CryoEM Structure with ATP Synthase Enables Late-Stage Diversification of Cruentaren†A.

Cruentaren A is a natural product that exhibits potent antiproliferative activity against various cancer cell lines, yet its binding site within ATP synthase remained unknown, thus limiting the development of improved analogues as anticancer agents. Herein, we report the cryogenic electron microscopy (cryoEM) structure of cruentaren A bound to ATP synthase, which allowed the design of new inhibitors through semisynthetic modification. Examples of cruentaren A derivatives include a trans-alkene isomer, which was found to exhibit similar activity to cruentaren A against three cancer cell lines as well as several other analogues that retained potent inhibitory activity. Together, these studies provide a foundation for the generation of cruentaren A derivatives as potential therapeutics for the treatment of cancer.

Recent advances toward the development of Hsp90 C-terminal inhibitors.

Heat shock protein 90 (Hsp90) is a dynamic protein which serves to ensure proper folding of nascent client proteins, regulate transcriptional responses to environmental stress and guide misfolded and damaged proteins to destruction via ubiquitin proteasome pathway. Recent advances in the field of Hsp90 have been made through development of isoform selective inhibitors, Hsp90 C-terminal inhibitors and disruption of protein-protein interactions. These approaches have led to alleviation of adverse off-target effects caused by pan-inhibition of Hsp90 using N-terminal inhibitors. In this review, we provide an overview of relevant advances on targeting the Hsp90 C-terminal Domain (CTD) and the development of Hsp90 C-terminal inhibitors (CTIs) since 2015.

The biology and inhibition of glucose-regulated protein 94/gp96.

The 94 kDa molecular chaperone, glucose-regulated protein 94 (Grp94), has garnered interest during the last decade due to its direct association with endoplasmic reticulum (ER) stress and disease. Grp94 belongs to the Hsp90 family of molecular chaperones and is a master regulator of ER homeostasis due to its ability to fold and stabilize proteins/receptors, and to chaperone misfolded proteins for degradation. Multiple studies have demonstrated that Grp94 knockdown or inhibition leads to the degradation of client protein substrates, which leads to disruption of disease-dependent signaling pathways. As a result, small molecule inhibitors of Grp94 have become a promising therapeutic approach to target a variety of disease states. Specifically, Grp94 has proven to be a promising target for cancer, glaucoma, immune-mediated inflammation, and viral infection. Moreover, Grp94-peptide complexes have been utilized effectively as adjuvants for vaccines against a variety of disease states. This work highlights the significance of Grp94 biology and the development of therapeutics that target this molecular chaperone in multiple disease states.

Syntheses of Symmetrical and Unsymmetrical Lysobisphosphatidic Acid Derivatives.

Recent years have witnessed significant achievements in the field of organic chemistry, which have led to new drugs and the discovery of new and biologically interesting molecules. Herein, we describe a practical and efficient approach to the synthesis of enantiomerically pure and diverse lysobisphosphatidic acid analogues. The key feature of the synthesis is a one-pot, sequential phosphorylation of a protected sn-2-O-oleoyl glycerol or sn-3-O-oleoyl glycerol with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, followed by oxidation.

Synthesis and Evaluation of Simplified Cruentaren A Analogues.

The 90 kDa heat shock protein (Hsp90) belongs to a group of molecular chaperones that regulate homeostasis via the folding of nascent polypeptides into their biologically active proteins, many of which are involved in cancer development and progression. As a result, inhibition of Hsp90 is an exciting area of research for the treatment of cancer. However, most of the 18 Hsp90 N-terminal inhibitors evaluated in clinical trials exhibited deleterious side effects and toxicities. Cruentaren A is a natural product that manifests potent anticancer activity against various human cancer cell lines via disruption of interactions between Hsp90α and F1FO ATP synthase, which does not induce the pro-survival, heat shock response, a major limitation associated with current Hsp90 inhibitors. However, the development of cruentaren A as a new anticancer agent has been hindered by its complex structure. Herein, we systematically removed the functionalities present in fragment 2 of cruentaren A and incorporated some key structural modifications from previous work, which produced 12 simplified analogues. Our studies determined that all functional groups present in fragment 2 are essential for cruentaren A's anticancer activity.

Synthesis and evaluation of 3'- and 4'-substituted cyclohexyl noviomimetics that modulate mitochondrial respiration.

