RESUMEN
Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of â¼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.
Asunto(s)
Dihidropiridinas , Rabdomiosarcoma , Humanos , Niño , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/química , Relación Estructura-Actividad , Antihipertensivos/farmacología , Canales de Calcio Tipo L/metabolismo , Rabdomiosarcoma/tratamiento farmacológico , Dihidropiridinas/farmacologíaRESUMEN
Gastric cancer is one of the most common and deadly types of cancer in the world, and poor prognosis with treatment failure is widely reported in the literature. In this context, kinases have been considered a relevant choice for targeted therapy in gastric cancer. Here, we explore the antiproliferative and antimigratory effects of the AURKA inhibitor and the prognostic and therapeutic value as a biomarker of gastric cancer. A total of 145 kinase inhibitors were screened to evaluate the cytotoxic or cytostatic effects in the gastric cancer cell line. Using the Alamar Blue assay, flow cytometry, quantitative polymerase chain reaction, and observation of caspase 3/7 activity and cell migration, we investigated the antiproliferative, proapoptotic, and antimigratory effects of the AURKA inhibitor. Moreover, AURKA overexpression was evaluated in the gastric cell lines and the gastric tumor tissue. Out of the 145 inhibitors, two presented the highest antiproliferative effect. Both molecules can induce apoptosis by the caspases 3/7 pathway in addition to inhibiting cancer cell migration, mainly the AURKA inhibitor. Moreover, molecular docking analysis revealed that GW779439X interacts in the active site of the AURKA enzyme with similar energy as a well-described inhibitor. Our study identified AURKA overexpression in the gastric cancer cell line and gastric tumor tissue, revealing that its overexpression in patients with cancer is correlated with low survival. Therefore, it is feasible to suggest AURKA as a potential marker of gastric cancer, besides providing robust information for diagnosis and estimated survival of patients. AURKA can be considered a new molecular target used in the prognosis and therapy of gastric cancer.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridazinas/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Apoptosis , Aurora Quinasa A/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Simulación del Acoplamiento Molecular , Pronóstico , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Tasa de SupervivenciaRESUMEN
PURPOSE: Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the field of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains understudied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer. METHODS: Stably overexpressing NEK5 cell lines (MCF7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immunofluorescence and quantification of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis. RESULTS: NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type-specific manner, respectively. CONCLUSION: While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating specific steps of metastatic progression.
Asunto(s)
Neoplasias de la Mama , Quinasas Relacionadas con NIMA , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Quinasas Relacionadas con NIMA/genética , Fenotipo , ARN Interferente PequeñoRESUMEN
We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Humanos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Estructura Molecular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Screening a series of 4-anilinoquinolines and 4-anilinoquinazolines enabled identification of potent novel inhibitors of dengue virus (DENV). Preparation of focused 4-anilinoquinoline/quinazoline scaffold arrays led to the identification of a series of high potency 6-substituted bromine and iodine derivatives. The most potent compound 6-iodo-4-((3,4,5-trimethoxyphenyl)amino)quinoline-3-carbonitrile (47) inhibited DENV infection with an EC50 = 79 nM. Crucially, these compounds showed very limited toxicity with CC50 values >10 µM in almost all cases. This new promising series provides an anchor point for further development to optimize compound properties.
Asunto(s)
Compuestos de Anilina/farmacología , Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Quinazolinas/farmacología , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Antivirales/síntesis química , Antivirales/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinazolinas/síntesis química , Quinazolinas/química , Relación Estructura-ActividadRESUMEN
We report the synthesis of several related 4-anilinoquinazolines as inhibitors of cardiac troponin I-interacting kinase (TNNi3K). These close structural analogs of 3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-(dimethylamino)-N-methylbenzenesulfonamide (GSK114) provide new understanding of structure-activity relationships between the 4-anilinoquinazoline scaffold and TNNi3K inhibition. Through a small focused library of inhibitors, we observed that the N-methylbenzenesulfonamide was driving the potency in addition to the more traditional quinazoline hinge-binding motif. We also identified a compound devoid of TNNi3K kinase activity due to the addition of a methyl group in the hinge binding region. This compound could serve as a negative control in the study of TNNi3K biology. Small molecule crystal structures of several quinazolines have been solved, supporting observations made about overall conformation and TNNi3K inhibition.
