RESUMEN
Tyrosine kinases are a subfamily of kinases with critical roles in cellular machinery. Dysregulation of their active or inactive forms is associated with diseases like cancer. This study aimed to holistically understand their flexibility-activity relationships, focusing on pockets and fluctuations. We studied 43 different tyrosine kinases by collecting 120 µs of molecular dynamics simulations, pocket and residue fluctuation analysis, and a complementary machine learning approach. We found that the inactive forms often have increased flexibility, particularly at the DFG motif level. Noteworthy, thanks to these long simulations combined with a decision tree, we identified a semiquantitative fluctuation threshold of the DGF+3 residue over which the kinase has a higher probability to be in the inactive form.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas Tirosina Quinasas , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.
Asunto(s)
Proteínas de Escherichia coli , Fosfoproteínas Fosfatasas , Proteínas Tirosina Quinasas , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismoRESUMEN
Flavonoids are widely used as phytomedicines. Here, we report on flavonoid phytomedicines with potential for development into prophylactics or therapeutics against coronavirus disease 2019 (COVID-19). These flavonoid-based phytomedicines include: caflanone, Equivir, hesperetin, myricetin, and Linebacker. Our in silico studies show that these flavonoid-based molecules can bind with high affinity to the spike protein, helicase, and protease sites on the ACE2 receptor used by the severe acute respiratory syndrome coronavirus 2 to infect cells and cause COVID-19. Meanwhile, in vitro studies show potential of caflanone to inhibit virus entry factors including, ABL-2, cathepsin L, cytokines (IL-1ß, IL-6, IL-8, Mip-1α, TNF-α), and PI4Kiiiß as well as AXL-2, which facilitates mother-to-fetus transmission of coronavirus. The potential for the use of smart drug delivery technologies like nanoparticle drones loaded with these phytomedicines to overcome bioavailability limitations and improve therapeutic efficacy are discussed.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Coronavirus Humano OC43/efectos de los fármacos , Flavonoides/farmacología , Peptidil-Dipeptidasa A/química , Neumonía Viral/tratamiento farmacológico , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2 , Animales , Antivirales/química , Betacoronavirus/química , Betacoronavirus/crecimiento & desarrollo , Sitios de Unión , COVID-19 , Cloroquina/química , Cloroquina/farmacología , Infecciones por Coronavirus/genética , Coronavirus Humano OC43/química , Coronavirus Humano OC43/crecimiento & desarrollo , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Flavonoides/química , Humanos , Interleucinas/antagonistas & inhibidores , Interleucinas/química , Interleucinas/genética , Interleucinas/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/virología , Ratones , Simulación del Acoplamiento Molecular , Nanopartículas/administración & dosificación , Nanopartículas/química , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Fitoterapia/métodos , Neumonía Viral/genética , Cultivo Primario de Células , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Termodinámica , Internalización del Virus/efectos de los fármacosRESUMEN
Epigallocatechin gallate (EGCG) is an inhibitor of DYRK1A, a serine/threonine kinase considered to be a major contributor of cognitive dysfunctions in Down syndrome (DS). Two clinical trials in adult patients with DS have shown the safety and efficacy to improve cognitive phenotypes using commercial green tea extract containing EGCG (45% content). In the present study, we performed a preclinical study using FontUp®, a new nutritional supplement with a chocolate taste specifically formulated for the nutritional needs of patients with DS and enriched with a standardized amount of EGCG in young mice overexpressing Dyrk1A (TgBACDyrk1A). This preparation is differential with previous one used, because its green tea extract has been purified to up 94% EGCG of total catechins. We analyzed the in vitro effect of green tea catechins not only for EGCG, but for others residually contained in FontUp®, on DYRK1A kinase activity. Like EGCG, epicatechin gallate was a noncompetitive inhibitor against ATP, molecular docking computations confirming these results. Oral FontUp® normalized brain and plasma biomarkers deregulated in TgBACDyrk1A, without negative effect on liver and cardiac functions. We compared the bioavailability of EGCG in plasma and brain of mice and have demonstrated that EGCG had well crossed the blood-brain barrier.
