Publications by authors named "Brian S J Blagg"

160 Publications

A novel C-terminal Hsp90 inhibitor KU758 synergizes efficacy in combination with BRAF or MEK inhibitors and targets drug-resistant pathways in BRAF-mutant melanomas.

Melanoma Res 2021 06;31(3):197-207

Department of Surgery, University of Michigan, Ann Arbor, Michigan.

Melanoma remains the most aggressive and fatal form of skin cancer, despite several FDA-approved targeted chemotherapies and immunotherapies for use in advanced disease. Of the 100 350 new patients diagnosed with melanoma in 2020 in the US, more than half will develop metastatic disease leading to a 5-year survival rate <30%, with a majority of these developing drug-resistance within the first year of treatment. These statistics underscore the critical need in the field to develop more durable therapeutics as well as those that can overcome chemotherapy-induced drug resistance from currently approved agents. Fortunately, several of the drug-resistance pathways in melanoma, including the proteins in those pathways, rely in part on Hsp90 chaperone function. This presents a unique and novel opportunity to simultaneously target multiple proteins and drug-resistant pathways in this disease via molecular chaperone inhibition. Taken together, we hypothesize that our novel C-terminal Hsp90 inhibitor, KU758, in combination with the current standard of care targeted therapies (e.g. vemurafenib and cobimetinib) can both synergize melanoma treatment efficacy in BRAF-mutant tumors, as well as target and overcome several major resistance pathways in this disease. Using in vitro proliferation and protein-based Western Blot analyses, our novel inhibitor, KU758, potently inhibited melanoma cell proliferation (without induction of the heat shock response) in vitro and synergized with both BRAF and MEK inhibitors in inhibition of cell migration and protein expression from resistance pathways. Overall, our work provides early support for further translation of C-terminal Hsp90 inhibitor and mitogen-activated protein kinase pathway inhibitor combinations as a novel therapeutic strategy for BRAF-mutant melanomas.
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http://dx.doi.org/10.1097/CMR.0000000000000734DOI Listing
June 2021

Biological Evaluation of 5'-(-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors.

ACS Med Chem Lett 2021 Mar 1;12(3):373-379. Epub 2021 Mar 1.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5'-(-Ethylcarboxamido)adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure-activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957913PMC
March 2021

Selective Inhibition of the Hsp90α Isoform.

Angew Chem Int Ed Engl 2021 05 26;60(19):10547-10551. Epub 2021 Mar 26.

Department of Chemistry and Biochemistry, The University of Notre Dame, 305 McCourtney Hall, Notre Dame, IN, 46556, USA.

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.
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http://dx.doi.org/10.1002/anie.202015422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086817PMC
May 2021

The Development of Hsp90β-Selective Inhibitors to Overcome Detriments Associated with -Hsp90 Inhibition.

J Med Chem 2021 02 11;64(3):1545-1557. Epub 2021 Jan 11.

Department of Chemistry and Biochemistry, The University of Notre Dame, 305 McCourtney Hall, Notre Dame, Indiana 46556, United States.

The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which 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 for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit -inhibitory activity against all four Hsp90 isoforms, which may lead to adverse effects. The development of Hsp90 isoform-selective inhibitors represents an alternative approach toward the treatment of cancer and may limit some of these detriments. Described herein, is a structure-based approach to develop isoform-selective inhibitors of Hsp90β, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90β-selective inhibitors as a method for overcoming the detriments associated with -inhibition.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01700DOI Listing
February 2021

Assay design and development strategies for finding Hsp90 inhibitors and their role in human diseases.

Pharmacol Ther 2021 May 24;221:107747. Epub 2020 Nov 24.

