular chaperone implicated in many diseases including cancer and neurodegenerative disease. Most inhibitors target the ATPase site in Hsp90’s N-terminal domain, with relatively few inhibitors of other domains reported to date. Here, we show that peptides derived from a short helix at the C-terminus of Hsp90 show micromolar activity as Hsp90 inhibitors in vitro. These inhibitors do not block the N-terminal domain’s ATP-binding site, and thus are likely to bind at the C-terminal domain. Substitutions and helix stapling were applied to demonstrate structure–activity relationships and improve activity. These helical peptides will help guide the design of a new class of inhibitors of Hsp90’s C-terminal domain.     

3D-QSAR,molecular docking,and molecular dynamic simulations for prediction of new Hsp90 inhibitors based on isoxazole scaffold

Heat shock protein 90(Hsp90), as a molecular chaperone, play a crucial role in folding and proper function of many proteins. Hsp90 inhibitors containing isoxazole scaffold are currently being used in the treatment of cancer as tumor suppressers. Here in the present studies, new compounds based on isoxazole scaffold were predicted using a combination of molecular modeling techniques including three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamic (MD) simulations. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were also done. The steric and electrostatic contour map of CoMFA and CoMSIA were created. Hydrophobic, hydrogen bond donor and acceptor of CoMSIA model also were generated, and new compounds were predicted by CoMFA and CoMSIA contour maps. To investigate the binding modes of the predicted compounds in the active site of Hsp90, a molecular docking simulation was carried out. MD simulations were also conducted to evaluate the obtained results on the best predicted compound and the best reported Hsp90 inhibitors in the 3D-QSAR model. Findings indicate that the predicted ligands were stable in the active site of Hsp90.     

An evaluation of Hsp90 as a mediator of cortical patterning in Tetrahymena

This study asks two questions: 1) whether Hsp90 is involved in the regulation of cortical patterning in Tetrahymena, and 2) if it is, whether specific defects in this regulation can be attributed to functional insufficiency of the Hsp90 molecule. To address question 1, we compared the effects of a specific inhibitor of Hsp90, geldanamycin, on population growth and on development of the oral apparatus in two Tetrahymena species, T. pyriformis and T. thermophila. We observed that geldanamycin inhibits population growth in both species at very low concentrations, and that it has far more severe effects on oral patterning in T. pyriformis than in T. thermophila. These effects are parallel to those of high temperature in the same two species, and provide a tentative affirmative answer to the first question. To address question 2, we ascertained the base sequence of the genes that encode the Hsp90 molecules which are induced at high temperatures in both Tetrahymena species, as well as corresponding sequences in Paramecium tetraurelia. Extensive comparative analyses of the deduced amino acid sequences of the Hsp90 molecules of the two Tetrahymena species indicate that on the basis of what we currently know about Hsp90 both proteins are equally likely to be functional. Phylogenetic analyses of Hsp90 amino acid sequences indicate that the two Tetrahymena Hsp90 molecules have undergone a similar number of amino acid substitutions from their most recent common ancestor, with none of these corresponding to any known functionally critical region of the molecule. Thus there is no evidence that the Hsp90 molecule of T. pyriformis is functionally impaired; the flaw in the control of cortical patterning is more likely to be caused by defects in mechanism(s) that mediate the response to Hsp90, as would be expected from the "Hsp90 capacitor" model of Rutherford and Lindquist.     

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.     

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.Electronic supplementary materialThe online version of this article (10.1007/s12192-020-01167-0) contains supplementary material, which is available to authorized users.     

Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B₃ (CVB₃), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC₅₀ values of 0.42 ± 0.18 μM and 0.60 ± 0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.