Publications by authors named "John Koren"

45 Publications

Broad Kinase Inhibition Mitigates Early Neuronal Dysfunction in Tauopathy.

Int J Mol Sci 2021 Jan 26;22(3). Epub 2021 Jan 26.

Department of Neuroscience & Center for Translational Research in Neurodegenerative Disease, University of Florida, Gainesville, FL 32669, USA.

Tauopathies are a group of more than twenty known disorders that involve progressive neurodegeneration, cognitive decline and pathological tau accumulation. Current therapeutic strategies provide only limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression. In this study, we treated early stage tau transgenic mice with a multi-target kinase inhibitor to identify novel substrates that contribute to cognitive impairment and exhibit therapeutic potential. Drug treatment significantly ameliorated brain atrophy and cognitive function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to determine the altered protein network during this early stage in tauopathy and compare this model with the human Alzheimer's disease (AD) proteome. We identified a cluster of preserved pathways shared with human tauopathy with striking potential for broad multi-target kinase intervention. We further report high confidence candidate proteins as novel therapeutically relevant targets for the treatment of tauopathy. Proteomics data are available via ProteomeXchange with identifier PXD023562.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22031186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865413PMC
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c01700DOI Listing
February 2021

UQCRH downregulation promotes Warburg effect in renal cell carcinoma cells.

Sci Rep 2020 09 14;10(1):15021. Epub 2020 Sep 14.

Department of Biological Sciences, Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, 193 Galvin Life Sciences Center, Notre Dame, IN, 46556, USA.

Ubiquinol-cytochrome c reductase hinge protein (UQCRH) is the hinge protein for the multi-subunit complex III of the mitochondrial electron transport chain and is involved in the electron transfer reaction between cytochrome c1 and c. Recent genome-wide transcriptomic and epigenomic profiling of clear cell renal cell carcinoma (ccRCC) by The Cancer Genome Atlas (TCGA) identified UQCRH as the top-ranked gene showing inverse correlation between DNA hypermethylation and mRNA downregulation. The function and underlying mechanism of UQCRH in the Warburg effect metabolism of ccRCC have not been characterized. Here, we verified the clinical association of low UQCRH expression and shorter survival of ccRCC patients through in silico analysis and identified KMRC2 as a highly relevant ccRCC cell line that displays hypermethylation-induced UQCRH extinction. Ectopic overexpression of UQCRH in KMRC2 restored mitochondrial membrane potential, increased oxygen consumption, and attenuated the Warburg effect at the cellular level. UQCRH overexpression in KMRC2 induced higher apoptosis and slowed down in vitro and in vivo tumor growth. UQCRH knockout by CRISPR/Cas9 had little impact on the metabolism and proliferation of 786O ccRCC cell line, suggesting the dispensable role of UQCRH in cells that have entered a Warburg-like state through other mechanisms. Together, our study suggests that loss of UQCRH expression by hypermethylation may promote kidney carcinogenesis through exacerbating the functional decline of mitochondria thus reinforcing the Warburg effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-72107-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490363PMC
September 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.
View Article and Find Full Text PDF

Download full-text PDF

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

Repeated repeat problems: Combinatorial effect of C9orf72-derived dipeptide repeat proteins.

Int J Biol Macromol 2019 Apr 9;127:136-145. Epub 2019 Jan 9.

Department of Molecular Medicine, College of Medicine, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL 33612, USA; Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia. Electronic address:

A microsatellite expansion mutation in C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). The expansion mutation leads to C9orf72 loss of function, RNA foci formation, and generation of five species of non-AUG RAN translated dipeptide repeat proteins (DPRs), such as poly(GA), poly(GP), poly(GR), poly(PA), and poly(PR). Although one cell can contain more than type of DPRs, information about interplay between different DPR species is limited. Here we show that the combined expression of distinct C9orf72-derived dipeptide repeat species produces cellular outcomes and structural differences that are unique compared to the expression of a single DPR species, suggesting the complex biological interactions that occur when multiple DPR variants are simultaneously expressed. Our data highlights the need for further analysis of how combined expression of different DPRs affects the disease state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2019.01.035DOI Listing
April 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06486-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195591PMC
October 2018

Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD.

