ABSTRACT
The intricate protein-chaperone network is vital for cellular function. Recent discoveries have unveiled the existence of specialized chaperone complexes called epichaperomes, protein assemblies orchestrating the reconfiguration of protein-protein interaction networks, enhancing cellular adaptability and proliferation. This study delves into the structural and regulatory aspects of epichaperomes, with a particular emphasis on the significance of post-translational modifications in shaping their formation and function. A central finding of this investigation is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 situated within an intrinsically disordered region, as critical determinants in epichaperome assembly. Our data demonstrate that the phosphorylation of these serine residues enhances HSP90's interaction with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Furthermore, this study establishes a direct link between epichaperome function and cellular physiology, especially in contexts where robust proliferation and adaptive behavior are essential, such as cancer and stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone complexes in diseases characterized by epichaperome dysregulation, bridging the gap between fundamental research and precision medicine.
ABSTRACT
Background: Immune checkpoint inhibitors (ICIs) and their combination with other therapies such as chemotherapy, fail in most cancer patients. We previously identified the PDZ-LIM domain-containing protein 2 (PDLIM2) as a bona fide tumor suppressor that is repressed in lung cancer to drive cancer and its chemo and immunotherapy resistance, suggesting a new target for lung cancer therapy improvement. Methods: Human clinical samples and data were used to investigate PDLIM2 genetic and epigenetic changes in lung cancer. Using an endogenous mouse lung cancer model faithfully recapitulating refractory human lung cancer and a clinically feasible nano-delivery system, we investigated the therapeutic efficacy, action mechanism, and safety of systemically administrated PDLIM2 expression plasmids encapsulated in nanoparticles (nanoPDLIM2) and its combination with PD-1 antibody and chemotherapeutic drugs. Results: PDLIM2 repression in human lung cancer involves both genetic deletion and epigenetic alteration. NanoPDLIM2 showed low toxicity, high tumor specificity, antitumor activity, and greatly improved the efficacy of anti-PD-1 and chemotherapeutic drugs, with complete tumor remission in most mice and substantial tumor reduction in the remaining mice by their triple combination. Mechanistically, nanoPDLIM2 increased major histocompatibility complex class I (MHC-I) expression, suppressed multi-drug resistance 1 (MDR1) induction and survival genes and other tumor-related genes expression in tumor cells, and enhanced lymphocyte tumor infiltration, turning the cold tumors hot and sensitive to ICIs and rendering them vulnerable to chemotherapeutic drugs and activated tumor-infiltrating lymphocytes (TILs) including those unleashed by ICIs. Conclusions: These studies established a clinically applicable PDLIM2-based combination therapy with great efficacy for lung cancer and possibly other cold cancers.
ABSTRACT
Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.
Subject(s)
Neoplasms , Protein Interaction Maps , Humans , Proteome/metabolism , Protein Interaction Mapping , Neoplasms/genetics , AcclimatizationABSTRACT
Protein-assembly defects due to an enrichment of aberrant conformational protein variants are emerging as a new frontier in therapeutics design. Understanding the structural elements that rewire the conformational dynamics of proteins and pathologically perturb functionally oriented ensembles is important for inhibitor development. Chaperones are hub proteins for the assembly of multiprotein complexes and an enrichment of aberrant conformers can affect the cellular proteome, and in turn, phenotypes. Here, we integrate computational and experimental tools to investigte how N-glycosylation of specific residues in glucose-regulated protein 94 (GRP94) modulates internal dynamics and alters the conformational fitness of regions fundamental for the interaction with ATP and synthetic ligands and impacts substructures important for the recognition of interacting proteins. N-glycosylation plays an active role in modulating the energy landscape of GRP94, and we provide support for leveraging the knowledge on distinct glycosylation variants to design molecules targeting GRP94 disease-associated conformational states and assemblies.