Department of Chemistry & Biochemistry, Warren Family Research Center for Drug Discovery and Development, 305 McCourtney Hall, University of Norte Dame, Norte Dame, IN 46656, USA. Electronic address:

Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the maturation of its client proteins including protein kinases, transcription factors, and steroid hormone receptors which are structurally and functionally diverse. These client proteins are involved in various cellular signaling pathways, and Hsp90 is implicated in various human diseases including cancer, inflammation, and diseases associated with protein misfolding; thus making Hsp90 a promising target for drug discovery. Some of its client proteins are well-known cancer targets. Instead of targeting these client proteins individually, however, targeting Hsp90 is more practical for cancer drug development. Efforts have been invested in recognizing potential drugs for clinical use that inhibit Hsp90 activity and result in the prevention of Hsp90 client maturation and dampening of subsequent signaling cascades. Here, we discuss current assays and technologies used to find and characterize Hsp90 inhibitors that include biophysical, biochemical, cell-based assays and computational modeling. This review highlights recent discoveries that N-terminal isoform-selective compounds and inhibitors that target the Hsp90 C-terminus that may offer the potential to overcome some of the detriments observed with pan Hsp90 inhibitors. The tools and assays summarized in this review should be used to develop Hsp90-targeting drugs with high specificity, potency, and drug-like properties that may prove immensely useful in the clinic.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107747DOI Listing
May 2021

Synthesis and Evaluation of Ginkgolic Acid Derivatives as SUMOylation Inhibitors.

ACS Med Chem Lett 2020 Nov 24;11(11):2221-2226. Epub 2020 Sep 24.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.

SUMOylation has emerged as an important post-translational modification that has been shown to modulate protein activity associated with various signaling pathways, and consequently, it has emerged as an important therapeutic target. While several natural products have been shown to inhibit enzymes involved in the SUMOylation process, there has been little progress toward the development of more selective and potent SUMOylation inhibitors. Ginkgolic acid was one of the first natural products discovered to inhibit the SUMO E1 enzyme. Despite its use to mechanistically investigate the SUMOylation process, ginkgolic acid also modulates other pathways as well. In this Letter, preliminary structure-activity relationships for ginkgolic acid as a SUMOylation inhibitor are presented.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667826PMC
November 2020

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

Int J Mol Sci 2020 Sep 20;21(18). Epub 2020 Sep 20.

Inte:Ligand Softwareentwicklungs- und Consulting GmbH, Mariahilferstrasse 74B, 1070 Vienna, Austria.

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 and displayed antiproliferative activities in MCF-7 and Hep G2 cancer cell lines. Compound inhibited Hsp90-dependent refolding of denatured luciferase and induced the degradation of Hsp90 clients without the concomitant induction of Hsp70 levels. Furthermore, compound 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.
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http://dx.doi.org/10.3390/ijms21186898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555175PMC
September 2020

Natural products and other inhibitors of FF ATP synthase.

Eur J Med Chem 2020 Dec 3;207:112779. Epub 2020 Sep 3.

Department of Chemistry and Biochemistry, 305 McCourtney Hall, University of Notre Dame, IN, 46545, United States. Electronic address:

FF ATP synthase is responsible for the production of >95% of all ATP synthesis within the cell. Dysregulation of its expression, activity or localization is linked to various human diseases including cancer, diabetes, and Alzheimer's and Parkinson's disease. In addition, ATP synthase is a novel and viable drug target for the development of antimicrobials as evidenced by bedaquiline, which was approved in 2012 for the treatment of tuberculosis. Historically, natural products have been a rich source of ATP synthase inhibitors that help unravel the role of FF ATP synthase in cellular bioenergetics. During the last decade, new modulators of ATP synthase have been discovered through the isolation of novel natural products as well as through a ligand-based drug design process. In addition, new data has been obtained with regards to the structure and function of ATP synthase under physiological and pathological conditions. Crystal structure studies have provided a significant insight into the rotary function of the enzyme and may provide additional opportunities to design a new generation of inhibitors. This review provides an update on recently discovered ATP synthase modulators as well as an update on existing scaffolds.
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http://dx.doi.org/10.1016/j.ejmech.2020.112779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891226PMC
December 2020

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

Med Res Rev 2021 01 25;41(1):202-222. Epub 2020 Aug 25.

