RESUMEN
Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.
Asunto(s)
Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency inâ vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.
Asunto(s)
Carbohidrato Epimerasas , Ácido N-Acetilneuramínico , Humanos , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Ácidos Siálicos/química , Carbohidratos , PolisacáridosRESUMEN
MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is-to the best of our knowledge-the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients.
Asunto(s)
Acetofenonas/uso terapéutico , Antineoplásicos/uso terapéutico , Benzopiranos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Factores de Transcripción/antagonistas & inhibidores , Desacopladores/uso terapéutico , Acetofenonas/efectos adversos , Acetofenonas/química , Acetofenonas/farmacología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antineoplásicos/farmacología , Benzopiranos/efectos adversos , Benzopiranos/química , Benzopiranos/farmacología , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Genes Reporteros/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Neoplasias Hepáticas Experimentales/prevención & control , Neoplasias Hepáticas Experimentales/secundario , Ratones SCID , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Distribución Aleatoria , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas , Transactivadores , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Carga Tumoral/efectos de los fármacos , Desacopladores/efectos adversos , Desacopladores/química , Desacopladores/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In drug development, late stage toxicity issues of a compound are the main cause of failure in clinical trials. In silico methods are therefore of high importance to guide the early design process to reduce time, costs and animal testing. Technical advances and the ever growing amount of available toxicity data enabled machine learning, especially neural networks, to impact the field of predictive toxicology. In this study, cytotoxicity prediction, one of the earliest handles in drug discovery, is investigated using a deep learning approach trained on a highly consistent in-house data set of over 34,000 compounds with a share of less than 5% of cytotoxic molecules. The model reached a balanced accuracy of over 70%, similar to previously reported studies using Random Forest. Albeit yielding good results, neural networks are often described as a black box lacking deeper mechanistic understanding of the underlying model. To overcome this absence of interpretability, a Deep Taylor Decomposition method is investigated to identify substructures that may be responsible for the cytotoxic effects, the so-called toxicophores. Furthermore, this study introduces cytotoxicity maps which provide a visual structural interpretation of the relevance of these substructures. Using this approach could be helpful in drug development to predict the potential toxicity of a compound as well as to generate new insights into the toxic mechanism. Moreover, it could also help to de-risk and optimize compounds.
Asunto(s)
Citotoxinas/química , Citotoxinas/toxicidad , Aprendizaje Profundo , Descubrimiento de Drogas/métodos , Supervivencia Celular/efectos de los fármacos , Diseño Asistido por Computadora , Diseño de Fármacos , Descubrimiento de Drogas/estadística & datos numéricos , Células HEK293 , Células Hep G2 , Humanos , Modelos Biológicos , Redes Neurales de la Computación , Bibliotecas de Moléculas Pequeñas , Programas Informáticos , Toxicología/estadística & datos numéricosRESUMEN
Classical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.
Asunto(s)
Antineoplásicos/farmacología , Linfocitos B/inmunología , Diferenciación Celular/efectos de los fármacos , Enfermedad de Hodgkin/inmunología , Transcriptoma/efectos de los fármacos , Antígenos CD19/inmunología , Antígenos CD20/inmunología , Linfocitos B/efectos de los fármacos , Inmunoprecipitación de Cromatina , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.
Asunto(s)
Inhibidores Enzimáticos/química , Factores Inmunológicos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ácidos Siálicos/química , Bibliotecas de Moléculas Pequeñas/química , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Dominio Catalítico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glicosilación , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Simulación del Acoplamiento Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The Staphylococcus aureus ClpXP protease is an important regulator of cell homeostasis and virulence. We utilized a high-throughput screen against the ClpXP complex and identified a specific inhibitor of the ClpX chaperone that disrupts its oligomeric state. Synthesis of 34 derivatives revealed that the molecular scaffold is restrictive for diversification, with only minor changes tolerated. Subsequent analysis of the most active compound revealed strong attenuation of S. aureus toxin production, which was quantified with a customized MS-based assay platform. Transcriptome and whole-proteome studies further confirmed the global reduction of virulence and revealed characteristic signatures of protein expression in the compound-treated cells. Although these partially matched the pattern of ClpX knockout cells, further depletion of toxins was observed, leading to the intriguing perspective that additional virulence pathways may be directly or indirectly addressed by the small molecule.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Endopeptidasa Clp/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Inhibidores de Proteasas/farmacología , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Endopeptidasa Clp/deficiencia , Endopeptidasa Clp/metabolismo , Ensayos Analíticos de Alto Rendimiento , Staphylococcus aureus Resistente a Meticilina/metabolismo , Estructura Molecular , Inhibidores de Proteasas/química , Relación Estructura-Actividad , VirulenciaRESUMEN
