Publications by authors named "Tony Taldone"

55 Publications

Measuring Tumor Epichaperome Expression Using [I] PU-H71 Positron Emission Tomography as a Biomarker of Response for PU-H71 Plus Nab-Paclitaxel in HER2-Negative Metastatic Breast Cancer.

JCO Precis Oncol 2020 17;4. Epub 2020 Nov 17.

Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY.

Purpose: Epichaperome network maintenance is vital to survival of tumors that express it. PU-H71 is an epichaperome inhibitor that binds to the ATP-binding site of HSP90 and has demonstrated antitumor activity in breast cancer xenograft models and clinical safety in patients. PU-positron emission tomography (PET) is a theragnostic imaging tool that allows visualization of the epichaperome target. In this phase Ib trial, we present safety and tolerability for PU-H71 plus nab-paclitaxel in HER2-negative patients with metastatic breast cancer (MBC) and the utility of PU-PET as a noninvasive predictive biomarker.

Methods: We performed a 3 + 3 dose-escalation study with escalating PU-H71 doses and standard nab-paclitaxel. The primary objective was to establish safety and determine maximum tolerated dose (MTD)/recommended phase 2 dose. Secondary objectives were to assess pharmacokinetics and clinical efficacy. Patients could enroll in a companion PU-PET protocol to measure epichaperome expression before treatment initiation to allow exploratory correlation with treatment benefit.

Results: Of the 12 patients enrolled, dose-limiting toxicity occurred in one patient (G3 neutropenic fever) at dose level 1; MTD of PU-H71 was 300 mg/m plus nab-paclitaxel 260 mg/m administered every 3 weeks. Common toxicities included diarrhea, fatigue, peripheral neuropathy, and nausea. PU-H71 systemic exposure was not altered by nab-paclitaxel administration. Two of 12 patients had partial response (overall response rate, 17%) and the clinical benefit rate was 42% (5 of 12). Time to progression was associated with baseline epichaperome positivity and PU-H71 peak standard uptake value (SUV), with more durable disease control observed with high epichaperome levels.

Conclusion: The combination of PU-H71 and nab-paclitaxel was well tolerated, with evidence of clinical activity. More durable disease control without progression was observed in patients with high baseline epichaperome expression. A phase II trial of this combination with PU-PET as a companion diagnostic for patient selection is currently planned.
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http://dx.doi.org/10.1200/PO.20.00273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713524PMC
November 2020

Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone.

Cell Rep 2020 06;31(13):107840

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.
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http://dx.doi.org/10.1016/j.celrep.2020.107840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372946PMC
June 2020

Chemical probes and methods for single-cell detection and quantification of epichaperomes in hematologic malignancies.

Methods Enzymol 2020 10;639:289-311. Epub 2020 May 10.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, United States; Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States. Electronic address:

Detection of protein connectivity dysfunctions in biological samples, i.e., informing on how protein-protein interactions change from a normal to a disease state, is important for both biomedical research and clinical development. The epichaperome is an executor of protein connectivity dysfunction in disease, and thus a surrogate for its detection. This chapter will detail on published methods for epichaperome detection and quantification that combine the advantages of multiparameter flow cytometry with those of the PU-FITC fluorescently labeled epichaperome detection probe. It will offer a comprehensive method description that includes the synthesis and characterization of an epichaperome detection probe and of the negative control probe, the preparation of the biospecimen for epichaperome analysis, the execution of the epichaperome detection and quantification assay and lastly, the data acquisition and analysis. The method provides, at single-cell level, the functional signature of cells, differentiating itself from other single-cell methods that provide a catalog of molecules.
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http://dx.doi.org/10.1016/bs.mie.2020.04.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397528PMC
May 2020

First-in-Human Trial of Epichaperome-Targeted PET in Patients with Cancer.

Clin Cancer Res 2020 Oct 4;26(19):5178-5187. Epub 2020 May 4.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: I-PU-H71 is an investigational first-in-class radiologic agent specific for imaging tumor epichaperome formations. The intracellular epichaperome forms under cellular stress and is a clinically validated oncotherapeutic target. We conducted a first-in-human study of microdose I-PU-H71 for PET to study biodistribution, pharmacokinetics, metabolism, and safety; and the feasibility of epichaperome-targeted tumor imaging.