ACS Chem Biol 2018 08 19;13(8):2288-2299. Epub 2018 Jun 19.

Department of Molecular Medicine , University of South Florida , Tampa , Florida , United States of America.

Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.8b00454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126901PMC
August 2018

Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma.

ACS Chem Biol 2018 04 20;13(4):933-941. Epub 2018 Feb 20.

School of Chemistry & Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.

Gain-of-function mutations within the olfactomedin (OLF) domain of myocilin result in its toxic intracellular accumulation and hasten the onset of open-angle glaucoma. The absence of myocilin does not cause disease; therefore, strategies aimed at eliminating myocilin could lead to a successful glaucoma treatment. The endoplasmic reticulum Hsp90 paralog Grp94 accelerates OLF aggregation. Knockdown or pharmacological inhibition of Grp94 in cells facilitates clearance of mutant myocilin via a non-proteasomal pathway. Here, we expanded our support for targeting Grp94 over cytosolic paralogs Hsp90α and Hsp90β. We then developed a high-throughput screening assay to identify new chemical matter capable of disrupting the Grp94/OLF interaction. When applied to a blind, focused library of 17 Hsp90 inhibitors, our miniaturized single-read in vitro thioflavin T -based kinetics aggregation assay exclusively identified compounds that target the chaperone N-terminal nucleotide binding site. In follow up studies, one compound (2) decreased the extent of co-aggregation of Grp94 with OLF in a dose-dependent manner in vitro, and enabled clearance of the aggregation-prone full-length myocilin variant I477N in cells without inducing the heat shock response or causing cytotoxicity. Comparison of the co-crystal structure of compound 2 and another non-selective hit in complex with the N-terminal domain of Grp94 reveals a docking mode tailored to Grp94 and explains its selectivity. A new lead compound has been identified, supporting a targeted chemical biology assay approach to develop a protein degradation-based therapy for myocilin-associated glaucoma by selectively inhibiting Grp94.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.7b01083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195314PMC
April 2018

Imbalances in the Hsp90 Chaperone Machinery: Implications for Tauopathies.

Front Neurosci 2017 22;11:724. Epub 2017 Dec 22.

Department of Molecular Medicine and USF Health Byrd Institute, University of South Florida, Tampa, FL, United States.

The ATP-dependent 90 kDa heat shock protein, Hsp90, is a major regulator of protein triage, from assisting in nascent protein folding to refolding or degrading aberrant proteins. Tau, a microtubule associated protein, aberrantly accumulates in Alzheimer's disease (AD) and other neurodegenerative diseases, deemed tauopathies. Hsp90 binds to and regulates tau fate in coordination with a diverse group of co-chaperones. Imbalances in chaperone levels and activity, as found in the aging brain, can contribute to disease onset and progression. For example, the levels of the Hsp90 co-chaperone, FK506-binding protein 51 kDa (FKBP51), progressively increase with age. and tau models demonstrated that FKBP51 synergizes with Hsp90 to increase neurotoxic tau oligomer production. Inversely, protein phosphatase 5 (PP5), which dephosphorylates tau to restore microtubule-binding function, is repressed with aging and activity is further repressed in AD. Similarly, levels of cyclophilin 40 (CyP40) are reduced in the aged brain and further repressed in AD. Interestingly, CyP40 was shown to breakup tau aggregates and prevent tau-induced neurotoxicity . Moreover, the only known stimulator of Hsp90 ATPase activity, Aha1, increases tau aggregation and toxicity. While the levels of Aha1 are not significantly altered with aging, increased levels have been found in AD brains. Overall, these changes in the Hsp90 heterocomplex could drive tau deposition and neurotoxicity. While the relationship of tau and Hsp90 in coordination with these co-chaperones is still under investigation, it is clear that imbalances in these proteins with aging can contribute to disease onset and progression. This review highlights the current understanding of how the Hsp90 family of molecular chaperones regulates tau or other misfolded proteins in neurodegenerative diseases with a particular emphasis on the impact of aging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnins.2017.00724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744016PMC
December 2017

Isoform-selective Hsp90 inhibition rescues model of hereditary open-angle glaucoma.