Subject(s)
Molecular Chaperones , Glycosylation , Ligands , Molecular Chaperones/chemistry , Protein Conformation , Protein BindingABSTRACT
The increasingly appreciated prevalence of complicated stressor-to-phenotype associations in human disease requires a greater understanding of how specific stressors affect systems or interactome properties. Many currently untreatable diseases arise due to variations in, and through a combination of, multiple stressors of genetic, epigenetic, and environmental nature. Unfortunately, how such stressors lead to a specific disease phenotype or inflict a vulnerability to some cells and tissues but not others remains largely unknown and unsatisfactorily addressed. Analysis of cell- and tissue-specific interactome networks may shed light on organization of biological systems and subsequently to disease vulnerabilities. However, deriving human interactomes across different cell and disease contexts remains a challenge. To this end, this opinion article links stressor-induced protein interactome network perturbations to the formation of pathologic scaffolds termed epichaperomes, revealing a viable and reproducible experimental solution to obtaining rigorous context-dependent interactomes. This article presents our views on how a specialized 'omics platform called epichaperomics may complement and enhance the currently available conventional approaches and aid the scientific community in defining, understanding, and ultimately controlling interactome networks of complex diseases such as Alzheimer's disease. Ultimately, this approach may aid the transition from a limited single-alteration perspective in disease to a comprehensive network-based mindset, which we posit will result in precision medicine paradigms for disease diagnosis and treatment.
Subject(s)
Alzheimer Disease , Alzheimer Disease/genetics , Humans , Phenotype , Precision Medicine , ProteinsABSTRACT
Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks.
Subject(s)
Epigenesis, Genetic , Genome , Molecular Chaperones/genetics , Neoplasms/genetics , Protein Interaction Mapping , Protein Interaction Maps , Animals , Female , Heterografts , Humans , Mice , Signal TransductionABSTRACT
Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.
Subject(s)
Central Nervous System/metabolism , Molecular Chaperones/metabolism , Protein Interaction Mapping/instrumentation , Proteome/metabolism , Animals , Biomarkers, Tumor/metabolism , Blood-Brain Barrier/metabolism , Brain Neoplasms/diagnosis , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Cell Survival/drug effects , Central Nervous System/drug effects , Glioblastoma/diagnosis , Glioblastoma/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/metabolism , Humans , Mice , Molecular Probes/chemistry , Molecular Probes/pharmacokinetics , Molecular Probes/pharmacology , Molecular Probes/therapeutic use , Positron-Emission TomographyABSTRACT
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.
Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Animals , Cell Line, Tumor , Cytosol/metabolism , Glycosylation , HSP70 Heat-Shock Proteins/chemistry , Humans , Membrane Proteins/chemistry , Mice, Inbred NOD , Molecular Weight , Neoplasms/metabolism , Oncogenes , Polysaccharides/metabolism , Protein ConformationABSTRACT
The chaperome is a large family of proteins composed of chaperones, co-chaperones and a multitude of other factors. Elegant studies in yeast and other organisms have paved the road to how we currently understand the complex organization of this large family into protein networks. The goal of this chapter is to provide an overview of chaperome networks in cancer cells, with a focus on two cellular states defined by chaperome network organization. One state characterized by chaperome networks working in isolation and with little overlap, contains global chaperome networks resembling those of normal, non-transformed, cells. We propose that in this state, redundancy in chaperome networks results in a tumor type unamenable for single-agent chaperome therapy. The second state comprises chaperome networks interconnected in response to cellular stress, such as MYC hyperactivation. This is a state where no redundant pathways can be deployed, and is a state of vulnerability, amenable for chaperome therapy. We conclude by proposing a change in how we discover and implement chaperome inhibitor strategies, and suggest an approach to chaperome therapy where the properties of chaperome networks, rather than genetics or client proteins, are used in chaperome inhibitor implementation.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Molecular Chaperones/antagonists & inhibitors , Molecular Chaperones/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Neoplasms/pathologyABSTRACT
We synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogs and identified several potential targets by proteomic analysis. The validation of the proteomic analysis identified the chemical probe as a specific inhibitor of glucose-regulated protein 94 (GRP94), a potential drug target for several types of cancers. Therefore, as a result of the use of bisubstrate-type chemical probes and a chemical-biology methodology, this work opens the way to the development of a new family of GRP94 inhibitors that could potentially be of therapeutic interest.
Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Affinity Labels/pharmacology , Cytosine/analogs & derivatives , Cytosine/pharmacology , Membrane Glycoproteins/antagonists & inhibitors , Adenosine/radiation effects , Affinity Labels/chemical synthesis , Affinity Labels/radiation effects , Cell Line, Tumor , Click Chemistry , Cytosine/radiation effects , Humans , Membrane Glycoproteins/chemistry , Proteome/chemistry , Proteomics , Ultraviolet RaysABSTRACT
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.
Subject(s)
Alzheimer Disease/metabolism , Hippocampus/metabolism , Neuronal Plasticity/physiology , Proteome/metabolism , Alzheimer Disease/pathology , Animals , Brain/pathology , Brain Mapping , Cognitive Dysfunction/metabolism , Executive Function/physiology , Female , Hippocampus/pathology , Humans , Male , Memory/physiology , Mice , Neural PathwaysABSTRACT
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.