Department of Chemistry and Biochemistry, Warren Family Research Center for Drug Discovery and Development, University of Notre Dame, Notre Dame, Indiana, USA.

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.
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http://dx.doi.org/10.1002/med.21729DOI Listing
January 2021

From Bacteria to Cancer: A Benzothiazole-Based DNA Gyrase B Inhibitor Redesigned for Hsp90 C-Terminal Inhibition.

ACS Med Chem Lett 2020 Aug 11;11(8):1535-1538. Epub 2020 Jun 11.

Department of Chemistry and Biochemistry, 305 McCourtney Hall, The University of Notre Dame, South Bend, Indiana 46545, United States.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for the folding and maturation of client proteins that are associated with all ten hallmarks of cancer. Hsp90 N-terminal pan inhibitors have experienced unfavorable results in clinical trials due to induction of the heat shock response (HSR), among other concerns. Novobiocin, a well characterized DNA gyrase B inhibitor, was identified as the first Hsp90 C-terminal inhibitor that manifested anticancer effects without induction of the HSR. In this letter, a library of Hsp90 C-terminal inhibitors derived from a benzothiazole-based scaffold, known to inhibit DNA gyrase B, was designed, synthesized, and evaluated. Several compounds were found to manifest low micromolar activity against both MCF-7 and SKBr3 breast cancer cell lines via Hsp90 C-terminal inhibition.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429967PMC
August 2020

Current Status of SUMOylation Inhibitors.

Curr Med Chem 2020 Aug 10. Epub 2020 Aug 10.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556. United States.

SUMOylation has emerged as an important post-translational modification that involves the covalent attachment of the Small Ubiquitin-like Modifier (SUMO) polypeptide to a lysine residue of a target protein. The enzymatic pathway of SUMOylation is very similar to ubiquitinylation and involves an activating enzyme, a conjugating enzyme, ligases and deconjugating enzymes. SUMOylation modulates the function of a number of proteins associated with various pathways, and in fact, dysregulation of the SUMOylation pathway is observed in both cancer and neurological diseases. In many cancers, the SUMO enzymes are upregulated and SUMO levels correlate directly with prognosis and disease progression. As a result, there has been an emphasis on the discovery and development of inhibitors of SUMOylation. In this review, the latest advances in SUMOylation inhibitors is described alongside the methods used to discover small molecule SUMOylation inhibitors, which include natural products, peptidomimetics, as well as synthetic derivatives identified via virtual screens.
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http://dx.doi.org/10.2174/0929867327666200810135039DOI Listing
August 2020

A novel heat shock protein inhibitor KU757 with efficacy in lenvatinib-resistant follicular thyroid cancer cells overcomes up-regulated glycolysis in drug-resistant cells in vitro.

Surgery 2021 01 24;169(1):34-42. Epub 2020 Jul 24.

Department of Surgery, University of Michigan, Ann Arbor, MI; Department of Pharmacology, University of Michigan, Ann Arbor, MI; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI. Electronic address:

Background: Patients with advanced differentiated thyroid cancer develop resistance to lenvatinib treatment from metabolic dysregulation. Heat shock protein 90 is a molecular chaperone that plays an important role in glycolysis and metabolic pathway regulation. We hypothesize that lenvatinib-resistant differentiated thyroid cancer cells will have an increased dependency on glycolysis and that a novel C-terminal heat shock protein 90 inhibitor (KU757) can effectively treat lenvatinib-resistant cells by targeting glycolysis.

Methods: Inhibitory concentration 50 values of thyroid cancer cells were determined by CellTiter-Glo assay (Promega Corp, Madison, WI). Glycolysis was measured through Seahorse experiments. Reverse transcription-polymerase chain reaction and Western blot evaluated glycolytic pathway genes/proteins. Exosomes were isolated/validated by nanoparticle tracking analysis and Western blot. Differentially expressed long non-coding ribonucleic acids in exosomes and cells were evaluated using quantitative polymerase chain reaction.

