RESUMEN
The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.
Asunto(s)
Inflamación/genética , Conformación Proteica , Ubiquitina Tiolesterasa/genética , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/genética , Humanos , Inflamación/patología , Mutación/genética , Unión Proteica/genética , Dominios Proteicos/genética , Multimerización de Proteína/genética , Proteínas Proto-Oncogénicas c-myb/química , Proteínas Proto-Oncogénicas c-myb/genética , Transducción de Señal/genética , Especificidad por Sustrato , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Ubiquitina/genética , Ubiquitina Tiolesterasa/químicaRESUMEN
Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Isoformas de Proteínas/metabolismo , Proliferación Celular/fisiología , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Linfocitos T/metabolismoRESUMEN
In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Ratones , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutagénesis Insercional , Mutación , Receptores ErbB , Exones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Asunto(s)
Piperidinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Animales , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Humanos , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Piperidinas/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/síntesis química , Pirimidinas/síntesis química , Especificidad por Sustrato , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.
Asunto(s)
Psoriasis , TYK2 Quinasa , Animales , Humanos , Quinasas Janus , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Psoriasis/tratamiento farmacológico , RoedoresRESUMEN
The discovery, structure-activity relationships, and optimization of a novel class of fatty acid synthase (FASN) inhibitors is reported. High throughput screening identified a series of substituted piperazines with structural features that enable interactions with many of the potency-driving regions of the FASN KR domain binding site. Derived from this series was FT113, a compound with potent biochemical and cellular activity, which translated into excellent activity in in vivo models.
Asunto(s)
Ácido Graso Sintasas/antagonistas & inhibidores , Piperazinas/química , Administración Oral , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Ácido Graso Sintasas/metabolismo , Semivida , Humanos , Malonil Coenzima A/metabolismo , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Piperazinas/administración & dosificación , Piperazinas/farmacocinética , Piperazinas/farmacología , Estructura Terciaria de Proteína , Ratas , Relación Estructura-ActividadRESUMEN
Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.
Asunto(s)
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Oogénesis/fisiología , Conformación Proteica , Estructura Terciaria de Proteína/genética , Animales , Cristalización , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Polarización de Fluorescencia , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genéticaRESUMEN
BACKGROUND: Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. OBJECTIVES: The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. METHODS: Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. RESULTS: We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. CONCLUSION: Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.
Asunto(s)
Receptores Androgénicos , Neoplasias de la Mama Triple Negativas , Masculino , Humanos , Receptores Androgénicos/metabolismo , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Unión Proteica , ARN/metabolismoRESUMEN
TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.
Asunto(s)
Artritis Reumatoide , Enfermedades Autoinmunes , Psoriasis , Humanos , TYK2 Quinasa , Estudio de Asociación del Genoma Completo , Enfermedades Autoinmunes/tratamiento farmacológico , Psoriasis/tratamiento farmacológicoRESUMEN
Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.
Asunto(s)
Adenosina Trifosfato/metabolismo , Resistencia a Medicamentos/genética , Receptores ErbB/genética , Mutación , Animales , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Gefitinib , Insectos , Cinética , Conformación Proteica , Quinazolinas/farmacologíaRESUMEN
Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2'. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.