KU-32 (2) and KU-596 (3), are first and second generation cytoprotective novologues that are derivatives of novobiocin (1), a heat shock protein 90 (Hsp90) C-terminal inhibitor. Although 2 and 3 improve mitochondrial bioenergetics and have demonstrated considerable cytoprotective activity, they contain a synthetically demanding noviose sugar. This issue was initially addressed by creating noviomimetics, such as KU-1202 (4), which replaced the noviose sugar with ether-linked cyclohexyl derivatives that retained some cytoprotective potential due to their ability to increase mitochondrial bioenergetics. Based on structure-activity relationship (SAR) studies of KU-1202 (4), the current study investigated 3'- and 4'-substituted cyclohexyl scaffolds as noviomimetics and determined their efficacy at increasing mitochondrial bioenergetic as a marker for cytoprotective potential.

The role and therapeutic potential of Hsp90, Hsp70, and smaller heat shock proteins in peripheral and central neuropathies.

Heat shock proteins (Hsps) are molecular chaperones that also play important roles in the activation of the heat shock response (HSR). The HSR is an evolutionary conserved and protective mechanism that is used to counter abnormal physiological conditions, stressors, and disease states, such as those exemplified in cancer and/or neurodegeneration. In normal cells, heat shock factor-1 (HSF-1), the transcription factor that regulates the HSR, remains in a dormant multiprotein complex that is formed upon association with chaperones (Hsp90, Hsp70, etc.), co-chaperones, and client proteins. However, under cellular stress, HSF-1 dissociates from Hsp90 and induces the transcriptional upregulation of Hsp70 to afford protection against the encountered cellular stress. As a consequence of both peripheral and central neuropathies, cellular stress occurs and results in the accumulation of unfolded and/or misfolded proteins, which can be counterbalanced by activation of the HSR. Since Hsp90 is the primary regulator of the HSR, modulation of Hsp90 by small molecules represents an attractive therapeutic approach against both peripheral and central neuropathies.

HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts.

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids, and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study, we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. The increase in other chaperones as a consequence of HSP90 improves folding of NPC1 protein and relieves cholesterol accumulation in NPC1 mutant fibroblasts.

Structure-Based Design, Synthesis, and Biological Evaluation of Hsp90ß-Selective Inhibitors.

The 90 kDa heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding and/or trafficking of ∼400 client proteins, many of which are directly associated with cancer progression. Consequently, inhibition of Hsp90 can exhibit similar activity as combination therapy as multiple signaling nodes can be targeted simultaneously. In fact, seventeen small-molecule inhibitors that bind the Hsp90 N-terminus entered clinical trials for the treatment of cancer, all of which exhibited pan-inhibitory activity against all four Hsp90 isoforms. Unfortunately, most demonstrated undesired effects alongside induction of the pro-survival heat shock response. As a result, isoform-selective inhibitors have been sought to overcome these detriments. Described herein is a structure-based approach to design Hsp90ß-selective inhibitors along with preliminary SAR. In the end, compound 5 was shown to manifest ∼370-fold selectivity for Hsp90ß versus Hsp90α, and induced the degradation of select Hsp90ß-dependent clients. These data support the development of Hsp90ß-selective inhibitors as a new paradigm to overcome the detriments associated with pan-inhibition of Hsp90.

Reaction between harmaline and vanillin to produce dimeric scaffoldsthat exhibit anti-proliferative activity.

Herbal medicine is used as a complement to modern medicine for the treatment of human diseases suchas cancer, inflammation, and diabetes. Nutraceutical components in foods such as vanillin produceantioxidant and anticancer activities and many of these produce minimal adverse effects in humans.Therefore, strategies that combine both herbal medicine and nutraceutical components could producecompounds that exhibit reduced toxicity. Recently, we developed GZ16.007, which is a combination ofharmaline and curcumin that is currently undergoing clinical evaluation for the treatment of cancer. Incontrast to the utilization of curcumin, we report herein the synthesis of a novel scaffold that utilizes har-maline and vanillin as nutraceutical components to form a newly identified anti-cancer scaffold. It wasdetermined that the inclusion of two molecules of harmaline and a single equivalent of vanillin produceda dimeric product that was active against various human cancer cell lines. The synthesis, evaluation andpreliminary SAR studies for the dimeric scaffold is discussed herein.

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.

Stimulation of heat shock protein 90 chaperone function through binding of a novobiocin analog KU-32.