Results: Extracellular acidification rate demonstrated >2-fold upregulation of glycolysis in lenvatinib-resistant cells versus parent cells and was downregulated after KU757 treatment. Lenvatinib-resistant cells showed increased expression of the glycolytic genes lactic acid dehydrogenase, pyruvate kinase M1/2, and hexokinase 2. KU757 treatment resulted in downregulation of these genes and proteins. Several long non-coding ribonucleic acids associated with glycolysis were significantly upregulated in WRO-lenvatinib-resistant cells and exosomes and downregulated after KU757 treatment.

Conclusion: Lenvatinib resistance leads to increased glycolysis, and KU757 effectively treats lenvatinib-resistant cells and overcomes this increased glycolysis by targeting key glycolytic genes, proteins, and long non-coding ribonucleic acids.
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http://dx.doi.org/10.1016/j.surg.2020.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736095PMC
January 2021

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

Bioorg Med Chem Lett 2020 08 2;30(16):127303. Epub 2020 Jun 2.

Department of Chemistry and Biochemistry, University of Notre Dame, IN, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmcl.2020.127303DOI Listing
August 2020

Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway.

Sci Signal 2020 05 5;13(630). Epub 2020 May 5.

Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.

Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating μ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.
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http://dx.doi.org/10.1126/scisignal.aaz1854DOI Listing
May 2020

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

Adv Exp Med Biol 2020 ;1243:135-146

Department of Chemistry and Biochemistry, The Warren Family Research Center for Drug Discovery and Development, The University of Notre Dame, Notre Dame, IN, USA.

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.
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http://dx.doi.org/10.1007/978-3-030-40204-4_9DOI Listing
June 2020

Novel C-terminal heat shock protein 90 inhibitors target breast cancer stem cells and block migration, self-renewal, and epithelial-mesenchymal transition.

Mol Oncol 2020 09 15;14(9):2058-2068. Epub 2020 May 15.

Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

In patients with triple-negative breast cancer (TNBC), evidence suggests that tumor-initiating cells (TIC) have stem cell-like properties, leading to invasion and metastasis. HSP90 plays a critical role in the conformational maintenance of many client proteins in TIC development. Therefore, we hypothesize that the novel C-terminal HSP90 inhibitors KU711 and KU758 can target TIC and represent a promising strategy for overcoming metastasis. Human breast cancer cells (MDA-MB-468LN, MDA-MB-231) treated with the HSP90 inhibitors KU711, KU758, and 17-AAG showed a 50-80% decrease in TIC markers CD44 and aldehyde dehydrogenase (P < 0.01) as assessed by flow cytometry. A decrease in sphere formation, which was used to assess self-renewal, was observed after the treatment of TNBC cells starting at 2.5 µm KU711 and 0.31 µm KU758. KU compounds also blocked the invasion and migration of TNBC cells in a dose-dependent manner. The knockdown of HSP90 clients was observed without any change in prosurvival HSP70 levels. In vivo, in a murine orthotopic breast cancer model, treatment with KU758 and KU711 yielded an approximately twofold and a fourfold reduction in tumor volumes versus control, respectively, without demonstrated toxicity. In conclusion, C-terminal HSP90 inhibitors are potent novel therapeutics against TNBC in vitro and in vivo as they target TICs and block invasion, EMT transition, and self-renewal.
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http://dx.doi.org/10.1002/1878-0261.12686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463342PMC
September 2020

Catalysis-Enabled Access to Cryptic Geldanamycin Oxides.