Experimental Design: Adult patients with cancer ( = 30) received I-PU-H71 tracer (201±12 MBq, <25 μg) intravenous bolus followed by PET/CT scans and blood radioassays.

Results: I-PU-H71 PET detected tumors of different cancer types (breast, lymphoma, neuroblastoma, genitourinary, gynecologic, sarcoma, and pancreas). I-PU-H71 was retained by tumors for several days while it cleared rapidly from bones, healthy soft tissues, and blood. Radiation dosimetry is favorable and patients suffered no adverse effects.

Conclusions: Our first-in-human results demonstrate the safety and feasibility of noninvasive detection of tumor epichaperomes using I-PU-H71 PET, supporting clinical development of PU-H71 and other epichaperome-targeted therapeutics.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541604PMC
October 2020

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Nat Commun 2020 01 16;11(1):319. Epub 2020 Jan 16.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
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http://dx.doi.org/10.1038/s41467-019-14082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965647PMC
January 2020

Paradigms for Precision Medicine in Epichaperome Cancer Therapy.

Cancer Cell 2019 11 24;36(5):559-573.e7. Epub 2019 Oct 24.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.
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http://dx.doi.org/10.1016/j.ccell.2019.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996250PMC
November 2019

NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90.

J Biol Chem 2019 11 9;294(44):16010-16019. Epub 2019 Sep 9.

Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.
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http://dx.doi.org/10.1074/jbc.RA119.009960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827299PMC
November 2019

Structures of Hsp90α and Hsp90β bound to a purine-scaffold inhibitor reveal an exploitable residue for drug selectivity.

Proteins 2019 10 12;87(10):869-877. Epub 2019 Jun 12.

Hauptman-Woodward Medical Research Institute, Buffalo, New York.

Hsp90α and Hsp90β are implicated in a number of cancers and neurodegenerative disorders but the lack of selective pharmacological probes confounds efforts to identify their individual roles. Here, we analyzed the binding of an Hsp90α-selective PU compound, PU-11-trans, to the two cytosolic paralogs. We determined the co-crystal structures of Hsp90α and Hsp90β bound to PU-11-trans, as well as the structure of the apo Hsp90β NTD. The two inhibitor-bound structures reveal that Ser52, a nonconserved residue in the ATP binding pocket in Hsp90α, provides additional stability to PU-11-trans through a water-mediated hydrogen-bonding network. Mutation of Ser52 to alanine, as found in Hsp90β, alters the dissociation constant of Hsp90α for PU-11-trans to match that of Hsp90β. Our results provide a structural explanation for the binding preference of PU inhibitors for Hsp90α and demonstrate that the single nonconserved residue in the ATP-binding pocket may be exploited for α/β selectivity.
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http://dx.doi.org/10.1002/prot.25750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718336PMC
October 2019

A Chemical Biology Approach to the Chaperome in Cancer-HSP90 and Beyond.

Cold Spring Harb Perspect Biol 2020 04 1;12(4). Epub 2020 Apr 1.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065.

Cancer is often associated with alterations in the chaperome, a collection of chaperones, cochaperones, and other cofactors. Changes in the expression levels of components of the chaperome, in the interaction strength among chaperome components, alterations in chaperome constituency, and in the cellular location of chaperome members, are all hallmarks of cancer. Here we aim to provide an overview on how chemical biology has played a role in deciphering such complexity in the biology of the chaperome in cancer and in other diseases. The focus here is narrow and on pathologic changes in the chaperome executed by enhancing the interaction strength between components of distinct chaperome pathways, specifically between those of HSP90 and HSP70 pathways. We will review chemical tools and chemical probe-based assays, with a focus on HSP90. We will discuss how kinetic binding, not classical equilibrium binding, is most appropriate in the development of drugs and probes for the chaperome in disease. We will then present our view on how chaperome inhibitors may become potential drugs and diagnostics in cancer.
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http://dx.doi.org/10.1101/cshperspect.a034116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773535PMC
April 2020

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Nat Commun 2018 10 19;9(1):4345. Epub 2018 Oct 19.