The layout of multipurpose screening libraries must address criteria for the compounds such as novelty, diversity potential, innovative design, and last but not least synthetic tractability. While academic compound collections are often innovative, novel, and highly divers, synthesis of analogs or larger substance quantities is often hampered by complex multistep syntheses with low overall yields. In addition, covalently binding compounds and interaction motifs designed to bind metal ions were discriminated against by the paradigm that these interaction types must almost inevitably lead to toxic effects. We would like to challenge this hypothesis. The lack of such interactions could be a reason for frequent failure in the disclosure of hits for hitherto undruggable target proteins using commercially available screening collections. Thus, easily synthesizable screening candidates equipped to bind covalently to nucleophiles or to metalloenzymes by chelation are under-represented in public access screening libraries. Within this work, we present the synthesis and deposition of 88 compounds with five distinct functional classes, each of which features under-represented screening motifs, for example, metal ion complexation, reversible covalent binding, or halogen bonding. The collection includes acetohydrazides, acylhydrazones, propylene glycol ethers, 2-cyanoacetamides, and 2-cyanoacrylamides. The rational for the synthesis of most of the compounds was recently published by our group and is now supplemented by additional compounds reported here for the first time. The public access disposition enables academic research groups to collectively expand the druggable space and interdisciplinary collaborate within the scientific field.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Técnicas de Química Sintética , Conducta Cooperativa , Compuestos Orgánicos/síntesis química , Compuestos Orgánicos/química , Compuestos Orgánicos/farmacología , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.
Asunto(s)
Acuaporina 2/metabolismo , Benzodioxoles/farmacología , Benzofuranos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/fisiología , Fluconazol/farmacología , Aparato de Golgi/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Microscopía Fluorescente , Fosforilación , Transporte de Proteínas/efectos de los fármacos , RatasRESUMEN
Digital media are indispensable in school, profession, family and leisure time. 1 to 6 % of all users show dsyfunctional ans addictive patterns, first of all in online and "social" media. In Switzerland over 80 % of young people own a smartphone and "pocket internet". Time of interaction with online-media (hours/day), as well as peer group pattern are markers for risk of addiction. Active music making and sports are protective factors. Family physicians are important in early recognition of "internet addictive disease". Care-givers with special experience in this field are often successful in reducing time of harmful interaction with the internet. Internet addictive disease is not yet classified in ICD and DSM-5 lists, even though it is an increasing reality.
Asunto(s)
Conducta Adictiva/diagnóstico , Conducta Adictiva/psicología , Internet , Adolescente , Adulto , Conducta Adictiva/epidemiología , Conducta Adictiva/rehabilitación , Comorbilidad , Consejo , Estudios Transversales , Diagnóstico Precoz , Intervención Médica Temprana , Medicina Familiar y Comunitaria , Femenino , Humanos , Masculino , Modelos Psicológicos , Motivación , Relaciones Médico-Paciente , Factores de Riesgo , Factores Sexuales , Suiza , Adulto JovenRESUMEN
Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease. Here, we established a Malachite green-based assay compatible with high throughput screening to monitor the liposome-stimulated ATPase of EHD4. In this way, we identified a drug-like molecule that inhibited EHD4's liposome-stimulated ATPase activity. Structure activity relationship (SAR) studies indicated sites of preferred substitutions for more potent inhibitor synthesis. Moreover, the assay optimization in this work can be applied to other dynamin family members showing a weak and liposome-dependent nucleotide hydrolysis activity.
Asunto(s)
Adenosina Trifosfatasas , Liposomas , Liposomas/metabolismo , Liposomas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Humanos , Relación Estructura-Actividad , Dinaminas/metabolismo , Dinaminas/antagonistas & inhibidores , Dinaminas/química , Colorantes de Rosanilina/química , HidrólisisRESUMEN
Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.
Vitamin B6 is an important nutrient for optimal brain function, with deficiencies linked to impaired memory, learning and mood in various mental disorders. In older people, vitamin B6 deficiency is also associated with declining memory and dementia. Although this has been known for years, the precise role of vitamin B6 in these disorders and whether supplements can be used to treat or prevent them remained unclear. This is partly because vitamin B6 is actually an umbrella term for a small number of very similar and interchangeable molecules. Only one of these is 'bioactive', meaning it has a biological role in cells. However, therapeutic strategies aimed at increasing only the bioactive form of vitamin B6 are lacking. Previous work showed that disrupting the gene for an enzyme called pyridoxal phosphatase, which breaks down vitamin B6, improves memory and learning in mice. To investigate whether these effects could be mimicked by drug-like compounds, Brenner, Zink, Witzinger et al. used several biochemical and structural biology approaches to search for molecules that bind to and inhibit pyridoxal phosphatase. The experiments showed that a molecule called 7,8-dihydroxyflavone which was previously found to improve memory and learning in laboratory animals with brain disorders binds to pyridoxal phosphatase and inhibits its activity. This led to increased bioactive vitamin B6 levels in mouse brain cells involved in memory and learning. The findings of Brenner et al. suggest that inhibiting pyridoxal phosphatase to increase vitamin B6 levels in the brain could be used together with supplements. The identification of 7,8-dihydroxyflavone as a promising candidate drug is a first step in the discovery of more efficient pyridoxal phosphatase inhibitors. These will be useful experimental tools to directly study whether increasing the levels of bioactive vitamin B6 in the brain may help those with mental health conditions associated with impaired memory, learning and mood.