ACS Cent Sci 2020 Mar 24;6(3):426-435. Epub 2020 Feb 24.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Catalytic, selective modifications of natural products can be a fertile platform for not only unveiling new natural product analogues with altered biological activity, but also for revealing new reactivity and selectivity hierarchies for embedded functional groups in complex environments. Motivated by these intersecting aims, we report site- and stereoselective oxidation reactions of geldanamycin facilitated by aspartyl-peptide catalysts. Through the isolation and characterization of four new geldanamycin oxides, we discovered a synergistic effect between lead peptide-based catalysts and geldanamycin, resulting in an unexpected reaction pathway. Curiously, our discoveries would likely not have been possible absent the attractive noncovalent interactions intrinsic to both the catalysts and the natural product. The result is a set of new "meta" catalytic reactions that deliver both unknown and previously incompletely characterized geldanamycin analogues. Enabled by the catalytic, site-selective epoxidation of geldanamycin, biological assays were carried out to document the bioactivities of the new compounds.
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http://dx.doi.org/10.1021/acscentsci.0c00024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099596PMC
March 2020

Pharmacologic dissection of the overlapping impact of heat shock protein family members on platelet function.

J Thromb Haemost 2020 05 30;18(5):1197-1209. Epub 2020 Mar 30.

Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Background: Platelets play a pivotal role in hemostasis, wound healing, and inflammation, and are thus implicated in a variety of diseases, including cancer. Platelet function is associated with release of granule content, cellular shape change, and upregulation of receptors that promote establishment of a thrombus and maintenance of hemostasis.

Objectives: The role of heat shock proteins (Hsps) in modulating platelet function has been studied for a number of years, but comparative roles of individual Hsps have not been thoroughly examined.

Methods: We utilized a panel of specific inhibitors of Hsp40, Hsp70, Hsp90, and Grp94 (the endoplasmic reticulum homolog of Hsp90) to assess their impact on several aspects of platelet function.

Results: Inhibition of each of the aforementioned Hsps reduced alpha granule release. In contrast, there was some selectivity in impacts on dense granule release. Thromboxane synthesis was impaired after exposure to inhibitors of Hsp40, Hsp90, and Grp94, but not after inhibition of Hsp70. Both expression of active glycoprotein IIb/IIIa (GPIIb/IIIa) and fibrinogen-induced platelet shape change were diminished by our inhibitors. In contrast, aggregation was selectively abrogated after inhibition of Hsp40 or Hsp90. Lastly, activated platelet-cancer cell interactions were reduced by inhibition of both Hsp70 and Grp94.

Conclusions: These data suggest the importance of Hsp networks in regulating platelet activity.
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http://dx.doi.org/10.1111/jth.14758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497839PMC
May 2020

The Alpha Isoform of Heat Shock Protein 90 and the Co-chaperones p23 and Cdc37 Promote Opioid Anti-nociception in the Brain.

Front Mol Neurosci 2019 29;12:294. Epub 2019 Nov 29.

Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, United States.

Opioid activation of the mu opioid receptor (MOR) promotes signaling cascades that evoke both analgesic responses to pain and side effects like addiction and dependence. Manipulation of these cascades, such as by biased agonism, has great promise to improve opioid therapy. However, the signaling cascades of the MOR are in general poorly understood, providing few targets for drug development. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel and crucial regulator of opioid anti-nociception in the brain by promoting ERK MAPK activation. In this study, we sought to identify the molecular isoforms and co-chaperones by which Hsp90 carried out this role, which could provide specific targets for future clinical intervention. We used novel selective small molecule inhibitors as well as CRISPR/Cas9 gene editing constructs delivered by the intracerebroventricular () route to the brains of adult CD-1 mice to target Hsp90 isoforms (Hsp90α/β, Grp94) and co-chaperones (p23, Cdc37, Aha1). We found that inhibition of the isoform Hsp90α fully blocked morphine anti-nociception in a model of post-surgical paw incision pain, while blocking ERK and JNK MAPK activation, suggesting Hsp90α as the main regulator of opioid response in the brain. We further found that inhibition of the co-chaperones p23 and Cdc37 blocked morphine anti-nociception, suggesting that these co-chaperones assist Hsp90α in promoting opioid anti-nociception. Lastly, we used cycloheximide treatment in the brain to demonstrate that rapid protein translation within 30 min of opioid treatment is required for Hsp90 regulation of opioid response. Together these studies provide insight into the molecular mechanisms by which Hsp90 promotes opioid anti-nociception. These findings thus both improve our basic science knowledge of MOR signal transduction and could provide future targets for clinical intervention to improve opioid therapy.
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http://dx.doi.org/10.3389/fnmol.2019.00294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895903PMC
November 2019