The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 256, New York, NY, 10065, USA.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41467-018-06486-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195591PMC
October 2018

Copper Mediated Coupling of 2-(Piperazine)-pyrimidine Iodides with Aryl Thiols using Cu(I)Thiophene-2-carboxylate.

Tetrahedron Lett 2017 Nov 23;58(48):4525-4531. Epub 2017 Oct 23.

Program in Chemical Biology, Sloan Kettering Institute, New York, NY 10065, USA.

A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including -substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.
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http://dx.doi.org/10.1016/j.tetlet.2017.10.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047360PMC
November 2017

Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has Antitumor Activity in Burkitt Lymphoma.

Mol Cancer Ther 2017 09 15;16(9):1779-1790. Epub 2017 Jun 15.

Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York.

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity and , including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition and Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587381PMC
September 2017

The epichaperome is an integrated chaperome network that facilitates tumour survival.

Nature 2016 Oct 5;538(7625):397-401. Epub 2016 Oct 5.

Program in Chemical Biology, Sloan Kettering Institute, New York, New York 10065, USA.

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.
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http://dx.doi.org/10.1038/nature19807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283383PMC
October 2016

Parkin and PINK1 Patient iPSC-Derived Midbrain Dopamine Neurons Exhibit Mitochondrial Dysfunction and α-Synuclein Accumulation.

Stem Cell Reports 2016 10 15;7(4):664-677. Epub 2016 Sep 15.

Soonchunhyang Institute of Medi-bio Science (SIMS), Soonchunhyang University, 25, Bongjeong-ro, Dongnam-gu, Cheonan-si 31151, Korea. Electronic address:

Parkinson's disease (PD) is characterized by the selective loss of dopamine neurons in the substantia nigra; however, the mechanism of neurodegeneration in PD remains unclear. A subset of familial PD is linked to mutations in PARK2 and PINK1, which lead to dysfunctional mitochondria-related proteins Parkin and PINK1, suggesting that pathways implicated in these monogenic forms could play a more general role in PD. We demonstrate that the identification of disease-related phenotypes in PD-patient-specific induced pluripotent stem cell (iPSC)-derived midbrain dopamine (mDA) neurons depends on the type of differentiation protocol utilized. In a floor-plate-based but not a neural-rosette-based directed differentiation strategy, iPSC-derived mDA neurons recapitulate PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. We propose that these form a pathogenic loop that contributes to disease. Our study illustrates the promise of iPSC technology for examining PD pathogenesis and identifying therapeutic targets.
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http://dx.doi.org/10.1016/j.stemcr.2016.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063469PMC
October 2016

Stability of Doxycycline in Feed and Water and Minimal Effective Doses in Tetracycline-Inducible Systems.

J Am Assoc Lab Anim Sci 2016 ;55(4):467-74

Tri-Institutional Training Program in Laboratory Animal Medicine and Science, Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, and The Rockefeller University, Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, New York, USA.

Despite the extensive use of doxycycline in tetracycline-inducible rodent models, little is known regarding its stability in feed or water or the most effective route or dose. We assessed the concentrations of doxycycline in reverse-osmosis-purified (RO; pH 6.0) and acidified RO (pH 2.6) water in untinted or green-tinted bottles. Doxycycline remained stable in all groups for 7 d and in acidified water in untinted bottles for 14 d. Fungal growth occurred in nonacidified water in tinted and untinted bottles by 12 and 14 d, respectively, and in tinted bottles containing acidified water on day 14, but not in untinted bottles with acidified water. Doxycycline concentrations were also assessed before and at various points after the pelleting of feed from 2 vendors. Each batch was divided for storage at 4 °C, at room temperature, or within ventilated mouse isolator cages and then sampled monthly for 6 mo. Drying caused the greatest decline in doxycycline concentration, whereas γ-irradiation plus shipping and storage condition had minimal effect. Two mouse lines with tetracycline-inducible promoters received 25, 150, or 467 μg/mL or 2 mg/mL doxycycline in water and 200 or 625 ppm in feed before analysis of GFP expression. GFP was expressed in Rosa-rtTA2 mice at 150 μg/mL, whereas Cags-rtTA3 mice required 25 μg/mL. These studies indicate that 1) doxycycline-compounded feed can be handled in the same manner as standard rodent feed, 2) tinted water bottles are not necessary for maintaining drug concentrations, and 3) concentrations lower than those used typically may be effective in lines with tetracycline-inducible promoters.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943619PMC
February 2017

Heat Shock Protein (HSP) Drug Discovery and Development: Targeting Heat Shock Proteins in Disease.