Asunto(s)
Encéfalo/efectos de los fármacos , Catequina/análogos & derivados , Síndrome de Down/dietoterapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Té/química , Animales , Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Disponibilidad Biológica , Biomarcadores/sangre , Biomarcadores/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Catequina/administración & dosificación , Catequina/efectos adversos , Catequina/química , Catequina/uso terapéutico , Suplementos Dietéticos , Síndrome de Down/sangre , Síndrome de Down/enzimología , Síndrome de Down/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Polifenoles/análisis , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Regulación hacia Arriba , Quinasas DyrKRESUMEN
Interleukin-2-inducible T cell kinase (ITK) is critical for T cell signaling and cytotoxicity, and control of Epstein-Barr virus (EBV). We identified a patient with a novel homozygous missense mutation (D540N) in a highly conserved residue in the kinase domain of ITK who presented with EBV-positive lymphomatoid granulomatosis. She was treated with interferon and chemotherapy and her disease went into remission; however, she has persistent elevation of EBV DNA in the blood, low CD4 T cells, low NK cells, and nearly absent iNKT cells. Molecular modeling predicts that the mutation increases the flexibility of the ITK kinase domain impairing phosphorylation of the protein. Stimulation of her T cells resulted in reduced phosphorylation of ITK, PLCγ, and PKC. The CD8 T cells were moderately impaired for cytotoxicity and degranulation. Importantly, addition of magnesium to her CD8 T cells in vitro restored cytotoxicity and degranulation to levels similar to controls. Supplemental magnesium in patients with mutations in another protein important for T cell signaling, MAGT1, was reported to restore EBV-specific cytotoxicity. Our findings highlight the critical role of ITK for T cell activation and suggest the potential for supplemental magnesium to treat patients with ITK deficiency.
Asunto(s)
Células Sanguíneas/inmunología , Células Sanguíneas/metabolismo , Susceptibilidad a Enfermedades , Magnesio/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Adulto , Análisis Mutacional de ADN , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Femenino , Homocigoto , Humanos , Granulomatosis Linfomatoide/diagnóstico , Granulomatosis Linfomatoide/etiología , Mutación Missense , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Tirosina Quinasas/química , Relación Estructura-Actividad , Secuenciación del ExomaRESUMEN
DYRK1A is one of five members of the dual-specificity tyrosine (Y) phosphorylation-regulated kinase (DYRK) family. The DYRK1A gene is located in the Down syndrome critical region and regulates cellular processes related to proliferation and differentiation of neuronal progenitor cells during early development. This has focused research on its role in neuronal degenerative diseases, including Alzheimer's and Down syndrome. Recent studies have also shown a possible role of DYRK1A in diabetes. Here we report a variety of scaffolds not generally known for DYRK1A inhibition, demonstrating their effects in in vitro assays and also in cell cultures. These inhibitors effectively block the tau phosphorylation that is a hallmark of Alzheimer's disease. The crystal structures of these inhibitors support the design of optimized and novel therapeutics.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Humanos , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad , Tirosina/metabolismo , Proteínas tau/metabolismo , Quinasas DyrKRESUMEN
Summary: Cysteine and histidine rich domains (CHORDs), implicated in immunity and disease resistance signaling in plants, and in development and signal transduction in muscles and tumorigenesis in animals, are seen to have a cylindrical three-dimensional structure stabilized by the tetrahedral chelation of two zinc ions. CHORDs are regarded as novel zinc-binding domains and classified independently in Pfam and ECOD. Our sequence and structure analysis reveals that both the zinc-binding sites in CHORD possess a zinc ribbon fold and are likely related to each other by duplication and circular permutation. Interestingly, we also detect an evolutionary relationship between each of the CHORD zinc fingers (ZFs) and the Bruton's tyrosine kinase (Btk)-type ZF of the zinc ribbon fold group. Btk_ZF is found in eukaryotic Tec kinase family proteins that are also implicated in signaling pathways in several lineages of hematopoietic cells involved in mammalian immunity. Our analysis suggests that the unique zinc-stabilized fold seen only in the CHORD and Btk_ZFs likely emerged specifically in eukaryotes to mediate diverse signaling pathways. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Evolución Molecular , Metaloproteínas/genética , Elementos Estructurales de las Proteínas/genética , Zinc/química , Agammaglobulinemia Tirosina Quinasa/química , Agammaglobulinemia Tirosina Quinasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cisteína , Eucariontes/genética , Eucariontes/metabolismo , Histidina , Humanos , Metaloproteínas/química , Metaloproteínas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Alineación de Secuencia , Transducción de Señal , Zinc/metabolismo , Dedos de Zinc/genéticaRESUMEN
Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors.