Old and New Approaches to Target the Hsp90 Chaperone.

Curr Cancer Drug Targets 2020 ;20(4):253-270

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, United States.

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that ensures cellular proteostasis by maintaining the folding, stabilization, activation, and degradation of over 400 client proteins. Hsp90 is not only critical for routine protein maintenance in healthy cells, but also during states of cellular stress, such as cancer and neurodegenerative diseases. Due to its ability to affect phosphorylation of numerous client proteins, inhibition of Hsp90 has been an attractive anticancer approach since the early 1990's, when researchers identified a druggable target on the amino terminus of Hsp90 for a variety of cancers. Since then, 17 Hsp90 inhibitors that target the chaperone's Nterminal domain, have entered clinical trials. None, however, have been approved thus far by the FDA as a cancer monotherapy. In these trials, a major limitation observed with Hsp90 inhibition at the N-terminal domain was dose-limiting toxicities and relatively poor pharmacokinetic profiles. Despite this, preclinical and clinical research continues to show that Hsp90 inhibitors effectively target cancer cell death and decrease tumor progression supporting the rationale for the development of novel Hsp90 inhibitors. Here, we present an in-depth overview of the Hsp90 inhibitors used in clinical trials. Finally, we present current shifts in the field related to targeting the carboxy-terminal domain of Hsp90 as well as to the development of isoform-selective inhibitors as a means to bypass the pitfalls of current Hsp90 inhibitors and improve clinical trial outcomes.
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http://dx.doi.org/10.2174/1568009619666191202101330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502213PMC
October 2020

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

Bioorg Med Chem Lett 2019 11 6;29(22):126676. Epub 2019 Sep 6.

Department of Chemistry and Biochemistry, The University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmcl.2019.126676DOI Listing
November 2019

A novel heat shock protein 90 inhibitor potently targets adrenocortical carcinoma tumor suppression.

Surgery 2020 01 24;167(1):233-240. Epub 2019 Sep 24.

Department of Surgery, University of Michigan, Ann Arbor, MI; Department of Pharmacology, University of Michigan, Ann Arbor, MI. Electronic address:

Introduction: Adrenocortical carcinoma is an aggressive cancer with a poor prognosis. Long noncoding RNAs are differentially expressed in cancer patients and contribute to cellular homeostasis, survival, and metastasis. We hypothesize that our novel C-terminal Hsp90 inhibitor KU758 can effectively target adrenocortical carcinoma cells and favorably alter long noncoding RNA expression.

Methods: Cell viability after KU758 treatment was measured in the adrenocortical carcinoma cell lines SW13, RL251, and NCI-H295R by MTS assay. Cellular mobility and metastatic potential after Hsp90 inhibition was measured through migration, invasion, and aggregate formation assays. β-catenin activity in NCI-H295R cells was determined by immunofluorescence and polymerase chain reaction. Long noncoding RNA expression was determined by polymerase chain reaction array after Hsp90 inhibition.

Results: KU758 is selective for adrenocortical carcinoma cells with IC50 values of 0.6 to 2.4 μM. KU758 treatment can effectively reduce migration, invasion, and aggregate formation in NCI-H295R and SW13 cells. β-catenin activity is decreased after treatment with KU758. Treatment with KU758 is associated with overall statistically significant upregulation of long noncoding RNA expression, including the tumor suppressor GAS5, which is implicated in the β-catenin and mammalian target of rapamycin pathways in adrenocortical carcinoma.