Curr Top Med Chem 2016 ;16(25):2753-64

Program in Chemical Biology, Sloan Kettering Institute, New York, NY 10021, USA.

Heat shock proteins (HSPs) present as a double edged sword. While they play an important role in maintaining protein homeostasis in a normal cell, cancer cells have evolved to co-opt HSP function to promote their own survival. As a result, HSPs such as HSP90 have attracted a great deal of interest as a potential anticancer target. These efforts have resulted in over 20 distinct compounds entering clinical evaluation for the treatment of cancer. However, despite the potent anticancer activity demonstrated in preclinical models, to date no HSP90 inhibitor has obtained regulatory approval. In this review we discuss the unique challenges faced in targeting HSPs that have likely contributed to their lack of progress in the clinic and suggest ways to overcome these so that the enormous potential of these compounds to benefit patients can finally be realized. We also provide a guideline for the future development of HSP-targeted agents based on the many lessons learned during the last two decades in developing HSP90 inhibitors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995156PMC
http://dx.doi.org/10.2174/1568026616666160413141911DOI Listing
February 2017

Radiosynthesis of the iodine-124 labeled Hsp90 inhibitor PU-H71.

J Labelled Comp Radiopharm 2016 Mar 25;59(3):129-32. Epub 2016 Jan 25.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone protein whose function is critical for maintaining several key proteins involved in survival and proliferation of cancer cells. PU-H71 (1), is a potent purine-scaffold based ATP pocket binding Hsp90 inhibitor which has been shown to have potent activity in a broad range of in vivo cancer models and is currently in Phase I clinical trials in patients with advanced solid malignancies, lymphomas, and myeloproliferative neoplasms. In this report, we describe the radiosynthesis of [(124)I]-PU-H71(5); this was synthesized from the corresponding Boc-protected stannane precursor 3 by iododestannylation with [(124)I]-NaI using chloramine-T as an oxidant for 2 min, followed by Boc deprotection with 6 N HCl at 50 °C for 30 min to yield the final compound. The final product 5 was purified using HPLC and was isolated with an overall yield of 55 ± 6% (n = 6, isolated) from 3, and >98% purity and an average specific activity of 980 mCi/µmol. Our report sets the stage for the introduction of [(124)I]-PU-H71 as a potential non-invasive probe for understanding biodistribution and pharmacokinetics of PU-H71 in living subjects using positron emission tomography imaging.
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http://dx.doi.org/10.1002/jlcr.3369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779400PMC
March 2016

A Hyperactive Signalosome in Acute Myeloid Leukemia Drives Addiction to a Tumor-Specific Hsp90 Species.

Cell Rep 2015 Dec 25;13(10):2159-73. Epub 2015 Nov 25.

Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY 10065, USA. Electronic address:

Acute myeloid leukemia (AML) is a heterogeneous and fatal disease with an urgent need for improved therapeutic regimens given that most patients die from relapsed disease. Irrespective of mutation status, the development of aggressive leukemias is enabled by increasing dependence on signaling networks. We demonstrate that a hyperactive signalosome drives addiction of AML cells to a tumor-specific Hsp90 species (teHsp90). Through genetic, environmental, and pharmacologic perturbations, we demonstrate a direct and quantitative link between hyperactivated signaling pathways and apoptotic sensitivity of AML to teHsp90 inhibition. Specifically, we find that hyperactive JAK-STAT and PI3K-AKT signaling networks are maintained by teHsp90 and, in fact, gradual activation of these networks drives tumors increasingly dependent on teHsp90. Thus, although clinically aggressive AML survives via signalosome activation, this addiction creates a vulnerability that can be exploited with Hsp90-directed therapy.
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http://dx.doi.org/10.1016/j.celrep.2015.10.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699804PMC
December 2015

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas.