Asunto(s)
Compuestos de Anilina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinazolinas/farmacología , Humanos , Miocitos Cardíacos/metabolismo , Troponina I/metabolismoRESUMEN
The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) activates CAMK1, CAMK4, AMPK, and AKT, leading to numerous physiological responses. The deregulation of CAMKK2 is linked to several diseases, suggesting the utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work, we demonstrate that STO-609, frequently described as a selective inhibitor for CAMKK2, potently inhibits a significant number of other kinases. Through an analysis of literature and public databases, we have identified other potent CAMKK2 inhibitors and verified their activities in differential scanning fluorimetry and enzyme inhibition assays. These inhibitors are potential starting points for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.
Asunto(s)
Bencimidazoles/química , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Naftalimidas/química , Inhibidores de Proteínas Quinasas/química , Animales , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/antagonistas & inhibidores , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/química , HumanosRESUMEN
The rapid emergence of multidrug resistance among bacterial pathogens has become a significant challenge to human health in our century. Therefore, development of next-generation antibacterial compounds is an urgent need. Two-component signal transduction systems (TCS) are stimulus-response coupling devices that allow bacteria to sense and elaborate adaptive responses to changing environmental conditions, including the challenges that pathogenic bacteria face inside the host. The differential presence of TCS, present in bacteria but absent in the animal kingdom, makes them attractive targets in the search for new antibacterial compounds. In Salmonella enterica, the PhoP/PhoQ two-component system controls the expression of crucial phenotypes that define the ability of the pathogen to establish infection in the host. We now report the screening of 686 compounds from a GlaxoSmithKline published kinase inhibitor set in a high-throughput whole-cell assay that targets Salmonella enterica serovar Typhimurium PhoP/PhoQ. We identified a series of quinazoline compounds that showed selective and potent downregulation of PhoP/PhoQ-activated genes and define structural attributes required for their efficacy. We demonstrate that their bioactivity is due to repression of the PhoQ sensor autokinase activity mediated by interaction with its catalytic domain, acting as competitive inhibitors of ATP binding. While noncytotoxic, the hit molecules exhibit antivirulence effect by blockage of S Typhimurium intramacrophage replication. Together, these features make these quinazoline compounds stand out as exciting leads to develop a therapeutic intervention to fight salmonellosis.
Asunto(s)
Quinazolinas/farmacología , Salmonella typhimurium/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Salmonella typhimurium/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Virulencia/genéticaRESUMEN
We demonstrate an innovative approach for optimization of kinase inhibitor potency and selectivity utilising kinase mini-panels and kinome-wide panels. We present a focused case study on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs.
Asunto(s)
Ciclina G/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/antagonistas & inhibidores , Humanos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
We screened a series of 4-anilinoquinolines and 4-anilinoquinazolines and identified novel inhibitors of Mycobacterium tuberculosis (Mtb). The focused 4-anilinoquinoline/quinazoline scaffold arrays yielded compounds with high potency and the identification of 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (34) with an MIC90 value of 0.63-1.25⯵M. We also defined a series of key structural features, including the benzyloxy aniline and the 6,7-dimethoxy quinoline ring, that are important for Mtb inhibition. Importantly the compounds showed very limited toxicity and scope for further improvement by iterative medicinal chemistry.
Asunto(s)
Compuestos de Anilina/farmacología , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Quinazolinas/farmacología , Quinolinas/farmacología , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Antituberculosos/síntesis química , Antituberculosos/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinazolinas/síntesis química , Quinazolinas/química , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.
Asunto(s)
Imidazoles/química , Proteínas de la Membrana , Péptidos/química , Proteínas Proto-Oncogénicas B-raf , Piridinas/química , Sulfotransferasas , Tirosina/química , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Sulfotransferasas/antagonistas & inhibidores , Sulfotransferasas/químicaRESUMEN
Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.