Sci Rep 2017 12 20;7(1):17951. Epub 2017 Dec 20.

Department of Molecular Medicine and Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL, 33613, USA.

The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-18344-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738387PMC
December 2017

Hsp90 and FKBP51: complex regulators of psychiatric diseases.

Philos Trans R Soc Lond B Biol Sci 2018 Jan;373(1738)

Department of Molecular Medicine, Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL 33613, USA

Mood disorders affect nearly a quarter of the world's population. Therefore, understanding the molecular mechanisms underlying these conditions is of great importance. FK-506 binding protein 5 () encodes the FKBP51 protein, a heat shock protein 90 kDa (Hsp90) co-chaperone, and is a risk factor for several affective disorders. FKBP51, in coordination with Hsp90, regulates glucocorticoid receptor (GR) activity via a short negative feedback loop. This signalling pathway rapidly restores homeostasis in the hypothalamic-pituitary-adrenal (HPA) axis following stress. Expression of increases with age through reduced DNA methylation. High levels of FKBP51 are linked to GR resistance and reduced stress coping behaviour. Moreover, common allelic variants in the gene are associated with increased risk of developing affective disorders like anxiety, depression and post-traumatic stress disorder (PTSD). This review highlights the current understanding of the Hsp90 co-chaperone, FKBP5, in disease from both human and animal studies. In addition, genetic implications in the clinic involving life stress exposure, gender differences and treatment outcomes are discussed.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rstb.2016.0532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717532PMC
January 2018

Hsp90 activator Aha1 drives production of pathological tau aggregates.

Proc Natl Acad Sci U S A 2017 09 21;114(36):9707-9712. Epub 2017 Aug 21.

Department of Molecular Medicine and Byrd Alzheimer's Institute, University of South Florida, Tampa, FL 33613.

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

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1707039114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594679PMC
September 2017

Human cyclophilin 40 unravels neurotoxic amyloids.

PLoS Biol 2017 Jun 27;15(6):e2001336. Epub 2017 Jun 27.

Department of Molecular Medicine and Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida, United States of America.

The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2001336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486962PMC
June 2017

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

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

Department of Epidemiology-Biostatistics, Memorial Sloan Kettering Cancer Center, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature19807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283383PMC
October 2016

RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures.

Nano Lett 2016 Oct 3;16(10):6099-6108. Epub 2016 Oct 3.

Department of Chemistry and Biochemistry, Seton Hall University , South Orange, New Jersey 07079, United States.

The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50-95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50-80%) and death (50-60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5-20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.nanolett.6b02274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378679PMC
October 2016

Stressing Out Hsp90 in Neurotoxic Proteinopathies.

Curr Top Med Chem 2016 ;16(25):2829-38

Program in Chemical Biology, Memorial Sloan-Kettering Cancer Center, New York, USA.

A toxic accumulation of proteins is the hallmark pathology of several neurodegenerative disorders. Protein accumulation is regularly prevented by the network of molecular chaperone proteins, including and especially Hsp90. For reasons not yet elucidated, Hsp90 and the molecular chaperones interact with, but do not degrade, these toxic proteins resulting in the pathogenic accumulation of proteins such as tau, in Alzheimer's Disease, and α-synuclein, in Parkinson's Disease. In this review, we describe the associations between Hsp90 and the pathogenic and driver proteins of several neurodegenerative disorders. We additionally describe how the inhibition of Hsp90 promotes the degradation of both mutant and pathogenic protein species in models of neurodegenerative diseases. We also examine the current state of Hsp90 inhibitors capable of crossing the blood-brain barrier; compounds which may be capable of slowing, preventing, and possible reversing neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995127PMC
http://dx.doi.org/10.2174/1568026616666160413141350DOI Listing
February 2017

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.10.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699804PMC
December 2015

HSP90 supports tumor growth and angiogenesis through PRKD2 protein stabilization.

Cancer Res 2014 Dec 8;74(23):7125-36. Epub 2014 Oct 8.

Center for Internal Medicine I, University of Ulm, Ulm, Germany.

