RESUMEN
Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.
Asunto(s)
Benzamidas/farmacología , Bencimidazoles/farmacología , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/inmunología , Lipopolisacáridos/inmunología , Quinasas Quinasa Quinasa PAM/inmunología , Modelos Moleculares , Transducción de Señal/efectos de los fármacos , Sinoviocitos/efectos de los fármacos , Sinoviocitos/inmunología , Células THP-1RESUMEN
The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.
Asunto(s)
Adenosina Trifosfato/metabolismo , Inflamasomas/metabolismo , Proteínas NLR/metabolismo , Células HEK293 , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , UbiquitinaciónRESUMEN
Malaria remains a global health burden partly due to Plasmodium parasite resistance to first-line therapeutics. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood-stage Plasmodium parasites, but details about its function during malaria's elusive liver stage are unclear. We used target-based screens to identify compounds that bind to Plasmodium falciparum and human Hsp90, which revealed insights into chemotypes with species-selective binding. Using cell-based malaria assays, we demonstrate that all identified Hsp90-binding compounds are liver- and blood-stage Plasmodium inhibitors. Additionally, the Hsp90 inhibitor SNX-0723 in combination with the phosphatidylinositol 3-kinase inhibitor PIK-75 synergistically reduces the liver-stage parasite load. Time course inhibition studies with the Hsp90 inhibitors and expression analysis support a role for Plasmodium Hsp90 in late-liver-stage parasite development. Our results suggest that Plasmodium Hsp90 is essential to liver- and blood-stage parasite infections and highlight an attractive route for development of species-selective PfHsp90 inhibitors that may act synergistically in combination therapies to prevent and treat malaria.
Asunto(s)
Antimaláricos/uso terapéutico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Benzamidas/uso terapéutico , Proteínas HSP90 de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Humanos , Hidrazonas/uso terapéutico , Indoles/uso terapéutico , Malaria/tratamiento farmacológico , Malaria/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Sulfonamidas/uso terapéutico , ortoaminobenzoatos/uso terapéuticoRESUMEN
The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Plasmodium falciparum/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Antimaláricos/química , Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares/metabolismoRESUMEN
Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFß activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically.
Asunto(s)
Apoptosis , Glioblastoma , Glioma , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Humanos , Apoptosis/genética , Citocinas , Glioblastoma/genética , Glioblastoma/inmunología , Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/genética , Glioma/inmunología , Glioma/metabolismo , Glioma/patología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta , Factor de Necrosis Tumoral alfaRESUMEN
Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.
Asunto(s)
Borrelia burgdorferi , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Verteporfina/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Indoles/síntesis química , Quinazolinas/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/química , Humanos , Indoles/farmacología , Modelos Moleculares , Unión Proteica , Quinazolinas/farmacología , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.
Asunto(s)
Cromatografía de Afinidad/instrumentación , Proteínas HSP90 de Choque Térmico/metabolismo , Resinas Sintéticas/química , Resinas Sintéticas/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Proteínas HSP90 de Choque Térmico/química , Humanos , Concentración de Iones de Hidrógeno , Ratones , Unión Proteica , Proteómica , Sensibilidad y Especificidad , PorcinosRESUMEN
BACKGROUND: We previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined. METHODS: Using unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity. In an attempt to achieve a stronger abscopal effect for distant tumors, the combination strategy with anti-PD-L1 antibody was tested. Tumor-infiltrating leukocytes were analyzed by single cell RNA-sequencing and receptor-ligand interactome analysis to characterize in more detailed the mechanisms of action of the treatment and key signaling pathways involved. RESULTS: HS201-PDT demonstrated greater tumor control and survival in immune competent mice than in immunocompromised mice, suggesting the role of induced antitumor immunity; however, survival was modest and an abscopal effect on distant implanted tumor was weak. A combination of HS201-PDT with anti-PD-L1 antibody demonstrated the greatest antigen-specific immune response, tumor growth suppression, prolonged mouse survival time and abscopal effect. The most significant increase of intratumoral, activated CD8+T cells and decrease of exhausted CD8+T cells occurred following combination treatment compared with HS201-PDT monotherapy. Receptor-ligand interactome analysis showed marked enhancement of several pathways, such as CXCL, GALECTIN, GITRL, PECAM1 and NOTCH, associated with CD8+T cell activation in the combination group. Notably, the expression of the CXCR3 gene signature was the highest in the combination group, possibly explaining the enhanced tumor infiltration by T cells. CONCLUSIONS: The increased antitumor activity and upregulated CXCR3 gene signature induced by the combination of anti-PD-L1 antibody with HS201-PDT warrants the clinical testing of HS201-PDT combined with PD-1/PD-L1 blockade in patients with breast cancer, and the use of the CXCR3 gene signature as a biomarker.
