RESUMEN
Epithelioid hemangioendothelioma (EHE) is a genetically homogenous vascular sarcoma that is a paradigm for TAZ dysregulation in cancer. EHE harbors a WWTR1(TAZ)-CAMTA1 gene fusion in >90% of cases, 45% of which have no other genetic alterations. In this study, we used a first of its kind approach to target the Wwtr1-Camta1 gene fusion to the Wwtr1 locus, to develop a conditional EHE mouse model whereby Wwtr1-Camta1 is controlled by the endogenous transcriptional regulators upon Cre activation. These mice develop EHE tumors that are indistinguishable from human EHE clinically, histologically, immunohistochemically, and genetically. Overall, these results demonstrate unequivocally that TAZ-CAMTA1 is sufficient to drive EHE formation with exquisite specificity, as no other tumor types were observed. Furthermore, we fully credential this unique EHE mouse model as a valid preclinical model for understanding the role of TAZ dysregulation in cancer formation and for testing therapies directed at TAZ-CAMTA1, TAZ, and YAP/TAZ signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al Calcio/metabolismo , Carcinogénesis/genética , Modelos Animales de Enfermedad , Fusión Génica , Hemangioendotelioma Epitelioide/genética , Hemangioendotelioma Epitelioide/patología , Transactivadores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Unión al Calcio/genética , Humanos , Ratones , Transducción de Señal/genética , Transactivadores/genéticaRESUMEN
The Hippo signaling pathway inhibits the activity of the oncogenic YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif)-TEAD (TEA/ATTS domain) transcriptional complex. In cancers, inactivating mutations in upstream Hippo components and/or enhanced activity of YAP/TAZ and TEAD have been observed. The activity of this transcriptional complex can be effectively inhibited by targeting the TEAD family of transcription factors. The development of TEAD inhibitors has been driven by the discovery that TEAD has druggable hydrophobic pockets, and is currently at the clinical development stage. Three small molecule TEAD inhibitors are currently being tested in Phase I clinical trials. In this review, we highlight the role of TEADs in cancer, discuss various avenues through which TEAD activity can be inhibited, and outline the opportunities for the administration of TEAD inhibitors.
Asunto(s)
Neoplasias , Factores de Transcripción de Dominio TEA , Humanos , Factores de Transcripción/metabolismo , Neoplasias/tratamiento farmacológico , Vía de Señalización HippoRESUMEN
The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.
Asunto(s)
Exocitosis , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafines/metabolismo , Humanos , Liposomas/metabolismo , Fusión de Membrana , Células PC12 , Dominios Homólogos a Pleckstrina , Unión Proteica , Ratas , Proteínas SNARE/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismo , Vertebrados , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Desarrollo de Músculos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones Noqueados , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Neoplasias/metabolismo , Unión Proteica , Factores de Transcripción de Dominio TEA , Transcriptoma/genéticaRESUMEN
The identification of protein-protein interaction disruptors (PPIDs) that disrupt the YAP/TAZ-TEAD interaction has gained considerable momentum. Several studies have shown that YAP/TAZ are no longer oncogenic when their interaction with the TEAD family of transcription factors is disrupted. The transcriptional co-regulator YAP (its homolog TAZ) interact with the surface pockets of TEADs. Peptidomimetic modalities like cystine-dense peptides and YAP cyclic and linear peptides exploit surface pockets (interface 2 and interface 3) on TEADs and function as PPIDs. The TEAD surface might pose a challenge for generating an effective small molecule PPID. Interestingly, TEADs also have a central pocket that is distinct from the surface pockets, and which small molecules leverage exclusively to disrupt the YAP/TAZ-TEAD interaction (allosteric PPIDs). Although small molecules that occupy the central pocket belong to diverse classes, they display certain common features. They are flexible, which allows them to adopt a palmitate-like conformation, and they have a predominant hydrophobic portion that contacts several hydrophobic residues and a small hydrophilic portion that faces the central pocket opening. Despite such progress, more selective PPIDs that also display favorable pharmacokinetic properties and show tolerable toxicity profiles are required to evaluate the feasibility of using these PPIDs for cancer therapy.