Asunto(s)
Glicoproteínas de Membrana/agonistas , Péptidos/farmacología , Secuencia de Aminoácidos , Bacteriófago T7 , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/química , Fosforilación , Unión Proteica , Multimerización de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Receptor trkBRESUMEN
Developing selective inhibitors for a particular kinase remains a major challenge in kinase-targeted drug discovery. Here we performed a multi-step virtual screening for dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) inhibitors by focusing on the selectivity for DYRK1A over cyclin-dependent kinase 5 (CDK5). To examine the key factors contributing to the selectivity, we constructed logistic regression models to discriminate between actives and inactives for DYRK1A and CDK5, respectively, using residue-based binding free energies. The residue-based parameters were calculated by molecular mechanics-generalized Born surface area (MM-GBSA) decomposition methods for kinase-ligand complexes modeled by computer ligand docking. Based on the findings from the logistic regression models, we built a three-dimensional (3D) pharmacophore model and chose filter criteria for the multi-step virtual screening. The virtual hit compounds obtained from the screening were assessed for their inhibitory activities against DYRK1A and CDK5 by in vitro assay. Our screening identified two novel selective DYRK1A inhibitors with IC50 values of several µM for DYRK1A and >100µM for CDK5, which can be further optimized to develop more potent selective DYRK1A inhibitors.
Asunto(s)
Evaluación Preclínica de Medicamentos , Inhibidores de Proteínas Quinasas/análisis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Bioensayo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Humanos , Ligandos , Modelos Logísticos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Máquina de Vectores de Soporte , Quinasas DyrKRESUMEN
Bruton's tyrosine kinase (BTK) belongs to the TEC family of nonreceptor tyrosine kinases and plays a critical role in multiple cell types responsible for numerous autoimmune diseases. This article will detail the structure-activity relationships (SARs) leading to a novel second generation series of potent and selective reversible carbazole inhibitors of BTK. With an excellent pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide 6 (BMS-935177) was selected to advance into clinical development.
Asunto(s)
Antirreumáticos/química , Carbazoles/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinonas/química , Administración Oral , Agammaglobulinemia Tirosina Quinasa , Animales , Antirreumáticos/síntesis química , Antirreumáticos/farmacocinética , Antirreumáticos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Disponibilidad Biológica , Carbazoles/síntesis química , Carbazoles/farmacocinética , Carbazoles/farmacología , Línea Celular , Cristalografía por Rayos X , Perros , Humanos , Macaca fascicularis , Ratones , Microsomas Hepáticos/metabolismo , Permeabilidad , Proteínas Tirosina Quinasas/química , Quinazolinonas/síntesis química , Quinazolinonas/farmacocinética , Quinazolinonas/farmacología , Relación Estructura-ActividadRESUMEN
The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/virología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/uso terapéutico , Línea Celular , Efecto Citopatogénico Viral/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Orden Génico , Vectores Genéticos/genética , Humanos , Gripe Humana/tratamiento farmacológico , Concentración 50 Inhibidora , Ligandos , Modelos Moleculares , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
D-Luciferin is widely used as a substrate in luciferase catalysed bioluminescence assays for in vitro studies. However, little is known about cross reactivity and potential interference of D-luciferin with other enzymes. We serendipitously found that firefly luciferin inhibited the CDK2/Cyclin A protein kinase. Inhibition profiling of D-luciferin over a 103-protein kinase panel showed significant inhibition of a small set of protein kinases, in particular the DYRK-family, but also other members of the CMGC-group, including ERK8 and CK2. Inhibition profiling on a 16-member focused library derived from D-luciferin confirms that D-luciferin represents a DYRK-selective chemotype of fragment-like molecular weight. Thus, observation of its inhibitory activity and the initial SAR information reported here promise to be useful for further design of protein kinase inhibitors with related scaffolds.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Luciferina de Luciérnaga/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/química , Diseño de Fármacos , Luciferina de Luciérnaga/análogos & derivados , Humanos , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Relación Estructura-Actividad , Quinasas DyrKRESUMEN
Nonreceptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb(3+)-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align, and select potential phosphorylation-dependent Tb(3+)-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to high-throughput screening (HTS) applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use HTS-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein-binding ligands.