Subject(s)
Gene Expression Regulation, Neoplastic , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Neoplasms/metabolism , Animals , Antineoplastic Agents/pharmacology , Biology , Decision Making , Drug Design , HEK293 Cells , Humans , K562 Cells , Kinetics , Mice , NIH 3T3 Cells , Neoplasms/drug therapy , Protein BindingABSTRACT
Most cancers are resistant to anti-PD-1/PD-L1 and chemotherapy. Herein we identify PDLIM2 as a tumor suppressor particularly important for lung cancer therapeutic responses. While PDLIM2 is epigenetically repressed in human lung cancer, associating with therapeutic resistance and poor prognosis, its global or lung epithelial-specific deletion in mice causes increased lung cancer development, chemoresistance, and complete resistance to anti-PD-1 and epigenetic drugs. PDLIM2 epigenetic restoration or ectopic expression shows antitumor activity, and synergizes with anti-PD-1, notably, with chemotherapy for complete remission of most lung cancers. Mechanistically, through repressing NF-κB/RelA and STAT3, PDLIM2 increases expression of genes involved in antigen presentation and T-cell activation while repressing multidrug resistance genes and cancer-related genes, thereby rendering cancer cells vulnerable to immune attacks and therapies. We identify PDLIM2-independent PD-L1 induction by chemotherapeutic and epigenetic drugs as another mechanism for their synergy with anti-PD-1. These findings establish a rationale to use combination therapies for cancer treatment.
Subject(s)
Epigenetic Repression/genetics , Gene Expression Regulation, Neoplastic , LIM Domain Proteins/genetics , Lung Neoplasms/genetics , Microfilament Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Animals , Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , Cell Line, Tumor , DNA Methylation , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Gene Knockdown Techniques , Genes, Tumor Suppressor , Humans , Lung Neoplasms/drug therapy , Mice , Mice, Knockout , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/genetics , STAT3 Transcription Factor/genetics , Transcription Factor RelA/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Agents/administration & dosage , Molecular Chaperones/antagonists & inhibitors , Neoplasms/drug therapy , Protein Interaction Maps/drug effects , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Administration Schedule , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Molecular Chaperones/metabolism , Molecular Imaging , Neoplasms/diagnostic imaging , Neoplasms/genetics , Neoplasms/pathology , Precision Medicine/methods , Protein Interaction Mapping/methods , Protein Interaction Maps/genetics , Theranostic Nanomedicine/methods , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Molecular Chaperones/metabolism , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Drug Discovery , Female , Genes, myc/genetics , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Humans , Mice , Molecular Chaperones/antagonists & inhibitors , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/chemistry , Neoplasms/drug therapy , Neoplasms/genetics , Organ SpecificityABSTRACT
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/ß 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.
Subject(s)
Adenine/analogs & derivatives , Endoplasmic Reticulum/metabolism , HSP90 Heat-Shock Proteins/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Purines/chemistry , Adenine/chemistry , Adenine/pharmacokinetics , Adenine/pharmacology , Allosteric Site , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Differentiation , Cell Line , Cell Line, Tumor , Female , Insulin-Like Growth Factor II/metabolism , Ligands , Membrane Glycoproteins/metabolism , Mice, Nude , Molecular Docking Simulation , Myoblasts/cytology , Myoblasts/drug effects , Myoblasts/metabolism , Protein Binding , Purines/pharmacokinetics , Purines/pharmacology , Receptor, ErbB-2/metabolism , Structure-Activity Relationship , Tissue Distribution , Toll-Like Receptor 9/metabolism , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
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.
Subject(s)
Affinity Labels , Chromatography, Affinity/methods , HSP70 Heat-Shock Proteins/metabolism , Neoplasms/metabolism , Cell Line, Tumor , HSP70 Heat-Shock Proteins/isolation & purification , Humans , Protein BindingABSTRACT
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.
Subject(s)
Allosteric Site/drug effects , HSP70 Heat-Shock Proteins/antagonists & inhibitors , HSP70 Heat-Shock Proteins/metabolism , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Drug Discovery , HSP70 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/metabolism , Heat Shock Transcription Factors , Humans , Models, Molecular , Protein Structure, Tertiary/drug effects , Structural Homology, Protein , Transcription Factors/metabolismABSTRACT
Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90ß, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.