Hsp90 Mediates Membrane Deformation and Exosome Release.

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

Delivering hydrophilic peptide inhibitors of heat shock protein 70 into cancer cells.

Heat shock protein 70 (Hsp70) plays a major role in protein folding and has emerged as an attractive target in a wide range of cancers. Here we used a polymer nanogel to deliver two hydrophilic peptide inhibitors that block the interaction between the C-terminus of Hsp70 and heat shock organizing protein (HOP). The nanogels are able to load ∼200 wt% of the peptide inhibitors from solution via simple agitation at pH 7, and release them after cell uptake. Delivery of Hsp70 inhibitors to HCT116 cancer cells produced a clear Hsp70 inhibition phenotype: downregulation of client proteins glucocorticoid receptor (GR), immunophilins (FKBP51 and FKBP52), the protein kinase Akt-1, as well as the co-chaperone CHIP, and they induce cancer cell death. These results showcase the advantages of using versatile nanogels for delivery of hydrophilic cargo such as peptides and demonstrate the viability of these peptide inhibitors for targeting the Hsp70-HOP interaction in a cellular system.

Cyclic Peptides as Drugs for Intracellular Targets: The Next Frontier in Peptide Therapeutic Development.

Developing macrocyclic peptides that can reach intracellular targets is a significant challenge. This review discusses the most recent strategies used to develop cell permeable cyclic peptides that maintain binding to their biological target inside the cell. Macrocyclic peptides are unique from small molecules because traditional calculated physical properties are unsuccessful for predicting cell membrane permeability. Peptide synthesis and experimental membrane permeability is the only strategy that effectively differentiates between cell permeable and cell impermeable molecules. Discussed are chemical strategies, including backbone N-methylation and stereochemical changes, which have produced molecular scaffolds with improved cell permeability. However, these improvements often come at the expense of biological activity as chemical modifications alter the peptide conformation, frequently impacting the compound's ability to bind to the target. Highlighted is the most promising approach, which involves side-chain alterations that improve cell permeability without impact binding events.

C-Terminal HSP90 Inhibitors Block the HIF-1 Hypoxic Response by Degrading HIF-1α through the Oxygen-Dependent Degradation Pathway.

BACKGROUND/AIMS: Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. METHODS: HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. RESULTS: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. CONCLUSION: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.

Delivering bioactive cyclic peptides that target Hsp90 as prodrugs.

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.

Redefining the Phenotype of Heat Shock Protein 90 (Hsp90) Inhibitors.

The phenotypes produced when cells are treated with the heat shock protein 90 (Hsp90) inhibitors AUY922 or 17-AAG (classical inhibitors) are different to those produced when cells are knocked down with Hsp90α. Pull-down assays using classical inhibitors suggest that these molecules bind to multiple targets other than Hsp90. Classical inhibitors also induce similar protein markers as other anti-cancer therapies cisplatin and bortezomib that do not target Hsp90. Together these data suggest that AUY922 and 17-AAG acts on multiple targets and likely kills cells through multiple mechanisms. Comparing these classical inhibitors to the effects seen when treating cells with C-terminal Hsp90 modulators reveals that C-terminal modulators effectively bind to Hsp90, and induce phenotypic markers consistent with the Hsp90α CRISPR knockdown data. Our findings challenge the current interpretation of Hsp90 inhibitors and suggest that a large body of literature that describes the Hsp90 phenotype and inhibitors is re-examined in this context.

A Novel Class of Hsp90 C-Terminal Modulators Have Pre-Clinical Efficacy in Prostate Tumor Cells Without Induction of a Heat Shock Response.

BACKGROUND: While there is compelling rationale to use heat shock protein 90 (Hsp90) inhibitors for treatment of advanced prostate cancer, agents that target the N-terminal ATP-binding site of Hsp90 have shown little clinical benefit. These N-terminal binding agents induce a heat shock response that activates compensatory heat shock proteins, which is believed to contribute in part to the agents' lack of efficacy. Here, we describe the functional characterization of two novel agents, SM253 and SM258, that bind the N-middle linker region of Hsp90, resulting in reduced client protein activation and preventing C-terminal co-chaperones and client proteins from binding to Hsp90. METHODS: Inhibition of Hsp90 activity in prostate cancer cells by SM253 and SM 258 was assessed by pull-down assays. Cell viability, proliferation and apoptosis were assayed in prostate cancer cell lines (LNCaP, 22Rv1, PC-3) cultured with N-terminal Hsp90 inhibitors (AUY922, 17-AAG), SM253 or SM258. Expression of HSR heat shock proteins, Hsp90 client proteins and co-chaperones was assessed by immunoblotting. Efficacy of the SM compounds was evaluated in human primary prostate tumors cultured ex vivo by immunohistochemical detection of Hsp70 and Ki67. RESULTS: SM253 and SM258 exhibit antiproliferative and pro-apoptotic activity in multiple prostate cancer cell lines (LNCaP, 22Rv1, and PC-3) at low micromolar concentrations. Unlike the N-terminal inhibitors AUY922 and 17-AAG, these SM agents do not induce expression of Hsp27, Hsp40, or Hsp70, proteins that are characteristic of the heat shock response, in any of the prostate cell lines analyzed. Notably, SM258 significantly reduced proliferation within 2 days in human primary prostate tumors cultured ex vivo, without the significant induction of Hsp70 that was caused by AUY922 in the tissues. CONCLUSIONS: Our findings provide the first evidence of efficacy of this class of C-terminal modulators of Hsp90 in human prostate tumors, and indicate that further evaluation of these promising new agents is warranted. Prostate 76:1546-1559, 2016. © 2016 Wiley Periodicals, Inc.

