RESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Chaperonas Moleculares/metabolismo , Mapeo de Interacción de Proteínas/instrumentación , Proteoma/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Supervivencia Celular/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Sondas Moleculares/farmacología , Sondas Moleculares/uso terapéutico , Tomografía de Emisión de PositronesRESUMEN
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.
Asunto(s)
Antineoplásicos/administración & dosificación , Chaperonas Moleculares/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Mapas de Interacción de Proteínas/efectos de los fármacos , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Chaperonas Moleculares/metabolismo , Imagen Molecular , Neoplasias/diagnóstico por imagen , Neoplasias/genética , Neoplasias/patología , Medicina de Precisión/métodos , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Nanomedicina Teranóstica/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Mesencéfalo/metabolismo , Técnicas Biosensibles , Proteínas HSP90 de Choque Térmico/fisiología , Mesencéfalo/patología , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Estrés FisiológicoRESUMEN
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.
Asunto(s)
Adenina/análogos & derivados , Retículo Endoplásmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Purinas/química , Adenina/química , Adenina/farmacocinética , Adenina/farmacología , Sitio Alostérico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Femenino , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones Desnudos , Simulación del Acoplamiento Molecular , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Unión Proteica , Purinas/farmacocinética , Purinas/farmacología , Receptor ErbB-2/metabolismo , Relación Estructura-Actividad , Distribución Tisular , Receptor Toll-Like 9/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Benzodioxoles/farmacología , Neoplasias Óseas/tratamiento farmacológico , Ácidos Borónicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias Experimentales/tratamiento farmacológico , Purinas/farmacología , Pirazinas/farmacología , Sarcoma de Ewing/tratamiento farmacológico , Animales , Benzodioxoles/agonistas , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Ácidos Borónicos/agonistas , Bortezomib , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Purinas/agonistas , Pirazinas/agonistas , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Genética/métodos , Janus Quinasa 2/genética , Terapia Molecular Dirigida/métodos , Trastornos Mieloproliferativos/tratamiento farmacológico , Sustitución de Aminoácidos , Animales , Neoplasias de la Médula Ósea/tratamiento farmacológico , Transformación Celular Neoplásica/genética , Terapia Combinada , Eliminación de Gen , Janus Quinasa 2/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Trombopoyetina/genética , Resultado del TratamientoRESUMEN
We here describe the first reported comprehensive analysis of Hsp90 paralogue affinity and selectivity in the clinical Hsp90 inhibitor chemotypes. This has been possible through the development of a versatile experimental assay based on a new FP-probe (16a) that we both describe here. The assay can test rapidly and accurately the binding affinity of all major Hsp90 chemotypes and has a testing range that spans low nanomolar to millimolar binding affinities. We couple this assay with a computational analysis that allows for rationalization of paralogue selectivity and defines not only the major binding modes that relay pan-paralogue binding or, conversely, paralogue selectivity, but also identifies molecular characteristics that impart such features. The methods developed here provide a blueprint for parsing out the contribution of the four Hsp90 paralogues to the perceived biological activity with the current Hsp90 chemotypes and set the ground for the development of paralogue selective inhibitors.