Conclusion: The novel C-terminal Hsp90 inhibitor KU758 is effective in the treatment of adrenocortical carcinoma cells and can significantly alter long noncoding RNA expression for tumor suppression.
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http://dx.doi.org/10.1016/j.surg.2019.06.052DOI Listing
January 2020

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

Biochemistry 2019 07 18;58(30):3225-3231. Epub 2019 Jul 18.

Department of Pharmacology and Toxicology, College of Pharmacy , University of Arizona , 1703 East Mabel Street , P.O. Box 210207, Tucson , Arizona 85721 , United States.

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 containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol () 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 to be an allosteric inhibitor of PTP1B with a submicromolar . Cellular analyses using C2C12 myoblasts indicated that restored insulin signaling and increased glucose uptake.
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http://dx.doi.org/10.1021/acs.biochem.9b00499DOI Listing
July 2019

The succinct synthesis of AT13387, a clinically relevant Hsp90 inhibitor.

Synth Commun 2019 23;49(11):1436-1443. Epub 2019 Apr 23.

Warren Family Research Center for Drug Discovery and Development, Department of Chemistry and Biochemistry,University of Notre Dame, Notre Dame, Indiana, USA.

AT13387 is an orally bioavailable clinical candidate developed to inhibit theheat shock protein 90 (Hsp90). This article describes a modified synthetic route for the multi-gram production of AT13387 in 46% overall yield. The modified synthetic route is short, avoids stringent reaction conditions and difficult purifications, which led to increase in an overall yield.
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http://dx.doi.org/10.1080/00397911.2019.1602654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577423PMC
April 2019

KU-596 decreases mitochondrial superoxide and improves bioenergetics following downregulation of manganese superoxide dismutase in diabetic sensory neurons.

Exp Neurol 2019 03 15;313:88-97. Epub 2018 Dec 15.

Department of Pharmacology and Toxicology, University of Kansas, 5064 Malott Hall, 1251 Wescoe Hall Dr., Lawrence, KS 66045, USA. Electronic address:

Neuronal mitochondrial dysfunction and oxidative stress are key pathophysiologic mechanisms of diabetic peripheral neuropathy (DPN). KU-596 is a small molecule modulator of heat shock protein 90 (Hsp90) that can reverse clinically relevant measures of DPN in diabetic animal models. Mechanistically, drug efficacy requires Hsp70 and correlates with improving mitochondrial maximal respiratory capacity (MRC) and decreasing oxidative stress in diabetic sensory neurons. The goal of this study was to determine if ex vivo treatment of diabetic neurons with KU-596 improves MRC by decreasing glucose-induced oxidative stress in an Hsp70-dependent manner. Sensory neurons were isolated from non-diabetic or diabetic mice wild type (WT) or Hsp70 knockout (Hsp70 KO) mice and treated with KU-596 in the presence of low or high glucose concentrations. In diabetic WT and Hsp70 KO neurons, hyperglycemia significantly increased superoxide levels, but KU-596 only decreased superoxide in WT neurons. Similarly, KU-596 significantly improved MRC in diabetic WT neurons maintained in high glucose but did not improve MRC in diabetic Hsp70 KO neurons under the same conditions. Since manganese superoxide dismutase (MnSOD) is the main mechanism to detoxify mitochondrial superoxide radicals, the cause and effect relationship between improved respiration and decreased oxidative stress was examined after knocking down MnSOD. Downregulating MnSOD in diabetic WT neurons increased hyperglycemia-induced superoxide levels, which was still significantly decreased by KU-596. However, KU-596 did not improve MRC following MnSOD knockdown. These data suggest that the ability of KU-596 to improve MRC is not necessarily dependent on decreasing mitochondrial superoxide in a MnSOD-dependent manner.
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http://dx.doi.org/10.1016/j.expneurol.2018.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582953PMC
March 2019

Molecular insights into the interaction of Hsp90 with allosteric inhibitors targeting the C-terminal domain.

