Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes.

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

Structural basis of the key residue W320 responsible for Hsp90 conformational change.

The 90-kDa heat shock protein (Hsp90) is a homodimeric molecular chaperone with ATPase activity, which has become an intensely studied target for the development of drugs for the treatment of cancer, neurodegenerative and infectious diseases. The equilibrium between Hsp90 dimers and oligomers is important for modulating its function. In the absence of ATP, the passive chaperone activity of Hsp90 dimers and oligomers has been shown to stabilize client proteins as a holdase, which enhances substrate binding and prevents irreversible aggregation and precipitation of the substrate proteins. In the presence of ATP and its associated cochaperones, Hsp90 homodimers act as foldases with the binding and hydrolysis of ATP driving conformational changes that mediate client folding. Crystal structures of both wild type and W320A mutant Hsp90αMC (middle/C-terminal domain) have been determined, which displayed a preference for hexameric and dimeric states, respectively. Structural analysis showed that W320 is a key residue for Hsp90 oligomerization by forming intermolecular interactions at the Hsp90 hexameric interface through cation-π interactions with R367. W320A substitution results in the formation of a more open conformation of Hsp90, which has not previously been reported, and the induction of a conformational change in the catalytic loop. The structures provide new insights into the mechanism by which W320 functions as a key switch for conformational changes in Hsp90 self-oligomerization, and binding cochaperones and client proteins.Communicated by Ramaswamy H. Sarma.

Structure-Activity Relationship Study of Tertiary Alcohol Hsp90α-Selective Inhibitors with Novel Binding Mode.

The heat shock protein 90 (Hsp90) family of molecular chaperones mediates the folding and activation of client proteins associated with all 10 hallmarks of cancer. Herein, the design, synthesis, and biological validation of Hsp90α-selective inhibitors that contain a tertiary alcohol are reported. Forty-one analogues were synthesized to modulate hydrogen-bonding interactions and to probe for steric and hydrophobic interactions within the Hsp90α binding site. Cocrystal structures of lead compound 23d (IC50 = 0.25 µM, 15-fold selective vs Hsp90ß) and a 5-fluoroisoindoline derivative (KUNA-111) revealed a novel binding mode that induced conformational changes within Hsp90α's N-terminal domain. The lead Hsp90α-selective inhibitors did not manifest significant antiproliferative activity, but they did result in selective and dose-dependent degradation of Hsp90α clients in the cellular environment. Additional studies will be sought to determine the effects of the novel conformational change induced by 23d.

Crystal structure of the middle and C-terminal domains of Hsp90α labeled with a coumarin derivative reveals a potential allosteric binding site as a drug target.

The 90 kDa heat-shock protein (Hsp90) is an abundant molecular chaperone that is essential to activate, stabilize and regulate the function of a plethora of client proteins. As drug targets for the treatment of cancer and neurodegenerative diseases, Hsp90 inhibitors that bind to the N-terminal ATP-binding site of Hsp90 have shown disappointing efficacy in clinical trials. Thus, allosteric regulation of the function of Hsp90 by compounds that interact with its middle and C-terminal (MC) domains is now being pursued as a mechanism to inhibit the ATPase activity and client protein-binding activity of Hsp90 without concomitant induction of the heat-shock response. Here, the crystal structure of the Hsp90αMC protein covalently linked to a coumarin derivative, MDCC {7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin}, which is located in a hydrophobic pocket that is formed at the Hsp90αMC hexamer interface, is reported. MDCC binding leads to the hexamerization of Hsp90, and the stabilization and conformational changes of three loops that are critical for its function. A fluorescence competition assay demonstrated that other characterized coumarin and isoflavone-containing Hsp90 inhibitors compete with MDCC binding, suggesting that they could bind at a common site or that they might allosterically alter the structure of the MDCC binding site. This study provides insights into the mechanism by which the coumarin class of allosteric inhibitors potentially disrupt the function of Hsp90 by regulating its oligomerization and the burial of interaction sites involved in the ATP-dependent folding of Hsp90 clients. The hydrophobic binding pocket characterized here will provide new structural information for future drug design.

Selective Inhibition of the Hsp90α Isoform.

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone that processes nascent polypeptides into their biologically active conformations. Many of these proteins contribute to the progression of cancer, and consequently, inhibition of the Hsp90 protein folding machinery represents an innovative approach toward cancer chemotherapy. However, clinical trials with Hsp90 N-terminal inhibitors have encountered deleterious side effects and toxicities, which appear to result from the pan-inhibition of all four Hsp90 isoforms. Therefore, the development of isoform-selective Hsp90 inhibitors is sought to delineate the pathological role played by each isoform. Herein, we describe a structure-based approach that was used to design the first Hsp90α-selective inhibitors, which exhibit >50-fold selectivity versus other Hsp90 isoforms.

Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds.

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine ß-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1-5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

High Throughput Screening for Natural Host Defense Peptide-Inducing Compounds as Novel Alternatives to Antibiotics.

A rise in antimicrobial resistance demands novel alternatives to antimicrobials for disease control and prevention. As an important component of innate immunity, host defense peptides (HDPs) are capable of killing a broad spectrum of pathogens and modulating a range of host immune responses. Enhancing the synthesis of endogenous HDPs has emerged as a novel host-directed antimicrobial therapeutic strategy. To facilitate the identification of natural products with a strong capacity to induce HDP synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian ß-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.

Structure-guided design of an Hsp90ß N-terminal isoform-selective inhibitor.