Reinventing Hsp90 Inhibitors: Blocking C-Terminal Binding Events to Hsp90 by Using Dimerized Inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone (90 kDa) that functions as a dimer. This protein facilitates the folding, assembly, and stabilization of more than 400 proteins that are responsible for cancer development and progression. Inhibiting Hsp90's function will shut down multiple cancer-driven pathways simultaneously because oncogenic clients rely heavily on Hsp90, which makes this chaperone a promising anticancer target. Classical inhibitors that block the binding of adenine triphosphate (ATP) to the N-terminus of Hsp90 are highly toxic to cells and trigger a resistance mechanism within cells. This resistance mechanism comprises a large increase in prosurvival proteins, namely, heat shock protein 70 (Hsp70), heat shock protein 27 (Hsp27), and heat shock factor 1 (HSF-1). Molecules that modulate the C-terminus of Hsp90 are effective at inducing cancer-cell death without activating the resistance mechanism. Herein, we describe the design, synthesis, and biological binding affinity for a series of dimerized C-terminal Hsp90 modulators. We show that dimers of these C-terminal modulators synergistically inhibit Hsp90 relative to monomers.

Regulating the master regulator: Controlling heat shock factor 1 as a chemotherapy approach.

Described is the role that heat shock factor 1 (HSF1) plays in regulating cellular stress. Focusing on the current state of the HSF1 field in chemotherapeutics we outline the cytoprotective role of HSF1 in the cell. Summarizing the mechanism by which HSF1 regulates the unfolded proteins that are generated under stress conditions provides the background on why HSF1, the master regulator, is such an important protein in cancer cell growth. Summarizing siRNA knockdown results and current inhibitors provides a comprehensive evaluation on HSF1 and its current state. One set of molecules stands out, in that they completely obliterate the levels of HSF1, while simultaneously inhibiting heat shock protein 90 (Hsp90). These molecules are extremely promising as chemotherapeutic agents and as tools that may ultimately provide the connection between Hsp90 inhibition and HSF1 protein levels.

The fungal natural product (1S,3S)-austrocortirubin induces DNA damage in HCT116 cells via a mechanism unique from other DNA damaging agents.

Screening a series of natural product-based tetrahydroanthraquinones led to the identification of a novel molecule, (1S,3S)-austrocortirubin (2), which acts via inducing DNA damage. Compound 2 has a GI50 of 3µM against HCT116 and induces apoptosis. Mechanism of action studies indicate that it causes significant DNA damage during G0/G1, S, and G2 cell cycle phases. Cells are stopped at the G2/M phase checkpoint, and do not reach mitosis due to large amounts of DNA damage. Thus, compound 2 exhibits a unique mechanism of action, one that is distinct from doxorubicin, despite the high degree of structural homology between these two quinone-based structures.

Thioimidazoline based compounds reverse glucocorticoid resistance in human acute lymphoblastic leukemia xenografts.

Glucocorticoids form a critical component of chemotherapy regimens for pediatric acute lymphoblastic leukemia (ALL) and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. A high-throughput screen identified four thioimidazoline-containing compounds that reversed dexamethasone resistance in an ALL xenograft derived from a chemoresistant pediatric ALL. The lead compound (1) was synergistic when used in combination with the glucocorticoids, dexamethasone or prednisolone. Synergy was observed in a range of dexamethasone-resistant xenografts representative of B-cell precursor ALL (BCP-ALL) and T-cell ALL. We describe here the synthesis of twenty compounds and biological evaluation of thirty two molecules that explore the structure-activity relationships (SAR) of this novel class of glucocorticoid sensitizing compounds. SAR analysis has identified that the most effective dexamethasone sensitizers contain a thioimidazoline acetamide substructure with a large hydrophobic moiety on the acetamide.

Predicting the unpredictable: Recent structure-activity studies on peptide-based macrocycles.

Heterocycle-containing macrocycles are an emerging class of molecules that have therapeutic efficacy. Many biologically active natural products that have interesting biological properties fall into this class of molecules. The highly specific and selective biological activity is often attributed to the unique conformation of these macrocycles, which is affected by the elements of the macrocycles as well as its surroundings in biological systems. In this review, the structure-activity relationship studies of several recently developed biologically active heterocycle-containing macrocycles have been discussed in order to facilitate an understanding on how unpredictable structures can be controlled.