The kinase PRKD2 (protein kinase D) is a crucial regulator of tumor cell-endothelial cell communication in gastrointestinal tumors and glioblastomas, but its mechanistic contributions to malignant development are not understood. Here, we report that the oncogenic chaperone HSP90 binds to and stabilizes PRKD2 in human cancer cells. Pharmacologic inhibition of HSP90 with structurally divergent small molecules currently in clinical development triggered proteasome-dependent degradation of PRKD2, augmenting apoptosis in human cancer cells of various tissue origins. Conversely, ectopic expression of PRKD2 protected cancer cells from the apoptotic effects of HSP90 abrogation, restoring blood vessel formation in two preclinical models of solid tumors. Mechanistic studies revealed that PRKD2 is essential for hypoxia-induced accumulation of hypoxia-inducible factor-1α (HIF1α) and activation of NF-κB in tumor cells. Notably, ectopic expression of PRKD2 was able to partially restore HIF1α and secreted VEGF-A levels in hypoxic cancer cells treated with HSP90 inhibitors. Taken together, our findings indicate that signals from hypoxia and HSP90 pathways are interconnected and funneled by PRKD2 into the NF-κB/VEGF-A signaling axis to promote tumor angiogenesis and tumor growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-14-1017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315623PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cb500256uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134716PMC
August 2014

Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions.

Expert Opin Investig Drugs 2014 May 26;23(5):611-28. Epub 2014 Mar 26.

New York University Cancer Institute, NYU Clinical Cancer Center, Division of Hematology/Medical Oncology , NY , USA.

Introduction: Heat shock protein 90 (HSP90) serves as a critical facilitator for oncogene addiction. There has been augmenting enthusiasm in pursuing HSP90 as an anticancer strategy. In fact, since the initial serendipitous discovery that geldanamycin (GM) inhibits HSP90, the field has rapidly moved from proof-of-concept clinical studies with GM derivatives to novel second-generation inhibitors.

Areas Covered: The authors highlight the current status of the second-generation HSP90 inhibitors in clinical development. Herein, the authors note the lessons learned from the completed clinical trials of first- and second-generation inhibitors and describe various assays attempting to serve for a more rational implementation of these agents to cancer treatment. Finally, the authors discuss the future perspectives for this promising class of agents.

Expert Opinion: The knowledge gained thus far provides perhaps only a glimpse at the potential of HSP90 for which there is still much work to be done. Lessons from the clinical trials suggest that HSP90 therapy would advance at a faster pace if patient selection and tumor pharmacokinetics of these drugs were better understood and applied to their clinical development. It is also evident that combining HSP90 inhibitors with other potent anticancer therapies holds great promise not only due to synergistic antitumor activity but also due to the potential of prolonging or preventing the development of drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/13543784.2014.902442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161020PMC
May 2014

Regulatory chaperone complexes in neurodegenerative diseases: a perspective on therapeutic intervention.

Curr Alzheimer Res 2014 Jan;11(1):59-68

Molecular Pharmacology & Chemistry, Sloan-Kettering Institute, Associate Attending, Breast Cancer Service, Department of Medicine, Memorial Hospital, Memorial Sloan- Kettering Cancer Center, ZRB2103, Associate Professor, Weill Graduate School of Medical Sciences, New York, USA.

Protein folding, protein degradation, and protein stability are regulated by the molecular chaperones. Under pathogenic conditions, aberrant proteins can be dysfunctional, unregulated, or pathogenically mutated. These aberrant proteins are triaged by the chaperone network for the maintenance of cellular homeostasis. These species, called chaperone client proteins, include the pathogenic factors of numerous neurodegenerative disorders, including tau in Alzheimer's disease, α-synuclein and LRRK2 in Parkinson's disease, SOD-1, TDP-43 and FUS in amyotrophic lateral sclerosis, and polyQ-expanded proteins such as huntingtin in Huntington's disease. In depth study of two molecular chaperones, Hsp90 and Hsc70, has led to a greater understanding of aberrant client fate and how retarding the chaperone system can promote clearance of these pathogenic clients. Here we discuss how chaperone interactions and small molecule inhibitors can regulate the burden of aberrant client signaling in these neurological disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1567205010666131119233044DOI Listing
January 2014

The role of FKBP5 in mood disorders: action of FKBP5 on steroid hormone receptors leads to questions about its evolutionary importance.