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Bibliotecas de Moléculas Pequeñas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The oncoprotein YAP (Yes-associated protein) requires the TEAD family of transcription factors for the up-regulation of genes important for cell proliferation. Disrupting YAP-TEAD interaction is an attractive strategy for cancer therapy. Targeting TEADs using small molecules that either bind to the YAP-binding pocket or the palmitate-binding pocket is proposed to disrupt the YAP-TEAD interaction. There is a need for methodologies to facilitate robust and reliable identification of compounds that occupy either YAP-binding pocket or palmitate-binding pocket. Here, using NMR spectroscopy, we validated compounds that bind to these pockets and also identify the residues in mouse TEAD4 (mTEAD4) that interact with these compounds. Flufenamic acid (FA) was used as a positive control for validation of palmitate-binding pocket-occupying compounds by NMR. Furthermore, we identify a hit from a fragment screen and show that it occupies a site close to YAP-binding pocket on the TEAD surface. Our results also indicate that purified mTEAD4 can catalyze autopalmitoylation. NMR studies on mTEAD4 revealed that exchanges exist in TEAD as NMR signal broadening was observed for residues close to the palmitoylation site. Mutating the palmitoylated cysteine (C360S mutant) abolished palmitoylation, while no significant changes in the NMR spectrum were observed for the mutant which still binds to YAP. We also show that FA inhibits TEAD autopalmitoylation. Our studies highlight the utility of NMR spectroscopy in identifying small molecules that bind to TEAD pockets and reinforce the notion that both palmitate-binding pocket and YAP-binding pocket are targetable.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Unión al ADN/química , Proteínas Musculares/química , Fosfoproteínas/química , Factores de Transcripción/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ácido Flufenámico/química , Lipoilación , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
PURPOSE: There are no effective treatment options for patients with aggressive epithelioid hemangioendothelioma (EHE) driven by the TAZ-CAMTA1 (TC) fusion gene. Here, we aimed to understand the regulation of TC using pharmacological tools and identify vulnerabilities that can potentially be exploited for the treatment of EHE. EXPERIMENTAL DESIGN: TC is a transcriptional co-regulator; we hypothesized that compounds that reduce TC nuclear levels, either through translocation of TC to the cytoplasm, or through degradation, would render TC less oncogenic. TC localization was monitored using immunofluorescence (IF) in an EHE tumor cell line. Two target-selective libraries were used to identify small molecules that reduce TC localization in the nucleus. The ability of the shortlisted hits to affect cell viability, apoptosis, and tumorigenesis was also evaluated. RESULTS: Basal TC remained 'immobile' in the nucleus; administration of cyclin-dependent kinase inhibitors (CDKi) such as CGP60474 and dinaciclib mobilized TC. 'Mobile' TC shuttled between the nucleus and cytoplasm; however, it was eventually degraded through proteasomes. This dramatically suppressed the levels of TC-regulated transcripts and cell viability, promoted apoptosis, and reduced the area of metastatic lesions in the allograft model of EHE. We specifically identified that the inhibition of CDK9, a transcriptional CDK, destabilizes TC. CONCLUSIONS: The CDK inhibitor dinaciclib exhibited anti-tumorigenic properties both in vitro and in vivo in EHE models. Dinaciclib has been rigorously tested in clinical trials and displayed an acceptable toxicity profile. Therefore, there is a potential therapeutic window for repurposing dinaciclib for the treatment of EHE.
RESUMEN
The Hippo signaling pathway is a highly conserved pathway that plays important roles in the regulation of cell proliferation and apoptosis. Transcription factors TEAD1-4 and transcriptional coregulators YAP/TAZ are the downstream effectors of the Hippo pathway and can modulate Hippo biology. Dysregulation of this pathway is implicated in tumorigenesis and acquired resistance to therapies. The emerging importance of YAP/TAZ-TEAD interaction in cancer development makes it a potential therapeutic target. In the past decade, disrupting YAP/TAZ-TEAD interaction as an effective approach for cancer treatment has achieved great progress. This approach followed a trajectory wherein peptidomimetic YAP-TEAD protein-protein interaction disruptors (PPIDs) were first designed, followed by the discovery of allosteric small molecule PPIDs, and currently, the development of direct small molecule PPIDs. YAP and TEAD form three interaction interfaces. Interfaces 2 and 3 are amenable for direct PPID design. One direct YAP-TEAD PPID (IAG933) that targets interface 3 has entered a clinical trial in 2021. However, in general, strategically designing effective small molecules PPIDs targeting TEAD interfaces 2 and 3 has been challenging compared with allosteric inhibitor development. This review focuses on the development of direct surface disruptors and discusses the challenges and opportunities for developing potent YAP/TAZ-TEAD inhibitors for the treatment of cancer.