Medchemcomm 2018 Aug 2;9(8):1323-1331. Epub 2018 Jul 2.

Department of Chemistry , University of South Florida , Tampa , FL 33620 , USA . Email:

Unique to targeting the C-terminal domain of Hsp90 (C-Hsp90) is the ability to uncouple the cytotoxic and cytoprotective outcomes of Hsp90 modulation. After the identification of novobiocin as a C-Hsp90 interacting ligand a diverse gamut of novologues emerged, from which KU-32 and KU-596 exhibited strong neuroprotective activity. However, further development of these ligands is hampered by the difficulty to obtain structural information on their complexes with Hsp90. Using saturation transfer difference (STD) NMR spectroscopy, we found that the primary binding epitopes of KU-32 and KU596 map at the ring systems of the ligands and specifically the coumarin and biphenyl structures, respectively. Based on both relative and absolute STD effects, we identified KU-596 sites that can be explored to design novel third-generation novologues. In addition, chemical shift perturbations obtained by methyl-TROSY reveal that novologues bind at the cryptic, C-Hsp90 ATP-binding pocket and produce global, long-range structural rearrangements to dimeric Hsp90.
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http://dx.doi.org/10.1039/c8md00151kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097425PMC
August 2018

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

Bioorg Med Chem Lett 2018 09 26;28(16):2701-2704. Epub 2018 Mar 26.

Department of Chemistry and Biochemistry, The University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119633PMC
September 2018

Exploiting polarity and chirality to probe the Hsp90 C-terminus.

Bioorg Med Chem 2018 07 13;26(12):3096-3110. Epub 2018 Apr 13.

Department of Chemistry and Biochemistry, University of Notre Dame, 305 McCourtney Hall, Notre Dame, IN 46556 USA. Electronic address:

Inhibition of the Hsp90 C-terminus is an attractive therapeutic approach for the treatment of cancer. Novobiocin, the first Hsp90 C-terminal inhibitor identified, contains a synthetically complex noviose sugar that has limited the generation of structure-activity relationships for this region of the molecule. The work described herein utilizes various ring systems as noviose surrogates to explore the size and nature of the surrounding binding pocket.
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http://dx.doi.org/10.1016/j.bmc.2018.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008240PMC
July 2018

Structure Based Design of a Grp94-Selective Inhibitor: Exploiting a Key Residue in Grp94 To Optimize Paralog-Selective Binding.

J Med Chem 2018 04 20;61(7):2793-2805. Epub 2018 Mar 20.

Hauptman-Woodward Medical Research Institute , Buffalo , New York 14203 , United States.

Grp94 and Hsp90, the ER and cytoplasmic hsp90 paralogs, share a conserved ATP-binding pocket that has been targeted for therapeutics. Paralog-selective inhibitors may lead to drugs with fewer side effects. Here, we analyzed 1 (BnIm), a benzyl imidazole resorcinylic inhibitor, for its mode of binding. The structures of 1 bound to Hsp90 and Grp94 reveal large conformational changes in Grp94 but not Hsp90 that expose site 2, a binding pocket adjacent to the central ATP cavity that is ordinarily blocked. The Grp94:1 structure reveals a flipped pose of the resorcinylic scaffold that inserts into the exposed site 2. We exploited this flipped binding pose to develop a Grp94-selective derivative of 1. Our structural analysis shows that the ability of the ligand to insert its benzyl imidazole substituent into site 1, a different side pocket off the ATP binding cavity, is the key to exposing site 2 in Grp94.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897183PMC
April 2018

Chaperone substrate provides missing link for cancer drug discovery.

J Biol Chem 2018 02;293(7):2381-2382

From the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46545.

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 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.
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http://dx.doi.org/10.1074/jbc.H118.001591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818199PMC
February 2018