Heat shock protein 90 (Hsp90) is a eukaryotic chaperone responsible for the folding and functional activation of numerous client proteins, many of which are oncoproteins. Thus, Hsp90 inhibition has been intensely pursued, resulting in the development of many potential Hsp90 inhibitors, not all of which are well-characterized. Hsp90 inhibitors not only abrogate its chaperone functions, but also could help us gain insight into the structure-function relationship of this chaperone. Here, using biochemical and cell-based assays along with isothermal titration calorimetry, we investigate KU-32, a derivative of the Hsp90 inhibitor novobiocin (NB), for its ability to modulate Hsp90 chaperone function. Although NB and KU-32 differ only slightly in structure, we found that upon binding, they induce completely opposite conformational changes in Hsp90. We observed that NB and KU-32 both bind to the C-terminal domain of Hsp90, but surprisingly, KU-32 stimulated the chaperone functions of Hsp90 via allosteric modulation of its N-terminal domain, responsible for the chaperone's ATPase activity. In vitro and in silico studies indicated that upon KU-32 binding, Hsp90 undergoes global structural changes leading to the formation of a "partially closed" intermediate that selectively binds ATP and increases ATPase activity. We also report that KU-32 promotes HeLa cell survival and enhances the refolding of an Hsp90 substrate inside the cell. This discovery explains the effectiveness of KU-32 analogs in the management of neuropathies and may facilitate the design of molecules that promote cell survival by enhancing Hsp90 chaperone function and reducing the load of misfolded proteins in cells.

Synthesis of paramagnetic ligands that target the C-terminal binding site of Hsp90.

Identification of a ligand binding site represents the starting point for a structure-based drug development program. Lack of a binding site hampers the development of improved ligands that modulate the protein of interest. In this letter, we describe the development of chemical tools that will allow for elucidation of the Hsp90 C-terminal ligand binding site. Our strategy is based on the preparation of paramagnetic analogs of KU-596, an investigational new drug that is currently undergoing clinical trials for the treatment of neuropathy and interacts with the Hsp90 C-terminal domain. In particular, we report the design and synthesis of three novel paramagnetic analogs of KU-596, which will be used to obtain long range distances for NMR structural studies of Hsp90 in complex with C-terminal ligands.

The Right Tool for the Job: An Overview of Hsp90 Inhibitors.

Molecular chaperones are responsible for maintaining intracellular protein quality control by facilitating the conformational maturation of new proteins as well as the refolding of denatured proteins. While there are several classes of molecular chaperones in the cell, this chapter will focus solely on the small molecule modulation of Hsp90, the 90 kDa heat shock protein. Hsp90 is not only responsible for folding nascent proteins, but it also regulates the triage of numerous client proteins through partnering with the ubiquitin-proteasome pathway. Consequently, Hsp90 plays critical role in maintaining the protein homeostasis (proteostasis) network within the cell and is required for the activation/maturation of more than 300 client protein substrates. Many of the clients that depend upon Hsp90 are overexpressed or mutated during malignant transformation. This often renders the clients thermodynamically unstable and dependent on Hsp90 for stability. This phenomenon results in an oncogenic 'addiction' to the Hsp90 protein folding machinery as Hsp90 maintains onco-client proteins. Furthermore, Hsp90-dependent substrates are associated with all ten hallmarks of cancer, making Hsp90 an attractive target for the development of cancer chemotherapeutics. In fact, 17 small molecule inhibitors of Hsp90 have been developed and clinically evaluated for the treatment of cancer. Unfortunately, most of these molecules have failed for various reasons, necessitating a new approach to modulate the Hsp90 protein folding machine.

Hsp90 activator Aha1 drives production of pathological tau aggregates.

The microtubule-associated protein tau (MAPT, tau) forms neurotoxic aggregates that promote cognitive deficits in tauopathies, the most common of which is Alzheimer's disease (AD). The 90-kDa heat shock protein (Hsp90) chaperone system affects the accumulation of these toxic tau species, which can be modulated with Hsp90 inhibitors. However, many Hsp90 inhibitors are not blood-brain barrier-permeable, and several present associated toxicities. Here, we find that the cochaperone, activator of Hsp90 ATPase homolog 1 (Aha1), dramatically increased the production of aggregated tau. Treatment with an Aha1 inhibitor, KU-177, dramatically reduced the accumulation of insoluble tau. Aha1 colocalized with tau pathology in human brain tissue, and this association positively correlated with AD progression. Aha1 overexpression in the rTg4510 tau transgenic mouse model promoted insoluble and oligomeric tau accumulation leading to a physiological deficit in cognitive function. Overall, these data demonstrate that Aha1 contributes to tau fibril formation and neurotoxicity through Hsp90. This suggests that therapeutics targeting Aha1 may reduce toxic tau oligomers and slow or prevent neurodegenerative disease progression.