Blood 2016 Feb 24;127(7):858-68. Epub 2015 Nov 24.

Hematology and Oncology Division.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.
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http://dx.doi.org/10.1182/blood-2015-05-645069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760090PMC
February 2016

Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma.

J Clin Invest 2015 Nov 3;125(12):4559-71. Epub 2015 Nov 3.

Rationally designed combinations of targeted therapies for refractory cancers, such as activated B cell-like diffuse large B cell lymphoma (ABC DLBCL), are likely required to achieve potent, durable responses. Here, we used a pharmacoproteomics approach to map the interactome of a tumor-enriched isoform of HSP90 (teHSP90). Specifically, we chemically precipitated teHSP90-client complexes from DLBCL cell lines with the small molecule PU-H71 and found that components of the proximal B cell receptor (BCR) signalosome were enriched within teHSP90 complexes. Functional assays revealed that teHSP90 facilitates BCR signaling dynamics by enabling phosphorylation of key BCR signalosome components, including the kinases SYK and BTK. Consequently, treatment of BCR-dependent ABC DLBCL cells with PU-H71 attenuated BCR signaling, calcium flux, and NF-κB signaling, ultimately leading to growth arrest. Combined exposure of ABC DLBCL cell lines to PU-H71 and ibrutinib, a BCR pathway inhibitor, more potently suppressed BCR signaling than either drug alone. Correspondingly, PU-H71 combined with ibrutinib induced synergistic killing of lymphoma cell lines, primary human lymphoma specimens ex vivo, and lymphoma xenografts in vivo, without notable toxicity. Together, our results demonstrate that a pharmacoproteome-driven rational combination therapy has potential to provide more potent BCR-directed therapy for ABC DLCBL patients.
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http://dx.doi.org/10.1172/JCI80714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665772PMC
November 2015

Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94.

J Med Chem 2015 May 22;58(9):3922-43. Epub 2015 Apr 22.

†Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, New York, New York 10021, United States.

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 μM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/β and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4518544PMC
May 2015

Selective targeting of the stress chaperome as a therapeutic strategy.

Trends Pharmacol Sci 2014 Nov 25;35(11):592-603. Epub 2014 Sep 25.

Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Normal cellular function is maintained by coordinated proteome machinery that performs a vast array of activities. Helping the proteome in such roles is the chaperome, a network of molecular chaperones and folding enzymes. The stressed cell contains, at any time, a complex mixture of chaperome complexes; a majority performs 'housekeeping functions' similarly to non-stressed, normal cells, but a finely-tuned fraction buffers the proteome altered by chronic stress. The stress chaperome is epigenetically distinct from its normal, housekeeping counterpart, providing a basis for its selective targeting by small molecules. We discuss here the development of chaperome inhibitors, and how agents targeting chaperome members in stressed cells are in fact being directed towards chaperome complexes, and their effect is therefore determined by their ability to sample and engage such complexes. A new approach is needed to target and implement chaperome modulators in the investigation of diseases, and we propose that the classical thinking in drug discovery needs adjustment when developing chaperome-targeting drugs.
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http://dx.doi.org/10.1016/j.tips.2014.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254259PMC
November 2014

Affinity purification probes of potential use to investigate the endogenous Hsp70 interactome in cancer.

ACS Chem Biol 2014 Aug 17;9(8):1698-705. Epub 2014 Jun 17.

Program in Molecular Pharmacology and Chemistry and Department of Medicine and §Program in Molecular Biology, Proteomics Core, Memorial Sloan-Kettering Cancer Center , New York, New York 10021, United States.

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.
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http://dx.doi.org/10.1021/cb500256uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134716PMC
August 2014

Protein chaperones: a composition of matter review (2008 - 2013).

Expert Opin Ther Pat 2014 May;24(5):501-18

Memorial Sloan-Kettering Cancer Center, Program in Molecular Pharmacology and Chemistry and Department of Medicine , NY , USA.

Introduction: Heat shock proteins (Hsps) are proteins with important functions in regulating disease phenotypes. Historically, Hsp90 has first received recognition as a target in cancer, with consequent efforts extending its potential role to other diseases. Hsp70 has also attracted interest as a therapeutic target for its role as a co-chaperone to Hsp90 as well as its own anti-apoptotic roles.