Asunto(s)
Inhibidores Enzimáticos , Monoéster Fosfórico Hidrolasas , Animales , Ratones , Humanos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfato de Piridoxal/metabolismo , Flavonas/farmacología , Flavonas/metabolismo , Flavonas/química , Ratones Endogámicos C57BLRESUMEN
Ectopic expression of the neuron-specific inositol-1,4,5-trisphosphate-3-kinase A (ITPKA) in lung cancer cells increases their metastatic potential because the protein exhibits two actin regulating activities; it bundles actin filaments and regulates inositol-1,4,5-trisphosphate (InsP3)-mediated calcium signals by phosphorylating InsP3. Thus, in order to inhibit the metastasis-promoting activity of ITPKA, both its actin bundling and its InsP3kinase activity has to be blocked. In this study, we performed a high throughput screen in order to identify specific and membrane-permeable substances against the InsP3kinase activity. Among 341,44 small molecules, 237 compounds (0.7%) were identified as potential InsP3kinase inhibitors. After determination of IC50-values, the three compounds with highest specificity and highest hydrophobicity (EPPC-3, BAMB-4, MEPTT-3) were further characterized. Only BAMB-4 was nearly completely taken up by H1299 cells and remained stable after cellular uptake, thus exhibiting a robust stability and a high membrane permeability. Determination of the inhibitor type revealed that BAMB-4 belongs to the group of mixed type inhibitors. Taken together, for the first time we identified a highly membrane-permeable inhibitor against the InsP3kinase activity of ITPKA providing the possibility to partly inhibit the metastasis-promoting effect of ITPKA in lung tumor cells.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Transcription factor NF-κB potently activates anti-apoptotic genes, and its inactivation significantly reduces tumor cell survival following genotoxic stresses. We identified two structurally distinct lead compounds that selectively inhibit NF-κB activation by DNA double-strand breaks, but not by other stimuli, such as TNFα. Our compounds do not directly inhibit previously identified regulators of this pathway, most critically including IκB kinase (IKK), but inhibit signal transmission in-between ATM, PARP1, and IKKγ. Deconvolution strategies, including derivatization and in vitro testing in multi-kinase panels, yielded shared targets, cdc-like kinase (CLK) 2 and 4, as essential regulators of DNA damage-induced IKK and NF-κB activity. Both leads sensitize to DNA damaging agents by increasing p53-induced apoptosis, thereby reducing cancer cell viability. We propose that our lead compounds and derivatives can be used in context of genotoxic therapy-induced or ongoing DNA damage to increase tumor cell apoptosis, which may be beneficial in cancer treatment.
Asunto(s)
FN-kappa B , Transducción de Señal , FN-kappa B/metabolismo , Daño del ADN , Regulación de la Expresión Génica , ADNRESUMEN
The architectural chromatin factor high-mobility group AT-hook 2 (HMGA2) is causally involved in several human malignancies and pathologies. HMGA2 is not expressed in most normal adult somatic cells, which renders the protein an attractive drug target. An established cell-based compound library screen identified the fibroblast growth factor receptor (FGFR) inhibitor PD173074 as an antagonist of HMGA2-mediated transcriptional reporter gene activation. We determined that PD173074 binds the C-terminus of HMGA2 and interferes with functional coordination of the three AT-hook DNA-binding domains mediated by the C-terminus. The HMGA2-antagonistic effect of PD173074 on transcriptional activation may therefore result from an induced altered DNA-binding mode of HMGA2. PD173074 as a novel HMGA2-specific antagonist could trigger the development of derivates with enhanced attributes and clinical potential.