Asunto(s)
Neoplasias de la Mama , Fotoquimioterapia , Animales , Línea Celular Tumoral , Femenino , Galectinas , Proteínas de Choque Térmico , Humanos , Inhibidores de Puntos de Control Inmunológico , Ligandos , Ratones , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Receptor de Muerte Celular Programada 1 , ARNRESUMEN
Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide , Activación de Linfocitos , Ratones , Animales , Humanos , Proteínas HSP90 de Choque Térmico/metabolismo , Linfocitos T , Artritis Reumatoide/tratamiento farmacológico , Modelos Animales de EnfermedadRESUMEN
A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/genética , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias de la Próstata/patologíaRESUMEN
Heat shock factor 1 (HSF1) is a cellular stress-protective transcription factor exploited by a wide range of cancers to drive proliferation, survival, invasion, and metastasis. Nuclear HSF1 abundance is a prognostic indicator for cancer severity, therapy resistance, and shortened patient survival. The HSF1 gene was amplified, and nuclear HSF1 abundance was markedly increased in prostate cancers and particularly in neuroendocrine prostate cancer (NEPC), for which there are no available treatment options. Despite genetic validation of HSF1 as a therapeutic target in a range of cancers, a direct and selective small-molecule HSF1 inhibitor has not been validated or developed for use in the clinic. We described the identification of a direct HSF1 inhibitor, Direct Targeted HSF1 InhiBitor (DTHIB), which physically engages HSF1 and selectively stimulates degradation of nuclear HSF1. DTHIB robustly inhibited the HSF1 cancer gene signature and prostate cancer cell proliferation. In addition, it potently attenuated tumor progression in four therapy-resistant prostate cancer animal models, including an NEPC model, where it caused profound tumor regression. This study reports the identification and validation of a direct HSF1 inhibitor and provides a path for the development of a small-molecule HSF1-targeted therapy for prostate cancers and other therapy-resistant cancers.
Asunto(s)
Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Neoplasias de la Próstata , Animales , Núcleo Celular/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genéticaRESUMEN
Photodynamic therapy (PDT) ablates malignancies by applying focused near-infrared (nIR) light onto a lesion of interest after systemic administration of a photosensitizer (PS); however, the accumulation of existing PS is not tumor-exclusive. We developed a tumor-localizing strategy for PDT, exploiting the high expression of heat shock protein 90 (Hsp90) in cancer cells to retain high concentrations of PS by tethering a small molecule Hsp90 inhibitor to a PS (verteporfin, VP) to create an Hsp90-targeted PS (HS201). HS201 accumulates to a greater extent than VP in breast cancer cells both in vitro and in vivo, resulting in increased treatment efficacy of HS201-PDT in various human breast cancer xenografts regardless of molecular and clinical subtypes. The therapeutic index achieved with Hsp90-targeted PDT would permit treatment not only of localized tumors, but also more diffusely infiltrating processes such as inflammatory breast cancer.