Discovery of Novel Hsp90 C-Terminal Inhibitors Using 3D-Pharmacophores Derived from Molecular Dynamics Simulations.

Hsp90 C-terminal domain (CTD) inhibitors are promising novel agents for cancer treatment, as they do not induce the heat shock response associated with Hsp90 N-terminal inhibitors. One challenge associated with CTD inhibitors is the lack of a co-crystallized complex, requiring the use of predicted allosteric apo pocket, limiting structure-based (SB) design approaches. To address this, a unique approach that enables the derivation and analysis of interactions between ligands and proteins from molecular dynamics (MD) trajectories was used to derive pharmacophore models for virtual screening (VS) and identify suitable binding sites for SB design. Furthermore, ligand-based (LB) pharmacophores were developed using a set of CTD inhibitors to compare VS performance with the MD derived models. Virtual hits identified by VS with both SB and LB models were tested for antiproliferative activity. Compounds 9 and 11 displayed antiproliferative activities in MCF-7 and Hep G2 cancer cell lines. Compound 11 inhibited Hsp90-dependent refolding of denatured luciferase and induced the degradation of Hsp90 clients without the concomitant induction of Hsp70 levels. Furthermore, compound 11 offers a unique scaffold that is promising for the further synthetic optimization and development of molecules needed for the evaluation of the Hsp90 CTD as a target for the development of anticancer drugs.

An Isoform-Selective PTP1B Inhibitor Derived from Nitrogen-Atom Augmentation of Radicicol.

A library of natural products and their derivatives was screened for inhibition of protein tyrosine phosphatase (PTP) 1B, which is a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising was compound 2 containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol (1) with hydrazine. This nitrogen-atom augmented radicicol derivative was found to be PTP1B selective relative to other highly homologous nonreceptor PTPs. Biochemical evaluation, molecular docking, and mutagenesis revealed 2 to be an allosteric inhibitor of PTP1B with a submicromolar Ki. Cellular analyses using C2C12 myoblasts indicated that 2 restored insulin signaling and increased glucose uptake.

Chaperone substrate provides missing link for cancer drug discovery.

Both Hsp70 and Hsp90 chaperones are overexpressed in cancer, making them relevant targets for the development of cancer chemotherapeutics, but a lack of biomolecular readouts for Hsp70 inhibition has limited the pursuit of specific inhibitors for this enzyme. A new study from Cesa et al. identifies two inhibitors of apoptosis proteins (IAPs) as specific client substrates of Hsp70. These results establish biomarkers that can be utilized to monitor Hsp70 inhibition and provide a framework for future efforts to deconvolute chaperone networks.

Mitochondrial-targeted Hsp90 C-terminal inhibitors manifest anti-proliferative activity.

The development of C-terminal heat shock protein 90 kDa (Hsp90) inhibitors has emerged as a potential treatment for cancer. Similarly, small molecules that target the mitochondria have proven to be efficacious towards cancer, as the reprogramming of mitochondrial function is often associated with oncogenic transformation. Herein, we report the development of triphenylphosphonium (TPP)-conjugated Hsp90 C-terminal inhibitors, their anti-proliferative activity, and accumulation in the mitochondria. In general, TPP-conjugated Hsp90 C-terminal inhibitors were found to manifest increased activity against various cancer cell lines when compared to the parent compounds.

Synthesis and evaluation of a ring-constrained Hsp90 C-terminal inhibitor that exhibits neuroprotective activity.

KU-596 is a second-generation C-terminal heat shock protein 90 KDa (Hsp90) modulator based on the natural product, novobiocin. KU-596 has been shown to induce Hsp70 levels and manifest neuroprotective activity through induction of the heat shock response. A ring-constrained analog of KU-596 was designed and synthesized to probe its binding orientation and ability to induce Hsp70 levels. Compound 2 was found to exhibit comparable or increased activity compared to KU-596, which is under clinical investigation for the treatment of neuropathy.