RESUMEN
PURPOSE: Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma caused by the WWTR1(TAZ)-CAMTA1 (TC) gene fusion. This fusion gene has been observed in almost all reported EHE cases and functions as a constitutively activated TAZ. Sequencing of human tumors has, however, identified additional secondary mutations in approximately 50% of EHE, most commonly the loss of tumor suppressor CDKN2A. In this study, the effect of loss of CDKN2A in EHE tumorigenesis was evaluated. EXPERIMENTAL DESIGN: Mice bearing a conditional TC allele were paired with a conditional Cdkn2a knockout allele and an endothelial-specific Cre. Histologic characterization and single-cell RNA-seq of the resultant tumors were performed. EHE cell lines were established through ex vivo culture of tumor cells and evaluated for sensitivity to TEAD inhibition and trametinib. RESULTS: Loss of Cdkn2a within EHE was associated with more aggressive disease, as displayed by earlier tumor-related morbidity/mortality and enhanced tumor cell proliferation. As no previous EHE cell lines exist, we attempted, successfully, to expand EHE tumor cells ex vivo and produced the first EHE cell lines. These cell lines are "addicted" to the TC oncoprotein, replicate the EHE transcriptional profile, and generate EHE tumors when injected into immunodeficient mice. CONCLUSIONS: CDKN2A loss enhances the tumorigenicity of EHE in vivo and enabled the generation of the first cell lines of this disease. These cell lines replicate key facets of the human disease phenotype. Therefore, these cell lines and allograft tumors generated after implantation serve as robust model systems for therapeutic testing of compounds directed at either EHE or other TAZ-driven cancers.
Asunto(s)
Hemangioendotelioma Epitelioide , Animales , Humanos , Ratones , Proteínas de Unión al Calcio/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Fusión Génica , Hemangioendotelioma Epitelioide/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Transactivadores/genética , Factores de Transcripción/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity of TAZ and YAP through physical interaction with Amot and AmotL1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Angiomotinas , División Celular/fisiología , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Citoplasma/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Fosfoproteínas/química , Fosfoproteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Estructura Terciaria de Proteína , Transactivadores , Factores de Transcripción , Transcripción Genética/fisiología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
The activities of YAP and TAZ, the end effectors of the Hippo pathway, are consistently altered in cancer, and this dysregulation drives aggressive tumor phenotypes. While the actions of these two proteins aid in tumorigenesis in the majority of cancers, the dysregulation of these proteins is rarely sufficient for initial tumor development. Herein, we present a unique TAZ-driven cancer, epithelioid hemangioendothelioma (EHE), which harbors a WWTR1(TAZ)-CAMTA1 gene fusion in at least 90% of cases. Recent investigations have elucidated the mechanisms by which YAP/TAP-fusion oncoproteins function and drive tumorigenesis. This review presents a critical evaluation of this recent work, with a particular focus on how the oncoproteins alter the normal activity of TAZ and YAP, and, concurrently, we generate a framework for how we can target the gene fusions in patients. Since EHE represents a paradigm of YAP/TAZ dysregulation in cancer, targeted therapies for EHE may also be effective against other YAP/TAZ-dependent cancers.
RESUMEN
Disrupting the formation of the oncogenic YAP/TAZ-TEAD transcriptional complex holds substantial therapeutic potential. However, the three protein interaction interfaces of this complex cannot be easily disrupted using small molecules. Here, we report that the pharmacologically active small molecule aurintricarboxylic acid (ATA) acts as a disruptor of the TAZ-TEAD complex. ATA was identified in a high-throughput screen using a TAZ-TEAD AlphaLISA assay that was tailored to identify disruptors of this transcriptional complex. We further used fluorescence polarization assays both to confirm disruption of the TAZ-TEAD complex and to demonstrate that ATA binds to interface 3. We have previously shown that cell-based models that express the oncogenic TAZ-CAMTA1 (TC) fusion protein display enhanced TEAD transcriptional activity because TC functions as an activated form of TAZ. Utilizing cell-based studies and our TC model system, we performed TC/TEAD reporter, RNA-Seq, and qPCR assays and found that ATA inhibits TC/TEAD transcriptional activity. Further, disruption of TC/TEAD and TAZ/TEAD interaction by ATA abrogated anchorage-independent growth, the phenotype most closely linked to dysregulated TAZ/TEAD activity. Therefore, this study demonstrates that ATA is a novel small molecule that has the ability to disrupt the undruggable TAZ-TEAD interface.