Asunto(s)
Uridina Fosforilasa/química , Dominio Catalítico , Clonación Molecular , Cristalización , Escherichia coli/enzimología , Floxuridina/farmacología , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfatos/farmacología , Ultracentrifugación , Uridina/farmacología , Uridina Fosforilasa/genética , Uridina Fosforilasa/metabolismoRESUMEN
Inhibition of mutant IDH1 is being evaluated clinically as a treatment option for oncology. Here we describe the structure-based design and optimization of quinoline lead compounds to identify FT-2102, a potent, orally bioavailable, brain penetrant, and selective mIDH1 inhibitor. FT-2102 has excellent ADME/PK properties and reduces 2-hydroxyglutarate levels in an mIDH1 xenograft tumor model. This compound has been selected as a candidate for clinical development in hematologic malignancies, solid tumors, and gliomas with mIDH1.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Piridinas/uso terapéutico , Quinolinas/uso terapéutico , Quinolonas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones Endogámicos BALB C , Estructura Molecular , Unión Proteica , Piridinas/síntesis química , Piridinas/metabolismo , Quinolinas/síntesis química , Quinolinas/metabolismo , Quinolonas/síntesis química , Quinolonas/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations at the arginine residue (R132) in isocitrate dehydrogenase 1 (IDH1) are frequently identified in various human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promising therapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimization of a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. The X-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allosteric site. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24 yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further development of quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Quinolonas/química , Quinolonas/farmacología , Sitio Alostérico/efectos de los fármacos , Animales , Disponibilidad Biológica , Línea Celular Tumoral , Cristalografía por Rayos X , Perros , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacocinética , Femenino , Humanos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Mutación Puntual , Quinolonas/farmacocinéticaRESUMEN
Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.
Asunto(s)
Janus Quinasa 2/química , Conformación Proteica , Dominios Homologos src , Cristalografía por Rayos X , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Modelos Moleculares , Mutación/genética , Fosforilación , Unión Proteica , Transducción de SeñalRESUMEN
In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Neoplasias Pulmonares/tratamiento farmacológico , Compuestos Organofosforados/farmacología , Fosfinas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Administración Oral , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones SCID , Conformación Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Compuestos Organofosforados/administración & dosificación , Compuestos Organofosforados/química , Fosfinas/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Pirimidinas/administración & dosificación , Pirimidinas/química , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-ActividadRESUMEN
The V617F mutation in the Jak2 pseudokinase domain causes myeloproliferative neoplasms, and the equivalent mutation in Jak1 (V658F) is found in T-cell leukemias. Crystal structures of wild-type and V658F-mutant human Jak1 pseudokinase reveal a conformational switch that remodels a linker segment encoded by exon 12, which is also a site of mutations in Jak2. This switch is required for V617F-mediated Jak2 activation and possibly for physiologic Jak activation.
Asunto(s)
Quinasas Janus/metabolismo , Oncogenes , Activación Enzimática , Humanos , Quinasas Janus/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKß, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Serina-Treonina Quinasas/química , Ubiquitinación , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Quinasa I-kappa B/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
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 small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.
Asunto(s)
Quinasas Asociadas a rho/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Tropomiosina/químicaRESUMEN
Formin proteins direct the nucleation and assembly of linear actin filaments in a variety of cellular processes using their conserved formin homology 2 (FH2) domain. Diaphanous-related formins (DRFs) are effectors of Rho-family GTPases, and in the absence of Rho activation they are maintained in an inactive state by intramolecular interactions between their regulatory N-terminal region and a C-terminal segment referred to as the DAD domain. Although structures are available for the isolated DAD segment in complex with the interacting region in the N-terminus, it remains unclear how this leads to inhibition of actin assembly by the FH2 domain. Here we describe the crystal structure of the N-terminal regulatory region of formin mDia1 in complex with a C-terminal fragment containing both the FH2 and DAD domains. In the crystal structure and in solution, these fragments form a tetrameric complex composed of two interlocking N+C dimers. Formation of the tetramer is likely a consequence of the particular N-terminal construct employed, as we show that a nearly full-length mDia1 protein is dimeric, as are other autoinhibited N+C complexes containing longer N-terminal fragments. The structure provides the first view of the intact C-terminus of a DRF, revealing the relationship of the DAD to the FH2 domain. Delineation of alternative dimeric N+C interactions within the tetramer provides two general models for autoinhibition in intact formins. In both models, engagement of the DAD by the N-terminus is incompatible with actin filament formation on the FH2, and in one model the actin binding surfaces of the FH2 domain are directly blocked by the N-terminus.