CNS Neurol Disord Drug Targets 2013 Dec;12(8):1157-62

Deaprtemtn of University of South Florida, 4001 E. Fletcher Ave MDC 36, FL 33613, USA.

Research on the FKBP5 gene and FKBP51 protein has more than doubled since the discovery that polymorphisms in this gene could alter treatment outcomes and depressive behavior in humans. This coincided with other data suggesting that the stress hormone axis contributes to the development of numerous mental illnesses. As a result, FKBP51 now lies at the heart of the research of many stress related psychiatric disorders, which has led to advances in the understanding of this protein and its role in humans and in animal models. Specifically, FKBP5-/- mice and a naturally existing overexpression of FKBP5 in 3 genera of new world monkeys have helped understand the effects of FKBP5 in vivo. This review will highlight these finding as well as discuss the current evolutionary need for the FKBP5 gene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4236834PMC
December 2013

Reconstructing the Hsp90/Tau Machine.

Curr Enzym Inhib 2013 Jan;9(1):41-45

Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, Tampa, Florida 33613.

Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Recent work demonstrates that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease (AD). The Hsp90 co-chaperone repertoire has diverse effects on tau stability; some co-chaperones stabilize tau while others facilitate its clearance. We propose that each of these proteins may be novel therapeutic targets. While targeting Hsp90 directly may be deleterious at the organismal level, perhaps targeting individual co-chaperone activities will be more tolerable.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1573408011309010006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689215PMC
January 2013

Allosteric heat shock protein 70 inhibitors rapidly rescue synaptic plasticity deficits by reducing aberrant tau.

Biol Psychiatry 2013 Sep 19;74(5):367-74. Epub 2013 Apr 19.

Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, 33613, USA.

Background: The microtubule-associated protein tau accumulates in neurodegenerative diseases known as tauopathies, the most common being Alzheimer's disease. One way to treat these disorders may be to reduce abnormal tau levels through chaperone manipulation, thus subverting synaptic plasticity defects caused by tau's toxic accretion.

Methods: Tauopathy models were used to study the impact of YM-01 on tau. YM-01 is an allosteric promoter of triage functions of the most abundant variant of the heat shock protein 70 (Hsp70) family in the brain, heat shock cognate 70 protein (Hsc70). The mechanisms by which YM-01 modified Hsc70 activity and tau stability were evaluated with biochemical methods, cell cultures, and primary neuronal cultures from tau transgenic mice. YM-01 was also administered to acute brain slices of tau mice; changes in tau stability and electrophysiological correlates of learning and memory were measured.

Results: Tau levels were rapidly and potently reduced in vitro and ex vivo upon treatment with nanomolar concentrations of YM-01. Consistent with Hsc70 having a key role in this process, overexpression of heat shock protein 40 (DNAJB2), an Hsp70 co-chaperone, suppressed YM-01 activity. In contrast to its effects in pathogenic tauopathy models, YM-01 had little activity in ex vivo brain slices from normal, wild-type mice unless microtubules were disrupted, suggesting that Hsc70 acts preferentially on abnormal pools of free tau. Finally, treatment with YM-01 increased long-term potentiation in tau transgenic brain slices.

Conclusions: Therapeutics that exploit the ability of chaperones to selectively target abnormal tau can rapidly and potently rescue the synaptic dysfunction that occurs in Alzheimer's disease and other tauopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biopsych.2013.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740016PMC
September 2013

Chaperone-dependent Neurodegeneration: A Molecular Perspective on Therapeutic Intervention.

J Alzheimers Dis Parkinsonism 2013 Apr;2013(Suppl 10)

Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Centre, New York, NY, USA.