Asunto(s)
Ácido Aurintricarboxílico , Factores de Transcripción , Proteínas de Fusión Oncogénica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: A consistent genetic alteration in vascular cancer epithelioid hemangioendothelioma (EHE) is the t(1;3)(p36;q25) chromosomal translocation, which generates a WWTR1(TAZ)-CAMTA1 (TC) fusion gene. TC is a transcriptional coactivator that drives EHE. Here, we aimed to identify the TC transcriptional targets and signaling mechanisms that underlie EHE tumorigenesis. EXPERIMENTAL DESIGN: We used NIH3T3 cells transformed with TC (NIH3T3/TC) as a model system to uncover TC-dependent oncogenic signaling. These cells proliferated in an anchorage-independent manner in suspension and soft agar. The findings of the cell-based studies were validated in a xenograft model. RESULTS: We identified connective tissue growth factor (CTGF) as a tumorigenic transcriptional target of TC. We show that CTGF binds to integrin αIIbß3, which is essential for sustaining the anchorage-independent proliferation of transformed NIH3T3/TC cells. NIH3T3/TC cells also have enhanced Ras and MAPK signaling, and the activity of these pathways is reduced upon CTGF knockdown, suggesting that CTGF signaling occurs via the Ras-MAPK cascade. Further, pharmacologic inhibition of MAPK signaling through PD 0325901 and trametinib abrogated TC-driven anchorage-independent growth. Likewise, for tumor growth in vivo, NIH3T3/TC cells require CTGF and MAPK signaling. NIH3T3/TC xenograft growth was profoundly reduced upon CTGF knockdown and after trametinib treatment. CONCLUSIONS: Collectively, our results demonstrated that CTGF and the Ras-MAPK signaling cascade are essential for TC-mediated tumorigenesis. These studies provided the preclinical rationale for SARC033 (NCI 10015-NCT03148275), a nonrandomized, open-label, phase II study of trametinib in patients with unresectable or metastatic EHE.
Asunto(s)
Hemangioendotelioma Epitelioide , Sarcoma , Adulto , Animales , Proteínas de Unión al Calcio/genética , Carcinogénesis/genética , Niño , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Hemangioendotelioma Epitelioide/tratamiento farmacológico , Hemangioendotelioma Epitelioide/genética , Humanos , Ratones , Células 3T3 NIH , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Activating mutations in KIT/PDGFRA receptor tyrosine kinases drive gastrointestinal stromal tumors (GIST). KIT/PDGFRA inhibitors, such as imatinib do not evoke an effective cytocidal response, leaving room for quiescence and development of multiple secondary resistance mutations. As the majority of the secondary resistance clones activate PI3K and MAPK pathways, we investigated whether combined targeting of KIT/PI3K/MAPK (KPM) pathways overcomes drug resistance and quiescence in GIST cells. We monitored the proliferation of imatinib-sensitive and-resistant GIST cell lines after treating them with various combinations of drugs to inhibit KPM pathways. Cytocidal response was evaluated through proliferation, apoptosis and colony outgrowth assays. Combined inhibition of KPM signaling pathways using a KPM inhibitor cocktail decreased the survival of drug-resistant GIST cells and dramatically reduced their proliferation. Downstream pathway analysis showed that the residual PI3K/MAPK signaling observed after KIT inhibitor treatment plays a role in mediating quiescence and drug resistance. The KPM inhibitor cocktail with sunitinib or regorafenib effectively induced apoptosis and prevented colony outgrowth after long-term drug removal, suggesting that it can be used as an effective strategy against quiescence and drug resistance in metastatic GIST.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Starting from our previously reported hit, a series of 1,5-diaryl-1,2,3-triazole-4-carbohydrazones were synthesized and evaluated as inhibitors of the YAP/TAZ-TEAD complex. Their binding to hTEAD2 was confirmed by nanodifferential scanning fluorimetry, and some of the compounds were also found to moderately disrupt the YAP-TEAD interaction, as assessed by a fluorescence polarization assay. A TEAD luciferase gene reporter assay performed in HEK293T cells and RTqPCR measurements in MDA-MB231 cells showed that these compounds inhibit YAP/TAZ-TEAD activity to cells in the micromolar range. In spite of the cytotoxic effects displayed by some of the compounds of this series, they are still good starting points and can be suitably modified into an effective and viable YAP-TEAD disruptor in the future.
Asunto(s)
Antineoplásicos/farmacología , Hidrazonas/farmacología , Factores de Transcripción de Dominio TEA/antagonistas & inhibidores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/antagonistas & inhibidores , Triazoles/farmacología , Proteínas Señalizadoras YAP/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Hidrazonas/síntesis química , Hidrazonas/química , Estructura Molecular , Relación Estructura-Actividad , Factores de Transcripción de Dominio TEA/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Triazoles/síntesis química , Triazoles/química , Proteínas Señalizadoras YAP/metabolismoRESUMEN
The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.
