RESUMEN
Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Interleucina-10/biosíntesis , Células Mieloides/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células Cultivadas , Quinasa 8 Dependiente de Ciclina/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-10/inmunología , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Células Mieloides/inmunología , Células Mieloides/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.
Asunto(s)
Dominio Catalítico , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión ProteicaAsunto(s)
Benzoatos/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Proteínas de Neoplasias/agonistas , Receptor alfa de Ácido Retinoico/agonistas , Transducción de Señal/efectos de los fármacos , Tetrahidronaftalenos/farmacología , Animales , Línea Celular Tumoral , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Ratones , Proteínas de Neoplasias/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Isolated protein motifs that are involved in interactions with their binding partners can be used to inhibit these interactions. However, peptides corresponding to protein fragments tend to have no defined secondary or tertiary structure in the absence of scaffolding by the rest of protein molecule. This results in low inhibitor potency. NMR and CD spectroscopy studies of lipopeptide inhibitors of the Hedgehog pathway revealed that membrane anchoring allows the cell membrane to function as a scaffold and facilitate the folding of short peptides. In addition, lipidation enhances cell permeability and increases the concentration of the compounds near the membrane, thus facilitating potent inhibition. The general applicability of this rational approach was further confirmed by the generation of selective antagonists of the insulin-like growth factor 1 receptor with GI(50) values in the nanomolar range. Lipopeptides corresponding to protein fragments were found to serve as potent and selective inhibitors of a number of nondruggable molecular targets.
Asunto(s)
Proteínas Hedgehog/antagonistas & inhibidores , Péptidos/química , Secuencia de Aminoácidos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dicroismo Circular , Proteínas Hedgehog/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos/farmacología , Estructura Terciaria de Proteína , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/metabolismoRESUMEN
Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Factores de Transcripción Forkhead/metabolismo , Factor de Transcripción GATA3/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Inmunofenotipificación , Isoquinolinas/farmacología , Adolescente , Adulto , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Ratones Endogámicos BALB C , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Despite a wealth of research on user participation, few studies offer insights into how to involve multi-organizational users in agile development methods. This paper is a case study of user involvement in developing a system for electronic laboratory requisitions using agile methodologies in a multi-organizational context. Building on an interpretive approach, we illuminate questions such as: How does collaboration between users and developers evolve and how might it be improved? What key motivational aspects are at play when users volunteer and continue contributing in the face of considerable added burdens? The study highlights how agile methods in themselves appear to facilitate mutually motivating collaboration between user groups and developers. Lessons learned for leveraging the advantages of agile development processes include acknowledging the substantial and ongoing contributions of users and their roles as co-designers of the system.
Asunto(s)
Motivación , Desarrollo de Programa , Sistemas de Información en Laboratorio Clínico , Sistemas de Información en Hospital , Humanos , Noruega , Estudios de Casos Organizacionales , Proyectos PilotoRESUMEN
Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Inhibidores de Proteínas Quinasas/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Cortistatin A is a natural product isolated from the marine sponge Corticium simplex and was found to be a potent and selective inhibitor of CDK8. Many synthetic groups have reported total syntheses of Cortistatin A; however, these syntheses require between 16 and 30 steps and report between 0.012-2% overall yields, which is not amenable to large-scale production. Owing to similarities between the complex core of Cortistatin A and the simple steroid core, we initiated a campaign to design simple, more easily prepared CDK8 inhibitors based on a steroid scaffold that would be more convenient for large-scale synthesis. Herein, we report the discovery and optimization of JH-VIII-49, a potent and selective inhibitor of CDK8 with a simple steroid core that has an eight-step synthesis with a 33% overall yield, making it suitable for large-scale preparation. Using this scaffold, we then developed a bivalent small molecule degrader, JH-XI-10-02, that can recruit the E3 ligase CRL4Cereblon to promote the ubiquitination and proteosomal degradation of CDK8.
RESUMEN
We conducted a systematic review of 15 relevant databases for articles about telemedicine. After eliminating articles that did not meet the inclusion criteria, 1615 remained for analysis. Three raters coded the articles to assess various theoretical and methodological variables. Only 5% (n = 85) of the telemedicine articles made mention of any theory or paradigmatic approach. Studies commonly reported the objectives (96%) but rarely stated a research question or hypothesis (11%). Randomized selection of the subjects was reported in 11% of patient studies and 4% of studies where providers were the subject. There was a wide range in the number of subjects employed, although the majority of studies were based on sample sizes of less than 100. Only 26% of the studies reported a time frame. Until the telemedicine field adheres to agreed standards of reporting methodological details it will be difficult to draw firm conclusions from review studies.
Asunto(s)
Investigación/normas , Telemedicina , HumanosRESUMEN
Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5 (YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50 for SIK2 inhibition for >16 h and reduce phosphorylation of a known SIK substrate in vivo. While in vivo active doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2 knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivo and further support the development of SIK inhibitors for treatment of inflammatory disorders.
Asunto(s)
Sondas Moleculares/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células Cultivadas , Concentración 50 Inhibidora , Ratones , Ratones Noqueados , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular , Humanos , Masculino , Ratones , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Northern Norwegian Health Net (the Net) is a closed network for social and health care institutions in North Norway. In its present form it was established in 2000, but the early start was in the late 1980s. This proceeding will give a brief history of the network, description of the technology used and the services offered. It also gives a summary of experiences with the Net and research conducted on the services. Most health care institutions in the region are connected in the Net, and the usage is increasing.
Asunto(s)
Redes de Comunicación de Computadores/organización & administración , Prestación Integrada de Atención de Salud , Aplicaciones de la Informática Médica , Humanos , Noruega , Salud Rural , TelemedicinaRESUMEN
PURPOSE: The purpose of this paper is to explore the making and scaling of information infrastructures, as well as how the conditions for scaling a component may change for the vendor. The first research question is how the making and scaling of a healthcare information infrastructure can be done and by whom. The second question is what scope for manoeuvre there might be for vendors aiming to expand their market. METHOD: This case study is based on an interpretive approach, whereby data is gathered through participant observation and semi-structured interviews. RESULTS: A case study of the making and scaling of an electronic system for general practitioners ordering laboratory services from hospitals is described as comprising two distinct phases. The first may be characterized as an evolving phase, when development, integration and implementation were achieved in small steps, and the vendor, together with end users, had considerable freedom to create the solution according to the users' needs. The second phase was characterized by a large-scale procurement process over which regional healthcare authorities exercised much more control and the needs of groups other than the end users influenced the design. CONCLUSION: The making and scaling of healthcare information infrastructures is not simply a process of evolution, in which the end users use and change the technology. It also consists of large steps, during which different actors, including vendors and healthcare authorities, may make substantial contributions. This process requires work, negotiation and strategies. The conditions for the vendor may change dramatically, from considerable freedom and close relationships with users and customers in the small-scale development, to losing control of the product and being required to engage in more formal relations with customers in the wider public healthcare market. Onerous procurement processes may be one of the reasons why large-scale implementation of information projects in healthcare is difficult and slow.