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response.

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.

Dimerization of a heat shock protein 90 inhibitor enhances inhibitory activity.

Heat shock protein 90 (hsp90) accounts for 1-2% of the total proteins in normal cells and it functions as a dimer. Hsp90 behaves as a molecular chaperone that folds, assembles, and stabilizes client proteins. We have developed a novel hsp90 inhibitor, and herein we describe the synthesis and biological activity of the dimerized variant of this inhibitor. Tethering a monomer inhibitor together produced a dimerized compound that more effectively inhibits hsp90 over the monomer.

Halting metastasis through CXCR4 inhibition.

Metastasis occurs when cancer cells leave the primary tumor site and migrate to distant parts of the body. The CXCR4-SDF-1 pathway facilitates this migration, and its expression has become the hallmark of several metastatic cancers. Targeted approaches are currently being developed to inhibit this pathway, and several candidates are now in clinical trials. Continued exploration of CXCR4 inhibitors will generate compounds that have improved activity over current candidates.

Heat shock proteins 27, 40, and 70 as combinational and dual therapeutic cancer targets.

The heat shock proteins are essential players in the development of cancer and they are prime therapeutic targets. Targeting multiple hsps in dual therapies decreases the likelihood of drug resistance compared to utilizing mono-therapies. Further, employing an hsp inhibitor in combination with another therapy has proven clinically successful. Examples of efficacious strategies include the inhibition of hsp27, which prevents protein aggregation, controlling hsp40's role as an ATPase modulator, and inhibiting hsp70 from acting as a molecular chaperone. While hsp40 therapies are just in the beginning stages, hsp27 and hsp70 therapies have been successfully used in dual inhibition treatments with hsp90 inhibitors and in combinational therapy with antineoplastic drugs. Both dual and combinatorial therapies show encouraging results when used in treating chemotherapeutically resistant diseases.

A potential rhodium cancer therapy: studies of a cytotoxic organorhodium(I) complex that binds DNA.

Described is a novel organorhodium(I) complex that is cytotoxic to the colon cancer cell line HCT116 and alters cell migration, DNA replication, and DNA condensation. Most importantly, the mechanism observed is not seen for the parent organorhodium dimer complex [{RhCl(COD)}2], RhCl3, or the free ligand/proligands (COD and 1-(n)butyl-3-methylimidazolium chloride). Thus, the activity of this organorhodium complex is attributable to its unique structure.

Psammaplysin F: a unique inhibitor of bacterial chromosomal partitioning.

Described is the antibiotic activity of a marine natural product. Psammaplysin F (1) inhibited the growth of four Gram-positive strains by >80% at 50µM, and the amine at position C-20 is responsible for the observed antibacterial activity. When tested against two strains of methicillin resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentrations (MICs) for psammaplysin F (40-80µM) were similar to the structurally-related alkaloid psammaplysin H (2). Psammaplysin F (1) increased membrane permeability by two to four-fold compared to psammaplysin H (2) or control-treated bacteria, respectively. Unlike psammaplysin H (2), we show that psammaplysin F (1) inhibits equal partitioning of DNA into each daughter cell, suggesting that this natural product is a unique prokaryotic cell division inhibitor.

Solid phase versus solution phase synthesis of heterocyclic macrocycles.

Comparing a solution phase route to a solid phase route in the synthesis of the cytotoxic natural product urukthapelstatin A (Ustat A) confirmed that a solid phase method is superior. The solution phase approach was tedious and involved cyclization of a ridged heterocyclic precursor, while solid phase allowed the rapid generation of a flexible linear peptide. Cyclization of the linear peptide was facile and subsequent generation of three oxazoles located within the structure of Ustat A proved relatively straightforward. Given the ease with which the oxazole Ustat A precursor is formed via our solid phase approach, this route is amenable to rapid analog synthesis.

Synthesis, structure-activity analysis, and biological evaluation of sanguinamide B analogues.

We report the first synthesis of sanguinamide B analogues. Substituting N-methylated (N-Me) amino acids, glycine (Gly), and L- or D-phenylalanine (Phe) into the backbone of sanguinamide B showed that only l- and d-Phe residues controlled the macrocycle conformation. The N-methylated and glycine analogues all had multiple conformations, whereas the L- and D-Phe derivatives only had a single conformation. Testing of all conformer analogues showed that inclusion of an L- or D-Phe was a superior design element than incorporating the N-Me moiety that is often utilized to control macrocyclic conformation. Finally, we show that there is an ideal Phe residue (in this case L-Phe) for generating compounds that have the greatest inhibitory effect on bacterial motility. Our data support the hypothesis that the macrocyclic conformation is dictated by the benzyl moiety requiring a "pseudoequatorial" position, and all other energy considerations are secondary.