Asunto(s)
Citoesqueleto/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/metabolismo , Citoesqueleto/metabolismo , Dimerización , Complejos de Clasificación Endosomal Requeridos para el Transporte , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación Missense/fisiología , Polímeros/metabolismo , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/química , Transporte de Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
The transcriptional co-regulators YAP and TAZ pair primarily with the TEAD family of transcription factors to elicit a gene expression signature that plays a prominent role in cancer development, progression and metastasis. YAP and TAZ endow cells with various oncogenic traits such that they sustain proliferation, inhibit apoptosis, maintain stemness, respond to mechanical stimuli, engineer metabolism, promote angiogenesis, suppress immune response and develop resistance to therapies. Therefore, inhibiting YAP/TAZ- TEAD is an attractive and viable option for novel cancer therapy. It is exciting to know that many drugs already in the clinic restrict YAP/TAZ activities and several novel YAP/TAZ inhibitors are currently under development. We have classified YAP/TAZ-inhibiting drugs into three groups. Group I drugs act on the upstream regulators that are stimulators of YAP/TAZ activities. Many of the Group I drugs have the potential to be repurposed as YAP/TAZ indirect inhibitors to treat various solid cancers. Group II modalities act directly on YAP/TAZ or TEADs and disrupt their interaction; targeting TEADs has emerged as a novel option to inhibit YAP/TAZ, as TEADs are major mediators of their oncogenic programs. TEADs can also be leveraged on using small molecules to activate YAP/TAZ-dependent gene expression for use in regenerative medicine. Group III drugs focus on targeting one of the oncogenic downstream YAP/TAZ transcriptional target genes. With the right strategy and impetus, it is not far-fetched to expect a repurposed group I drug or a novel group II drug to combat YAP and TAZ in cancers in the near future.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Neoplasias/terapia , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/farmacología , Antineoplásicos/farmacología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Proteínas de Unión al ADN/farmacología , Diseño de Fármacos , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/efectos de los fármacos , Proteínas Oncogénicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/farmacología , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
Transcriptional enhanced associate domain (TEAD) transcription factors together with coactivators and corepressors modulate the expression of genes that regulate fundamental processes, such as organogenesis and cell growth, and elevated TEAD activity is associated with tumorigenesis. Hence, novel modulators of TEAD and methods for their identification are in high demand. We describe the development of a new "thiol conjugation assay" for identification of novel small molecules that bind to the TEAD central pocket. The assay monitors prevention of covalent binding of a fluorescence turn-on probe to a cysteine in the central pocket by small molecules. Screening of a collection of compounds revealed kojic acid analogues as TEAD inhibitors, which covalently target the cysteine in the central pocket, block the interaction with coactivator yes-associated protein with nanomolar apparent IC50 values, and reduce TEAD target gene expression. This methodology promises to enable new medicinal chemistry programs aimed at the modulation of TEAD activity.
Asunto(s)
Descubrimiento de Drogas , Pironas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Compuestos de Sulfhidrilo/farmacología , Factores de Transcripción/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Fluorescencia , Humanos , Modelos Moleculares , Estructura Molecular , Pironas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Factores de Transcripción/genéticaRESUMEN
Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.
Asunto(s)
Peptidomiméticos , Factores de Transcripción/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Vía de Señalización Hippo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The AAA (ATPase associated with various cellular activities) proteins participate in membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA Vps (vacuolar protein sorting) 4 is central to traffic to lysosomes, retroviral budding and mammalian cell division. It dissociates ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes, enabling their recycling to the cytosol, and plays a role in fission of intraluminal vesicles within MVBs (multivesicular bodies). The mechanism of Vps4-catalysed disassembly of ESCRT networks is unknown; however, it requires interaction between Vps4 and ESCRT-III subunits. The 30 C-terminal residues of Vps2 and Vps46 (Did2) subunits are both necessary and sufficient for interaction with the Vps4 N-terminal MIT (microtubule-interacting and transport) domain, and the crystal structure of the Vps2 C-terminus in a complex with the Vps4 MIT domain shows that MIT helices alpha2 and alpha3 recognize a (D/E)XXLXXRLXXL(K/R) MIM (MIT-interacting motif). These Vps2-MIT interactions are essential for vacuolar sorting and for Vps4-catalysed disassembly of ESCRT-III networks in vitro. Electron microscopy of ESCRT-III filaments assembled in vitro has enabled us to identify surfaces of the Vps24 subunit that are critical for protein sorting in vivo. The ESCRT-III-Vps4 interaction predates the divergence of Archaea and Eukarya. The Crenarchaea have three classes of ESCRT-III-like subunits, and one of these subunits interacts with an archaeal Vps4-like protein in a manner closely related to the human Vps4-human ESCRT-III subunit Vps20 interaction. This archaeal Vps4-ESCRT-III interaction appears to have a fundamental role in cell division in the Crenarchaea.