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone responsible for folding proteins that are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms. pan-Inhibition of Hsp90 appears to be detrimental as toxicities have been reported alongside induction of the pro-survival heat shock response. The development of Hsp90 isoform-selective inhibitors represents an alternative approach towards the treatment of cancer that may limit some of the detriments. Described herein is a structure-based approach to design isoform-selective inhibitors of Hsp90ß, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90ß-selective inhibitors as a method to overcome the detriments associated with pan-inhibition.

A High-Throughput Screen for Inhibitors of the Hsp90-Chaperone Machine.

Hsp90 has emerged as a key chemotherapeutic target for the development of drugs for the treatment of cancer and neurodegenerative diseases. The shortcomings of many of the Hsp90 inhibitors that have made it to clinical trials have bolstered the need to identify new lead compounds with superior properties. Here, we describe a high-throughput screen for the identification of Hsp90 inhibitors based on the refolding of thermally denatured firefly luciferase.

Proteomic Profiling of Hsp90 Inhibitors.

Mass spectrometry assays demonstrate that Hsp90 inhibitors alter the expression of approximately one-quarter of the assayable proteome in mammalian cells. These changes are extraordinarily robust and reproducible, making "proteomics profiling" the gold standard for validating the effects of new Hsp90 inhibitors on cultured cells. Proteomics assays can also suggest novel hypotheses regarding drug mechanisms. To assist investigators in adopting this approach, this Chapter provides detailed protocols for conducting simple proteomics assays of Hsp90 inhibition. The protocols present a robust label-free approach that utilizes pre-fractionation of protein samples by SDS-PAGE, thereby providing reasonably good penetration into the proteome while addressing common issues with sample quality. The actual programming and operation of liquid chromatography-tandem mass spectrometers is not covered, but expectations for achievable performance are discussed, as are alternative approaches, common challenges, and software for data analysis.

Identification of proteins associated with Aha1 in HeLa cells by quantitative proteomics.

The identification of the activator of heat shock protein 90 (Hsp90) ATPase's (Aha1) protein-protein interaction (PPI) network will provide critical insights into the relationship of Aha1 with multi-molecular complexes and shed light onto Aha1's interconnections with Hsp90-regulated biological functions. Flag-tagged Aha1 was over-expressed in HeLa cells and isolated by anti-Flag affinity pull downs, followed by trypsin digestion and identification co-adsorbing proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). A probability-based identification of Aha1 PPIs was generated from the LC-MS/MS analysis by using a relative quantification strategy, spectral counting (SC). By comparing the SC-based protein levels between Aha1 pull-down samples and negative controls, 164 Aha1-interacting proteins were identified that were quantitatively enriched in the pull-down samples over the controls. The identified Aha1-interacting proteins are involved in a wide number of intracellular bioprocesses, including DNA maintenance, chromatin structure, RNA processing, translation, nucleocytoplasmic and vesicle transport, among others. The interactions of 33 of the identified proteins with Aha1 were further confirmed by Western blotting, demonstrating the reliability of our affinity-purification-coupled quantitative SC-MS strategy. Our proteomic data suggests that Aha1 may participate in diverse biological pathways to facilitate Hsp90 chaperone functions in response to stress.

High-throughput screen of natural product libraries for hsp90 inhibitors.

Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine.

Hsp90-dependent assembly of the DBC2/RhoBTB2-Cullin3 E3-ligase complex.

The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes.

Development of a high-throughput screening cancer cell-based luciferase refolding assay for identifying Hsp90 inhibitors.

The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.

The anticancer drug AUY922 generates a proteomics fingerprint that is highly conserved among structurally diverse Hsp90 inhibitors.

AUY922 is a potent synthetic Hsp90 antagonist that is moving steadily through clinical trials against a small range of cancers. To identify protein markers that might measure the drug's effects, and to gain understanding of mechanisms by which AUY922 might inhibit the proliferation of leukemia cells, we characterized AUY922's impacts on the proteomes of cultured Jurkat cells. We describe a robust and readily assayed proteomics fingerprint that AUY922 shares with the flagship Hsp90 inhibitors 17-DMAG and radicicol. We also extend our proteomics findings, demonstrating that an unrelated antagonist of protein folding potentiates the antiproliferative effects of AUY922. Results provide a set of candidate biomarkers for responses to AUY922 in leukemia cells and suggest new modalities for enhancing AUY922's anticancer activities.

Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.

Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins.

The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.

Approaches for defining the Hsp90-dependent proteome.

Hsp90 is the target of ongoing drug discovery studies seeking new compounds to treat cancer, neurodegenerative diseases, and protein folding disorders. To better understand Hsp90's roles in cellular pathologies and in normal cells, numerous studies have utilized proteomics assays and related high-throughput tools to characterize its physical and functional protein partnerships. This review surveys these studies, and summarizes the strengths and limitations of the individual attacks. We also include downloadable spreadsheets compiling all of the Hsp90-interacting proteins identified in more than 23 studies. These tools include cross-references among gene aliases, human homologues of yeast Hsp90-interacting proteins, hyperlinks to database entries, summaries of canonical pathways that are enriched in the Hsp90 interactome, and additional bioinformatic annotations. In addition to summarizing Hsp90 proteomics studies performed to date and the insights they have provided, we identify gaps in our current understanding of Hsp90-mediated proteostasis. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells.

BACKGROUND: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. METHODS: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. RESULTS: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. CONCLUSIONS: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Elucidation of the Hsp90 C-terminal inhibitor binding site.

The Hsp90 chaperone machine is required for the folding, activation, and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains; however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein.