RESUMEN
The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.
Asunto(s)
ADN Helicasas , Tiadiazoles , Urea , ADN Helicasas/metabolismo , Helicasa del Síndrome de Werner/metabolismoRESUMEN
The yes-associated protein (YAP) regulates the transcriptional activity of the TEAD transcription factors that are key in the control of organ morphogenesis. YAP interacts with TEAD via three secondary structure elements: a ß-strand, an α-helix, and an Ω-loop. Earlier results have shown that the ß-strand has only a marginal contribution in the YAP:TEAD interaction, but we show here that it significantly enhances the affinity of YAP for the Drosophila homolog of TEAD, scalloped (Sd). Nuclear magnetic resonance shows that the ß-strand adopts a more rigid conformation once bound to Sd; pre-steady state kinetic measurements show that the YAP:Sd complex is more stable. Although the crystal structures of the YAP:TEAD and YAP:Sd complexes reveal no differences at the binding interface that could explain these results. Molecular Dynamics simulations are in line with our experimental findings regarding ß-strand stability and overall binding affinity of YAP to TEAD and Sd. In particular, RMSF, correlated motion and MMGBSA analyses suggest that ß-sheet fluctuations play a relevant role in YAP53-57 ß-strand dissociation from TEAD4 and contribute to the lower affinity of YAP for TEAD4. Identifying a clear mechanism leading to the difference in YAP's ß-strand stability proved to be challenging, pointing to the potential relevance of multiple modest structural changes or fluctuations for regulation of binding affinity.
Asunto(s)
Factores de Transcripción de Dominio TEA , Factores de Transcripción , Factores de Transcripción/química , Proteínas de Unión al ADN/química , Conformación Proteica en Lámina beta , Regulación de la Expresión Génica , Unión ProteicaRESUMEN
The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. In this report, we show that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352GlnSd) that is predominantly acylated on the lysine (Lys350Sd). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, our findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells.
Asunto(s)
Proteínas de Drosophila , Factores de Transcripción de Dominio TEA , Animales , Cisteína/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoilación , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Transactivadores/metabolismoRESUMEN
The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full-length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high-affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high-affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA.
Asunto(s)
Proteínas Portadoras/química , ADN/química , Proteínas de Drosophila/química , Proteínas Intrínsecamente Desordenadas/química , Factores de Transcripción de Dominio TEA/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN/genética , ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Vía de Señalización Hippo/genética , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Proteínas Inmovilizadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción de Dominio TEA/genética , Factores de Transcripción de Dominio TEA/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína del Locus del Complejo MDS1 y EV11/genética , Neoplasias Ováricas/genética , Factor de Transcripción PAX8/genética , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Factor de Transcripción PAX8/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The Hippo signaling pathway, which plays a central role in the control of organ size in animals, is well conserved in metazoans. The most downstream elements of this pathway are the TEAD transcription factors that are regulated by their association with the transcriptional coactivator YAP. Therefore, the creation of the binding interface that ensures the formation of the YAP:TEAD complex is a critical molecular recognition event essential for the development/survival of many living organisms. In this report, using the available structural information on the YAP:TEAD complex, we study the TEAD-binding domain of YAP from different animal species. This analysis of more than 400 amino acid sequences reveals that the residues from YAP involved in the formation of the two main contact regions with TEAD are very well conserved. Therefore, the binding interface between YAP and TEAD, as found in humans, probably appeared at an early evolutionary stage in metazoans. We find that, in contrast to most other animal species, several Actinopterygii species possess YAP variants with a different TEAD-binding domain. However, these variants bind to TEAD with a similar affinity. Our studies show that the protein identified as a YAP homolog in Caenorhabditis elegans does not contain the TEAD-binding domain found in YAP of other metazoans. Finally, we do not identify in non-metazoan species, amino acid sequences containing both a TEAD-binding domain, as in metazoan YAP, and WW domain(s).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans , Evolución Molecular , Factores de Transcripción/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Caenorhabditis elegans/genética , Humanos , Dominios Proteicos , Especificidad de la Especie , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Musculares/química , Proteínas Nucleares/química , Factores de Transcripción/química , Animales , Sitios de Unión , Drosophila , Células HEK293 , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Dominios Proteicos , Factores de Transcripción de Dominio TEARESUMEN
The Hippo pathway is an evolutionarily conserved signaling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While much is known about how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.
Asunto(s)
Proteínas de Ciclo Celular/química , Complejos Multiproteicos/química , Factores de Transcripción/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfaRESUMEN
The Hippo pathway is a key signaling pathway in the control of organ size and development. The most distal elements of this pathway, the TEAD transcription factors, are regulated by several proteins, such as YAP (Yes-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and VGLL1-4 (Vestigial-like members 1-4). In this article, combining structural data and motif searches in protein databases, we identify two new TEAD interactors: FAM181A and FAM181B. Our structural data show that they bind to TEAD via an Ω-loop as YAP/TAZ do, but only FAM181B possesses the LxxLF motif (x any amino acid) found in YAP/TAZ. The affinity of different FAM181A/B fragments for TEAD is in the low micromolar range and full-length FAM181A/B proteins interact with TEAD in cells. These findings, together with a recent report showing that FAM181A/B proteins have a role in nervous system development, suggest a potential new involvement of the TEAD transcription factors in the development of this tissue.
Asunto(s)
Factores de Transcripción/química , Factores de Transcripción/metabolismo , Bases de Datos de Proteínas , Células HEK293 , Humanos , Conformación Proteica , Factores de Transcripción/genéticaRESUMEN
Four TEAD transcription factors (TEAD1-4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson's chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol-1 . We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H-bonds and the loss of van der Waals contacts, which explains why the Tyr421HisTEAD1 mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex. DATABASE: The crystallographic data have been deposited at the RSCB Protein Data Bank (PDB, www.pdb.org) with the access codes: 6HIL (wtYAP :Tyr421HisTEAD1 ), 6HIK (wtYAP :Tyr429HisTEAD4 ).
Asunto(s)
Proteínas de Ciclo Celular/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Mapas de Interacción de Proteínas/genética , Degeneración Retiniana/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Aciltransferasas , Proteínas de Ciclo Celular/química , Distrofias Hereditarias de la Córnea/patología , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Mutación/genética , Proteínas Nucleares/genética , Unión Proteica/genética , Degeneración Retiniana/patología , Transducción de Señal/genética , Factores de Transcripción de Dominio TEARESUMEN
Many interactions between proteins are mediated by intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) do not adopt a stable three-dimensional structure in their unbound form, but they become more structured upon binding to their partners. In this communication, we study how a bound IDR adapts to mutations, preventing the formation of hydrogen bonds at the binding interface that needs a precise positioning of the interacting residues to be formed. We use as a model the YAP:TEAD interface, where one YAP (IDP) and two TEAD residues form hydrogen bonds via their side chain. Our study shows that the conformational flexibility of bound YAP and the reorganization of water molecules at the interface help to reduce the energetic constraints created by the loss of H-bonds at the interface. The residual flexibility/dynamic of bound IDRs and water might, therefore, be a key for the adaptation of IDPs to different interface landscapes and to mutations occurring at binding interfaces.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Mutación , Unión Proteica , Conformación Proteica , Termodinámica , Agua/químicaRESUMEN
The Hippo pathway is deregulated in various cancers, and the discovery of molecules that modulate this pathway may open new therapeutic avenues in oncology. TEA/ATTS domain (TEAD) transcription factors are the most distal elements of the Hippo pathway and their transcriptional activity is regulated by the Yes-associated protein (YAP). Amongst the various possibilities for targeting this pathway, inhibition of the YAP:TEAD interaction is an attractive strategy. It has been shown recently that TEAD proteins are covalently linked via a conserved cysteine to a fatty acid molecule (palmitate) that binds to a deep hydrophobic cavity present in these proteins. This acylation of TEAD seems to be required for efficient binding to YAP, and understanding how it modulates the YAP:TEAD interaction may provide useful information on the regulation of TEAD function. In this report we have studied the effect of TEAD4 acylation on its interaction with YAP and the other co-activator transcriptional co-activator with PDZ-binding motif (TAZ). We show in our biochemical and cellular assays that YAP and TAZ bind in a similar manner to acylated and non-acylated TEAD4. This indicates that TEAD4 acylation is not a prerequisite for its interaction with YAP or TAZ. However, we observed that TEAD4 acylation significantly enhances its stability, suggesting that it may help this transcription factor to acquire and/or maintain its active conformation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Acilación/fisiología , Humanos , Modelos Moleculares , Transducción de Señal/fisiología , Factores de Transcripción de Dominio TEA , Transactivadores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
TEAD (TEA/ATTS domain) transcription factors are the most distal effectors of the Hippo pathway. YAP (Yes-associated protein) is a coactivator protein which, upon binding to TEAD proteins, stimulates their transcriptional activity. Since the Hippo pathway is deregulated in various cancers, designing inhibitors of the YAP:TEAD interaction is an attractive therapeutic strategy for oncology. Understanding the molecular events that take place at the YAP:TEAD interface is therefore important not only to devise drug discovery approaches, but also to gain knowledge on TEAD regulation. In this report, combining single site-directed mutagenesis and double mutant analyses, we conduct a detailed analysis on the role of several residues located at the YAP:TEAD interface. Our results provide quantitative understanding of the interactions taking place at the YAP:TEAD interface and give insights into the formation of the YAP:TEAD complex and more particularly on the interaction between TEAD and the Ω-loop found in YAP.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Mapas de Interacción de Proteínas , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas Musculares/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfoproteínas/genética , Unión Proteica , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Tiazoles/farmacología , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Ratas , Tiazoles/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Hippo signaling pathway, which controls organ size in animals, is altered in various human cancers. The TEAD transcription factors, the most downstream elements in this pathway, are regulated by different cofactors, such as the Vgll (vestigial-like) proteins. Having studied the interaction between Vgll1-derived peptides and human TEAD4, we show that, although it lacks a key secondary structure element required for tight binding by two other TEAD cofactors (YAP and TAZ), Vgll1-derived peptides bind to TEAD with nanomolar affinity. We identify a ß-strand:loop:α-helix motif as the minimal Vgll binding site. Finally, we reveal an unexpected difference between mouse and human Vgll1-derived peptides.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Musculares/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Péptidos/química , Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The Hippo pathway controls cell homeostasis, and its deregulation can lead to human diseases. In this pathway, the YAP and TAZ transcriptional cofactors play a key role in stimulating gene transcription through their interaction with the TEAD transcriptional factors. Our study of YAP and TAZ peptides in biochemical and biophysical assays shows that both proteins have essentially the same affinity for TEAD. Molecular modeling and structural biology data suggest that they also bind to the same site on TEAD. However, this apparent similarity hides differences in the ways in which the two proteins interact with TEAD. The secondary structure elements of their TEAD binding site do not contribute equally to the overall affinity, and critical interactions with TEAD are made through different residues. This convergent optimization of the YAP/TAZ TEAD binding site suggests that the similarity in the affinities of binding of YAP to TEAD and of TAZ to TEAD is important for Hippo pathway functionality.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Aciltransferasas , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Vía de Señalización Hippo , Humanos , Inmunohistoquímica , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor-like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100-1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/farmacología , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Leucemia de Células B/enzimología , Trastornos Mieloproliferativos/enzimología , Resorcinoles/farmacología , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular , Cartilla de ADN/genética , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Isoxazoles/uso terapéutico , Janus Quinasa 2/metabolismo , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/genética , Luciferasas , Ratones , Ratones Endogámicos BALB C , Mutagénesis , Mutación Missense/genética , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Fosforilación , ARN Interferente Pequeño/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Resorcinoles/uso terapéutico , Microtomografía por Rayos XRESUMEN
The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.
Asunto(s)
Adenosina Trifosfato/metabolismo , Magnesio/metabolismo , Fragmentos de Péptidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Receptor IGF Tipo 1/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Conformación Proteica/efectos de los fármacos , Pirimidinas/farmacología , Pirroles/farmacología , Estaurosporina/farmacología , Especificidad por SustratoRESUMEN
The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.
Asunto(s)
Bioensayo , Descubrimiento de Drogas , Fluorescencia , Termodinámica , Calorimetría , Cinética , Unión Proteica/fisiología , Desnaturalización Proteica , Receptor IGF Tipo 1/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , TemperaturaRESUMEN
The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.