RESUMO
Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed ß-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Serina-Treonina Quinases , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/química , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Domínios Proteicos , Cristalografia por Raios X , Células HEK293RESUMO
The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Medicina de Precisão , Fatores de Transcrição/metabolismo , Transdução de SinaisRESUMO
The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.
Assuntos
Lipídeos/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Lipoilação , Camundongos , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/agonistas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.
Assuntos
Inflamação/enzimologia , Neoplasias/enzimologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Animais , Artrite/enzimologia , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Colite/etiologia , Colite/prevenção & controle , Dermatite/enzimologia , Feminino , Técnicas de Introdução de Genes , Humanos , Ileíte/etiologia , Ileíte/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Melanoma Experimental/patologia , Camundongos , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologiaRESUMO
Receptor-interacting protein kinase 1 (RIPK1), a key component of the cellular necroptosis pathway, has gained recognition as an important therapeutic target. Pharmacologic inhibition or genetic inactivation of RIPK1 has shown promise in animal models of disease ranging from acute ischemic conditions, chronic inflammation, and neurodegeneration. We present here a class of RIPK1 inhibitors that is distinguished by a lack of a lipophilic aromatic group present in most literature inhibitors that typically occupies a hydrophobic back pocket of the protein active site. Despite not having this ubiquitous feature of many known RIPK1 inhibitors, we were able to obtain compounds with good potency, kinase selectivity, and pharmacokinetic properties in rats. The use of the lipophilic yet metabolically stable pentafluoroethyl group was critical to balancing the potency and properties of optimized analogs.
Assuntos
Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Humanos , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Relação Estrutura-AtividadeRESUMO
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and ß-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or ß form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the ß-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three ß-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with ß-WTA, fulfill two recognition principles: binding to the ß-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.
Assuntos
Antibacterianos/química , Anticorpos Antibacterianos/química , Complexo Antígeno-Anticorpo/metabolismo , Parede Celular/química , Regiões Determinantes de Complementaridade/química , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/imunologia , Ácidos Teicoicos/química , Antibacterianos/sangue , Anticorpos Antibacterianos/sangue , Complexo Antígeno-Anticorpo/genética , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Humanos , Imunidade Humoral , Ligação Proteica , Conformação Proteica , Infecções Estafilocócicas/terapia , Relação Estrutura-Atividade , Ácidos Teicoicos/metabolismo , Transgenes/genéticaRESUMO
This corrects the article DOI: 10.1038/ncomms14234.
RESUMO
Influenza B virus (IBV) causes annual influenza epidemics around the world. Here we use an in vivo plasmablast enrichment technique to isolate a human monoclonal antibody, 46B8 that neutralizes all IBVs tested in vitro and protects mice against lethal challenge of all IBVs tested when administered 72 h post infection. 46B8 demonstrates a superior therapeutic benefit over Tamiflu and has an additive antiviral effect in combination with Tamiflu. 46B8 binds to a conserved epitope in the vestigial esterase domain of hemagglutinin (HA) and blocks HA-mediated membrane fusion. After passage of the B/Brisbane/60/2008 virus in the presence of 46B8, we isolated three resistant clones, all harbouring the same mutation (Ser301Phe) in HA that abolishes 46B8 binding to HA at low pH. Interestingly, 46B8 is still able to protect mice against lethal challenge of the mutant viruses, possibly owing to its ability to mediate antibody-dependent cellular cytotoxicity (ADCC).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Imunoglobulina G/uso terapêutico , Vírus da Influenza B , Infecções por Orthomyxoviridae/terapia , Animais , Anticorpos Neutralizantes/imunologia , Epitopos , Hemaglutininas , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/imunologia , Camundongos , Modelos Moleculares , Infecções por Orthomyxoviridae/virologia , Oseltamivir , Conformação ProteicaRESUMO
Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Nicotina/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Nicotina/síntese química , Nicotina/química , Nicotina/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.
Assuntos
Antibacterianos/farmacologia , Bacteriemia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Espaço Intracelular/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Animais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Portador Sadio/tratamento farmacológico , Portador Sadio/microbiologia , Desenho de Fármacos , Feminino , Imunoconjugados/química , Espaço Intracelular/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Vancomicina/uso terapêuticoRESUMO
Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/química , Histonas/química , Acetilação , Benzamidas/química , Ligação Competitiva , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/química , Concentração Inibidora 50 , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Piridinas/química , Transcrição Gênica , VorinostatRESUMO
Recent advances enabling the cloning of human immunoglobulin G genes have proven effective for discovering monoclonal antibodies with therapeutic potential. However, these antibody-discovery methods are often arduous and identify only a few candidates from numerous antibody-secreting plasma cells or plasmablasts. We describe an in vivo enrichment technique that identifies broadly neutralizing human antibodies with high frequency. For this technique, human peripheral blood mononuclear cells from vaccinated donors are activated and enriched in an antigen-specific manner for the production of numerous antigen-specific plasmablasts. Using this technology, we identified four broadly neutralizing influenza A antibodies by screening only 840 human antibodies. Two of these antibodies neutralize every influenza A human isolate tested and perform better than the current anti-influenza A therapeutic, oseltamivir, in treating severe influenza infection in mice and ferrets. Furthermore, these antibodies elicit robust in vivo synergism when combined with oseltamivir, thus highlighting treatment strategies that could benefit influenza-infected patients.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/tratamento farmacológico , Testes de Neutralização/métodos , Plasmócitos/imunologia , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/uso terapêutico , Feminino , Furões , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos , Camundongos Endogâmicos DBARESUMO
The identification of a novel fused triazolo-pyrrolopyridine scaffold, optimized derivatives of which display nanomolar inhibition of Janus kinase 1, is described. Prototypical example 3 demonstrated lower cell potency shift, better permeability in cells and higher oral exposure in rat than the corresponding, previously reported, imidazo-pyrrolopyridine analogue 2. Examples 6, 7 and 18 were subsequently identified from an optimization campaign and demonstrated modest selectivity over JAK2, moderate to good oral bioavailability in rat with overall pharmacokinetic profiles comparable to that reported for an approved pan-JAK inhibitor (tofacitinib).
Assuntos
Janus Quinase 1/antagonistas & inibidores , Piridinas/farmacologia , Animais , Cristalografia por Raios X , Janus Quinase 1/química , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/química , Cinética , Modelos Moleculares , Piridinas/química , Pirróis/química , Pirróis/farmacologia , RatosRESUMO
This paper presents a pH-sensitive bifunctional crosslinker that enables facile conjugation of small molecule therapeutics to macromolecular carriers for use in drug delivery systems. This N-ethoxybenzylimidazole (NEBI) bifunctional crosslinker was designed to exploit mildly acidic, subcellular environments to trigger the release of therapeutics upon internalization in cells. We demonstrate that an analog of doxorubicin (a representative example of an anticancer therapeutic) conjugated to human serum albumin (HSA, a representative example of a macromolecular carrier) via this NEBI crosslinker can internalize and localize into acidic lysosomes of ovarian cancer cells. Fluorescence imaging and cell viability studies demonstrate that the HSA-NEBI-doxorubicin conjugate exhibited improved uptake and cytotoxic activity compared to the unconjugated doxorubicin analog. The pH-sensitive NEBI group was also shown to be relatively stable to biologically-relevant metal Lewis acids and to serum proteins, supporting that these bifunctional crosslinkers may be useful for constructing drug delivery systems that will be stable in biological fluids such as blood.