Areas Covered: Herein, patents from 2008 to 2013 are reviewed to identify those that disclose composition of matter claimed to inhibit Hsp90 or Hsp70.

Expert Opinion: For Hsp90, there has been considerable creativity in the discovery of novel pharmacophores that fall outside the three initially discovered scaffolds (i.e., ansamycins, resorcinols and purines). Nonetheless, much of the patent literature appears to build on previously reported structure activity relationship through slight modifications of Hsp90 inhibitor space by finding weaknesses in existing patents. The major goal of future development of Hsp90 inhibitors is not necessarily identifying better molecules but rather understanding how to rationally use these agents in the clinic. The development of Hsp70 inhibitors has lagged behind. It will require a more concerted effort from the drug discovery community in order to begin to realize the potential of this target.
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http://dx.doi.org/10.1517/13543776.2014.887681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4124460PMC
May 2014

Heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.

J Med Chem 2014 Feb 18;57(4):1208-24. Epub 2014 Feb 18.

Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center , New York, New York 10021, United States.

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.
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http://dx.doi.org/10.1021/jm401552yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983364PMC
February 2014

Heat shock protein 70 inhibitors. 1. 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides as irreversible binders to an allosteric site on heat shock protein 70.

J Med Chem 2014 Feb 18;57(4):1188-207. Epub 2014 Feb 18.

Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center , New York, New York 10021, United States.

Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.
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http://dx.doi.org/10.1021/jm401551nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983365PMC
February 2014

Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms.

Blood 2014 Mar 27;123(13):2075-83. Epub 2014 Jan 27.

Human Oncology and Pathogenesis Program.

The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK-signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting.
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http://dx.doi.org/10.1182/blood-2014-01-547760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968390PMC
March 2014

Pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor, alone and in combination with bortezomib in Ewing sarcoma.

Mol Oncol 2014 Mar 15;8(2):323-36. Epub 2013 Dec 15.

Department of Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein 90 (HSP90) is a critical component of the multi-chaperone complexes that regulate the disposition and activity of a large number of proteins involved in cell-signaling systems. We tested the efficacy of PU-H71, a novel HSP90 inhibitor in Ewing sarcoma cell lines, primary samples, benign mesenchymal stromal cells and hematopoietic stem cells. We performed cell cycle analysis, clonogenic assay, immunoblot analysis and reverse phase protein array in Ewing cell lines and in vivo experiments in NSG and nude mice using the A673 cell line. We noted a significant therapeutic window in the activity of PU-H71 against Ewing cell lines and benign cells. PU-H71 treatment resulted in G2/M phase arrest. Exposure to PU-H71 resulted in depletion of critical proteins including AKT, pERK, RAF-1, c-MYC, c-KIT, IGF1R, hTERT and EWS-FLI1 in Ewing cell lines. Our results indicated that Ewing sarcoma tumor growth and the metastatic burden were significantly reduced in the mice injected with PU-H71 compared to the control mice. We also investigated the effects of bortezomib, a proteasome inhibitor, alone and in combination with PU-H71 in Ewing sarcoma. Combination index (CI)-Fa plots and normalized isobolograms indicated synergism between PU-H71 and bortezomib. Ewing sarcoma xenografts were significantly inhibited when mice were treated with the combination compared to vehicle or either drug alone. This provides a strong rationale for clinical evaluation of PU-H71 alone and in combination with bortezomib in Ewing sarcoma.
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http://dx.doi.org/10.1016/j.molonc.2013.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982393PMC
March 2014

Human iPSC-based modeling of late-onset disease via progerin-induced aging.

Cell Stem Cell 2013 Dec;13(6):691-705

The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10065, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10065, USA; Gerstner Sloan-Kettering Graduate School, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10065, USA.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
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http://dx.doi.org/10.1016/j.stem.2013.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4153390PMC
December 2013

Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein.

Chem Biol 2013 Dec 14;20(12):1469-80. Epub 2013 Nov 14.

Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Electronic address:

Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.
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http://dx.doi.org/10.1016/j.chembiol.2013.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985611PMC
December 2013