Maintenance of cellular homeostasis is regulated by the molecular chaperones. Under pathogenic conditions, aberrant proteins are triaged by the chaperone network. These aberrant proteins, known as "clients," have major roles in the pathogenesis of numerous neurological disorders, including tau in Alzheimer's disease, α-synuclein and LRRK2 in Parkinson's disease, SOD-1, TDP-43 and FUS in amyotrophic lateral sclerosis, and polyQ-expanded proteins such as huntingtin in Huntington's disease. Recent work has demonstrated that the use of chemical compounds which inhibit the activity of molecular chaperones subsequently alter the fate of aberrant clients. Inhibition of Hsp90 and Hsc70, two major molecular chaperones, has led to a greater understanding of how chaperone triage decisions are made and how perturbing the chaperone system can promote clearance of these pathogenic clients. Described here are major pathways and components of several prominent neurological disorders. Also discussed is how treatment with chaperone inhibitors, predominately Hsp90 inhibitors which are selective for a diseased state, can relieve the burden of aberrant client signaling in these neurological disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4172/2161-0460.S10-007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172285PMC
April 2013

Glucose-regulated protein 94 triage of mutant myocilin through endoplasmic reticulum-associated degradation subverts a more efficient autophagic clearance mechanism.

J Biol Chem 2012 Nov 3;287(48):40661-9. Epub 2012 Oct 3.

Department of Molecular Medicine and Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL 33613, USA.

Background: Mutant myocilin accumulates in the endoplasmic reticulum for unknown reasons.

Results: Glucose-regulated protein (Grp) 94 depletion reduces mutant myocilin by engaging autophagy.

Conclusion: Grp94 triages mutant myocilin through ER-associated degradation, subverting autophagy.

Significance: Treating glaucoma could be possible by inhibiting Grp94 and reducing its novel client, mutant myocilin. Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that β-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.384800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504779PMC
November 2012

Exploiting the diversity of the heat-shock protein family for primary and secondary tauopathy therapeutics.

Curr Neuropharmacol 2011 Dec;9(4):623-31

Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, Tampa, FL 33613, USA.

The heat shock protein (Hsp) family is an evolutionarily conserved system that is charged with preventing unfolded or misfolded proteins in the cell from aggregating. In Alzheimer's disease, extracellular accumulation of the amyloid β peptide (Aβ) and intracellular aggregation of the microtubule associated protein tau may result from mechanisms involving chaperone proteins like the Hsps. Due to the ability of Hsps to regulate aberrantly accumulating proteins like Aβ and tau, therapeutic strategies are emerging that target this family of chaperones to modulate their pathobiology. This article focuses on the use of Hsp-based therapeutics for treating primary and secondary tauopathies like Alzheimer's disease. It will particularly focus on the pharmacological targeting of the Hsp70/90 system and the value of manipulating Hsp27 for treating Alzheimer's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/157015911798376226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3263456PMC
December 2011

Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance.

PLoS One 2012 26;7(4):e35566. Epub 2012 Apr 26.

Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, College of Medicine, University of South Florida, Tampa, Florida, United States of America.

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035566PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338522PMC
September 2012

The earliest tau dysfunction in Alzheimer's disease? Tau phosphorylated at s422 as a toxic seed.

Am J Pathol 2011 Nov 28;179(5):2148-51. Epub 2011 Sep 28.

USF Health Byrd Alzheimer's Institute, Department of Molecular Medicine, University of South Florida, Tampa, Florida, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajpath.2011.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204013PMC
November 2011

Molecular chaperones and regulation of tau quality control: strategies for drug discovery in tauopathies.

Future Med Chem 2011 Sep;3(12):1523-37

Department of Pathology and the Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.

Tau is a microtubule-associated protein that accumulates in at least 15 different neurodegenerative disorders, which are collectively referred to as tauopathies. In these diseases, tau is often hyperphosphorylated and found in aggregates, including paired helical filaments, neurofibrillary tangles and other abnormal oligomers. Tau aggregates are associated with neuron loss and cognitive decline, which suggests that this protein can somehow evade normal quality control allowing it to aberrantly accumulate and become proteotoxic. Consistent with this idea, recent studies have shown that molecular chaperones, such as heat shock protein 70 and heat shock protein 90, counteract tau accumulation and neurodegeneration in disease models. These molecular chaperones are major components of the protein quality control systems and they are specifically involved in the decision to retain or degrade many proteins, including tau and its modified variants. Thus, one potential way to treat tauopathies might be to either accelerate interactions of abnormal tau with these quality control factors or tip the balance of triage towards tau degradation. In this review, we summarize recent findings and suggest models for therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/fmc.11.88DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190966PMC
September 2011