Asunto(s)
Pruebas de Enzimas/métodos , Proteínas Tirosina Quinasas/metabolismo , Terbio/química , Secuencia de Aminoácidos , Técnicas Biosensibles , Simulación por Computador , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Células K562 , Mediciones Luminiscentes , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.
Asunto(s)
Quinasa Idelta de la Caseína/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/química , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica/efectos de los fármacos , Proteínas Tirosina Quinasas/química , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
INTRODUCTION: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy. METHODS: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA). RESULTS: A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation. CONCLUSIONS: Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.
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Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Administración Oral , Aminopiridinas/química , Aminopiridinas/farmacología , Animales , Artritis Experimental/metabolismo , Artritis Experimental/prevención & control , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Modelos Moleculares , Estructura Molecular , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Piridazinas/química , Piridazinas/farmacología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Quinasa SykRESUMEN
IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.
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Asma/tratamiento farmacológico , Cisteína/química , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/química , Animales , Cristalografía por Rayos X , Citocinas/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Regulación Enzimológica de la Expresión Génica , Humanos , Células Jurkat , Leucocitos Mononucleares/efectos de los fármacos , Ligandos , Masculino , Tamaño de la Partícula , Unión Proteica , Proteínas Tirosina Quinasas/química , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
We describe and apply a scaffold-focused virtual screen based upon scaffold trees to the mitotic kinase TTK (MPS1). Using level 1 of the scaffold tree, we perform both 2D and 3D similarity searches between a query scaffold and a level 1 scaffold library derived from a 2 million compound library; 98 compounds from 27 unique top-ranked level 1 scaffolds are selected for biochemical screening. We show that this scaffold-focused virtual screen prospectively identifies eight confirmed active compounds that are structurally differentiated from the query compound. In comparison, 100 compounds were selected for biochemical screening using a virtual screen based upon whole molecule similarity resulting in 12 confirmed active compounds that are structurally similar to the query compound. We elucidated the binding mode for four of the eight confirmed scaffold hops to TTK by determining their protein-ligand crystal structures; each represents a ligand-efficient scaffold for inhibitor design.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Interfaz Usuario-Computador , Proteínas de Ciclo Celular/química , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel ß-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.
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Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Sulfatos/metabolismo , Sulfotransferasas/química , Sulfotransferasas/metabolismo , Tirosina/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Sitios de Unión , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptidos/química , Péptidos/metabolismo , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas/química , Electricidad Estática , Homología Estructural de Proteína , Especificidad por Sustrato , Tirosina/químicaAsunto(s)
Antiinflamatorios/química , Antineoplásicos/química , Inflamación/tratamiento farmacológico , Leucemia de Células B/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa , Animales , Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Dominio Catalítico , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Industria Farmacéutica , Humanos , Inflamación/enzimología , Leucemia de Células B/enzimología , Modelos Moleculares , Conformación Proteica , Proteínas Tirosina Quinasas/química , Relación Estructura-ActividadRESUMEN
Bone marrow kinase on chromosome X (BMX) is a cytosolic tyrosine kinase and a member of the TEC kinase family. BMX is expressed in hematopoietic cells of the myeloid lineage where it participates in the immune response. It is also involved in the response to ischemia and pressure overload in the endocardium and the cardiac endothelium. Moreover, BMX is expressed in several types of cancers and very recently has been shown to mediate the survival and tumorigenicity of glioblastoma cancer stem cells. In the inflammatory response BMX regulates the secretion of proinflammatory cytokines induced by TNFα, IL-1ß, and TLR agonists. It is required for the activation of the MAP kinase and NFκB pathways and acts at the level of the essential TAK1/TAB complex. Cellular regulation of the IL-8 promoter by BMX is dependent on membrane localization mediated by its pleckstrin homology domain, as well as on BMX kinase activity. BMX deficiency confers protection from arthritis in a mouse model known to be dependent on macrophages and IL-1ß. Genetic replacement of BMX with a kinase-inactive allele surprisingly restored susceptibility to arthritis, suggesting that in vivo BMX kinase activity can be dispensable. This review summarizes recent advances in the knowledge of BMX biology and their relevance for translational medicine.