Asunto(s)
Proteínas Aviares/química , Heparitina Sulfato/química , Oligosacáridos/química , Inhibidores de Proteínas Quinasas/química , Sulfotransferasas/química , Quinasas raf/antagonistas & inhibidores , Animales , Proteínas Aviares/genética , Pollos , Heparitina Sulfato/farmacología , Humanos , Oligosacáridos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Sulfotransferasas/genética , Porcinos , Quinasas raf/químicaRESUMEN
SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 µM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/química , Análisis por Conglomerados , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-ActividadRESUMEN
Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to ß-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to ß-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various ß-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate ß-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.
Asunto(s)
Antibacterianos/farmacocinética , Proteínas Bacterianas/antagonistas & inhibidores , Listeria monocytogenes/enzimología , Oxadiazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antibacterianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evaluación Preclínica de Medicamentos , Listeria monocytogenes/genética , Oxadiazoles/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Staphylococcus aureus/enzimologíaRESUMEN
New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic-like Ser/Thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically useful microbiologic activity has been elusive. Additionally, PknB kinetics are not fully described, nor are structures with specific inhibitors available to inform inhibitor design. We used computational modeling with available structural information to identify human kinase inhibitors predicted to bind PknB, and we selected hits based on drug-like characteristics intended to increase the likelihood of cell entry. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We performed an in-depth characterization of PknB and found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel X-ray structure confirmed that the inhibitors bound as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity toward mycobacteria and nocardia. We demonstrated that the inhibitors are uniquely potentiated by ß-lactams but not antibiotics traditionally used to treat mycobacteria, consistent with PknB's role in sensing cell wall stress. This is the first demonstration in the phylum actinobacteria that some ß-lactam antibiotics could be more effective if paired with a PknB inhibitor. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can act with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.
Asunto(s)
Descubrimiento de Drogas/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , beta-Lactamas/farmacología , Cristalografía por Rayos X , Sinergismo Farmacológico , Pruebas de Enzimas , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Tiadiazoles/síntesis química , Tiadiazoles/farmacología , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Tiadiazoles/química , Agua/químicaRESUMEN
The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Proteínas Quinasas Dependientes de GMP Cíclico , Retículo Endoplásmico , Proteínas Protozoarias , Purinonas/farmacología , Toxoplasma , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high-throughput strategy to screen a GlaxoSmithKline Published Kinase Inhibitor Set. This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen, we identified compounds within chemotypes that impede HCMV protein production and identified kinase proteins associated with inhibition of HCMV protein production that are potential novel anti-HCMV targets. We focused our study on a top 'hit' in our screen, SB-734117, which we found inhibits productive replication of several HCMV strains. Kinase selectivity data indicated that SB-734117 exhibited polypharmacology and was an inhibitor of several proteins from the AGC and CMCG kinase groups. Using Western blotting, we found that SB-734711 inhibited accumulation of HCMV immediate-early proteins, phosphorylation of cellular proteins involved in immediate-early protein production (cAMP response element-binding protein and histone H3) and histone H3 lysine 36 trimethylation (H3K36me3). Therefore, we identified SB-734117 as a novel anti-HCMV compound and found that inhibition of AGC and CMCG kinase proteins during productive HCMV replication was associated with inhibition of viral protein production and prevented post-translational modification of cellular factors associated with viral protein production.
Asunto(s)
Antivirales/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citomegalovirus/efectos de los fármacos , Histonas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Western Blotting , Citomegalovirus/fisiología , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well-characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth-inhibitory effect. Their half-maximal effective concentration (EC50 ) values were determined in chordoma cells and normal fibroblasts. Twenty-seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho-EGFR and its downstream pathways in a dose-dependent manner. Analysis of substituent patterns suggested that EGFR-inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U-CH1 xenograft and a patient-derived xenograft, SF8894). One of the resistant cell lines (U-CH2) was shown to express high levels of phospho-MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p-)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.