Asunto(s)
Antineoplásicos/administración & dosificación , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Fotoquimioterapia/estadística & datos numéricos , Fármacos Fotosensibilizantes/administración & dosificación , Verteporfina/administración & dosificación , Línea Celular Tumoral , Proteínas HSP90 de Choque Térmico/administración & dosificación , Proteínas HSP90 de Choque Térmico/efectos de la radiación , Humanos , Células MCF-7RESUMEN
Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.
Asunto(s)
Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Presión Sanguínea/efectos de los fármacos , Cristalografía por Rayos X , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Contracción Muscular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/química , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Ratas Sprague-Dawley , Alineación de SecuenciaRESUMEN
Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers.Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models.Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography.Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 23(24); 7531-42. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Péptidos Cíclicos/administración & dosificación , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Proteínas HSP90 de Choque Térmico/genética , Humanos , Ligandos , Ratones , Péptidos Cíclicos/química , Transducción de Señal/efectos de los fármacos , Espectroscopía Infrarroja Corta , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by overexpressing p110HER2. Whole body cryoslicing, imaging, and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an "Achilles heel" for the early diagnosis of metastatic disease and targeted drug delivery.
Asunto(s)
Neoplasias de la Mama/patología , Colorantes Fluorescentes/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Endocitosis , Espacio Extracelular/metabolismo , Genes erbB-2 , Xenoinjertos , Humanos , RatonesRESUMEN
Tumor necrosis factor alpha (TNF-α) has both positive and negative roles in human disease. In certain cancers, TNF-α is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF-α antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNF-α-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNF-α stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated and non-phosphorylated TAK1 that binds within the ATP-binding pocket and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNF-α-induced cell death, with general implications for cancer and autoimmune disease treatment.
Asunto(s)
Benzamidas/química , Bencimidazoles/química , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Benzamidas/metabolismo , Benzamidas/farmacología , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Sitios de Unión , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Concentración 50 Inhibidora , Interleucina-6/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Sinoviocitos/citología , Sinoviocitos/efectos de los fármacos , Sinoviocitos/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
An estimated three billion people are at risk of Dengue virus (DENV) infection worldwide and there are currently no approved therapeutic interventions for DENV infection. Due to the relatively small size of the DENV genome, DENV is reliant on host factors throughout the viral life cycle. The inducible form of Heat Shock Protein 70 (Hsp70i) has been implicated as a host factor in DENV pathogenesis, however the complete role remains to be elucidated. Here we further illustrate the importance of Hsp70i in dengue virus pathogenesis and describe the antiviral activity of the allosteric small molecule inhibitor that is selective for Hsp70i, called HS-72. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained. HS-72 works to reduce DENV infection by inhibiting the entry stage of the viral life cycle, through disrupting the association of Hsp70i with the DENV receptor complex. This work highlights Hsp70i as an antiviral target and HS-72 as a potential anti-DENV therapeutic agent.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Dengue/metabolismo , Dengue/virología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Animales , Bencimidazoles/farmacología , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Dengue/tratamiento farmacológico , Virus del Dengue/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Humanos , Ácidos Nipecóticos/farmacología , Unión Proteica/efectos de los fármacos , Transporte de Proteínas , Proteoma , Proteómica/métodos , Receptores Virales/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Antineoplásicos/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Proteínas Portadoras/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Péptidos y Proteínas de Señalización Intracelular , Chaperonas Moleculares/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
A novel piperidine series of farnesyltransferase (FTase) inhibitors is described. Systematic medicinal chemistry studies starting with the lead compound, discovered from a 5-nitropiperidin-2-one combinatorial library, resulted in a potent series of novel FTase inhibitors. We found that all of four substituents of the piperidine core played an important role for FTase inhibition. A 10-fold increase in potency was observed by changing the piperidine-2-one core to the corresponding piperidine core. This class of compounds was found to inhibit farnesyltransferase in a Ras competitive manner. Optical resolution of several potent inhibitors revealed that the (+)-enantiomers showed potent farnesyltransferase inhibition. (+)-8 inhibited FTase with an IC(50) of 1.9 nM.