Asunto(s)
Neoplasias , Receptores de Factores de Crecimiento de Fibroblastos , Adulto , Humanos , Activación Transcripcional , Cromatina , ADN/metabolismo , Proteína HMGA2/genética , Proteína HMGA2/metabolismoRESUMEN
Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato) , Ácido Fítico , Animales , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Fosfatos de Inositol/metabolismo , Mamíferos/metabolismoRESUMEN
Microglia are the immune effector cells of the central nervous system (CNS) and react to pathologic events with a complex process including the release of nitric oxide (NO). NO is a free radical, which is toxic for all cells at high concentrations. To target an exaggerated NO release, we tested a library of 16 544 chemical compounds for their effect on lipopolysaccharide (LPS)-induced NO release in cell line and primary neonatal microglia. We identified a compound (C1) which significantly reduced NO release in a dose-dependent manner, with a low IC50 (252 nM) and no toxic side effects in vitro or in vivo. Target finding strategies such as in silico modelling and mass spectroscopy hint towards a direct interaction between C1 and the nitric oxide synthase making C1 a great candidate for specific intra-cellular interaction with the NO producing machinery.
Asunto(s)
Microglía , Óxido Nítrico , Recién Nacido , Humanos , Microglía/metabolismo , Óxido Nítrico/metabolismo , Enfermedades Neuroinflamatorias , Óxido Nítrico Sintasa de Tipo II/metabolismo , Línea Celular , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismoRESUMEN
Class II phosphoinositide-3-kinases (PI3Ks) play central roles in cell signaling, division, migration, and survival. Despite evidence that all PI3K class II isoforms serve unique cellular functions, the lack of isoform-selective inhibitors severely hampers the systematic investigation of their potential relevance as pharmacological targets. Here, we report the structural evaluation and molecular determinants for selective PI3K-C2α inhibition by a structure-activity relationship study based on a pteridinone scaffold, leading to the discovery of selective PI3K-C2α inhibitors called PITCOINs. Cocrystal structures and docking experiments supported the rationalization of the structural determinants essential for inhibitor activity and high selectivity. Profiling of PITCOINs in a panel of more than 118 diverse kinases showed no off-target kinase inhibition. Notably, by addressing a selectivity pocket, PITCOIN4 showed nanomolar inhibition of PI3K-C2α and >100-fold selectivity in a general kinase panel. Our study paves the way for the development of novel therapies for diseases related to PI3K-C2α function.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II , Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Isoformas de Proteínas , FosfatidilinositolesRESUMEN
A sub-library of 88 information-rich lead-like purine derivatives were prepared and deposited in an open access academic screening facility. The rationale for the synthesis of these rigid low complexity structures was the privileged character of the purine heterocycle associated with its inherent probability of interactions with multiple adenine-related targets. Although generally expected to be weak binders in many assays, such fragment-like compounds are estimated to match diverse binding sites. It is suggested that heterocycles with many anchor points for hydrogen bonds can be anticipated to undergo very specific interactions to produce more negative enthalpies and thus provide superior starting points for lead optimization than compounds that owe their activity to entropic effects. The in vitro cytotoxicity of the small compounds on a panel of human cancer cell lines has been investigated and some of them showed marked unselective or selective toxicity. This data may be useful if these fragments are to be incorporated into drug-like structures via metabolically cleavable connections. The sub-library will be implemented as part of the ChemBioNet ( www.chembionet.info ) library, and it is open to screening campaigns of academic research groups striving for a fragment-based approach in their biological assays.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Tecnología Química Verde/métodos , Purinas/síntesis química , Purinas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Automatización , Línea Celular Tumoral , Técnicas de Química Sintética , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Conformación Molecular , Purinas/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
A-kinase anchoring proteins (AKAPs) are a family of multivalent scaffolding proteins. They engage in direct protein-protein interactions with protein kinases, kinase substrates and further signaling molecules. Each AKAP interacts with a specific set of protein interaction partners and such sets can vary between different cellular compartments and cells. Thus, AKAPs can coordinate signal transduction processes spatially and temporally in defined cellular environments. AKAP-dependent protein-protein interactions are involved in a plethora of physiological processes, including processes in the cardiovascular, nervous, and immune system. Dysregulation of AKAPs and their interactions is associated with or causes widespread diseases, for example, cardiac diseases such as heart failure. However, there are profound shortcomings in understanding functions of specific AKAP-dependent protein-protein interactions. In part, this is due to the lack of agents for specifically targeting defined protein-protein interactions. Peptidic and non-peptidic inhibitors are invaluable molecular tools for elucidating the functions of AKAP-dependent protein-protein interactions. In addition, such interaction disruptors may pave the way to new concepts for the treatment of diseases where AKAP-dependent protein-protein interactions constitute potential drug targets.Here we describe screening approaches for the identification of small molecule disruptors of AKAP-dependent protein-protein interactions. Examples include interactions of AKAP18 and protein kinase A (PKA) and of AKAP-Lbc and RhoA. We discuss a homogenous time-resolved fluorescence (HTRF) and an AlphaScreen® assay for small molecule library screening and human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a cell system for the characterization of identified hits.