RESUMEN
To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high-content screening assays. By using automated image analysis with single-cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. Additionally, we developed a high-throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.
Asunto(s)
Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/análisis , Indoles/farmacología , Osteosarcoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Antineoplásicos/química , Técnicas Biosensibles , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Indoles/química , Modelos Moleculares , Estructura Molecular , Osteosarcoma/patología , Análisis de la Célula IndividualRESUMEN
The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Proteínas Señalizadoras YAP , Animales , Humanos , Ratones , Proliferación Celular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP/agonistas , Proteínas Señalizadoras YAP/efectos de los fármacos , Proteínas Señalizadoras YAP/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
[This corrects the article DOI: 10.1021/acsmedchemlett.7b00485.].
RESUMEN
Three-dimensional (3D) spheroid models are rapidly gaining favor for drug discovery applications due to their improved morphological characteristics, cellular complexity, long lifespan in culture, and higher physiological relevance relative to two-dimensional (2D) cell culture models. High-content imaging (HCI) of 3D spheroid models has the potential to provide valuable information to help researchers untangle disease pathophysiology and assess novel therapies more effectively. The transition from 2D monolayer models to dense 3D spheroids in HCI applications is not trivial, however, and requires 3D-optimized protocols, instrumentation, and resources. Here, we discuss considerations for moving from 2D to 3D models and present a framework for HCI and analysis of 3D spheroid models in a drug discovery setting. We combined scaffold-free, multicellular spheroid models with scalable, automation-compatible plate technology enabling image-based applications ranging from high-throughput screening to more complex, lower-throughput microphysiological systems of organ networks. We used this framework in three case studies: investigation of lipid droplet accumulation in a human liver nonalcoholic steatohepatitis (NASH) model, real-time immune cell interactions in a multicellular 3D lung cancer model, and a high-throughput screening application using a 3D co-culture model of gastric carcinoma to assess dose-dependent drug efficacy and specificity. The results of these proof-of-concept studies demonstrate the potential for high-resolution image-based analysis of 3D spheroid models for drug discovery applications, and confirm that cell-level and temporal-spatial analyses that fully exploit multicellular features of spheroid models are not only possible but soon will be routine practice in drug discovery workflows.
Asunto(s)
Descubrimiento de Drogas , Imagenología Tridimensional/tendencias , Imagen Molecular/tendencias , Enfermedad del Hígado Graso no Alcohólico/diagnóstico por imagen , Humanos , Gotas Lipídicas/ultraestructura , Hígado/efectos de los fármacos , Hígado/patología , Hígado/ultraestructura , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/ultraestructura , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patologíaRESUMEN
MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.
Asunto(s)
Inhibidores de Caspasas/uso terapéutico , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Urea/análogos & derivados , Urea/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/farmacología , Descubrimiento de Drogas , Femenino , Humanos , Inmunidad Humoral/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Urea/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
FGF19 signaling through the FGFR4/ß-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.
Asunto(s)
Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisteína/química , Perros , Diseño de Fármacos , Semivida , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and it is the third leading cause of cancer-related deaths worldwide. Recently, aberrant signaling through the FGF19/FGFR4 axis has been implicated in HCC. Here, we describe the development of FGF401, a highly potent and selective, first in class, reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4. FGF401 is exquisitely selective for FGFR4 versus the other FGFR paralogues FGFR1, FGFR2, FGFR3, and all other kinases in the kinome. FGF401 has excellent drug-like properties showing a robust pharmacokinetic/pharmacodynamics/efficacy relationship, driven by a fraction of time above the phospho-FGFR4 IC90 value. FGF401 has remarkable antitumor activity in mice bearing HCC tumor xenografts and patient-derived xenograft models that are positive for FGF19, FGFR4, and KLB. FGF401 is the first FGFR4 inhibitor to enter clinical trials, and a phase I/II study is currently ongoing in HCC and other solid malignancies.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Transducción de SeñalRESUMEN
As part of a project to identify FGFR4 selective inhibitors, scaffold morphing of a 2-formylquinoline amide hit identified series of 2-formylpyridine ureas (2-FPUs) with improved potency and physicochemical properties. In particular, tetrahydronaphthyridine urea analogues with cellular activities below 30 nM have been identified. Consistent with the hypothesized reversible-covalent mechanism of inhibition, the 2-FPUs exhibited slow binding kinetics, and the aldehyde, as the putative electrophile, could be demonstrated to be a key structural element for activity.
RESUMEN
A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diaminopyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a hemithioacetal adduct with a 2-formyl naphthalene. In addition, the introduction of an acrylamide into a known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family members.
RESUMEN
[Structure: see text] 12-hydroxydaphnetoxins, members of the structurally fascinating daphnane diterpene family, exhibit a wide range of significant biological activities. A general route to the BC-ring system of 12-hydroxy daphnetoxins is reported based on D-ribose. Depending on the choice of protecting groups and solvent, the oxidopyrylium-alkene [5+2] cycloaddition originating from A provides cycloadduct diastereomer B or C with good to excellent selectivity.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/síntesis química , Diterpenos/síntesis química , Ciclización , Estructura MolecularRESUMEN
As a result of our efforts to discover novel p53:MDM2 protein-protein interaction inhibitors useful for treating cancer, the potent and selective MDM2 inhibitor NVP-CGM097 (1) with an excellent in vivo profile was selected as a clinical candidate and is currently in phase 1 clinical development. This article provides an overview of the discovery of this new clinical p53:MDM2 inhibitor. The following aspects are addressed: mechanism of action, scientific rationale, binding mode, medicinal chemistry, pharmacokinetic and pharmacodynamic properties, and in vivo pharmacology/toxicology in preclinical species.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Isoquinolinas/síntesis química , Isoquinolinas/farmacología , Piperazinas/síntesis química , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Ensayos Clínicos Fase I como Asunto , Descubrimiento de Drogas , Humanos , Isoquinolinas/farmacocinética , Piperazinas/farmacocinética , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The daphnane diterpene orthoesters constitute a structurally fascinating family of natural products that exhibit a remarkable range of potent biological activities. Although partial activity information is available for some natural daphnanes, little information exists for non-natural congeners or on how changes in structure affect mode of action, function, potency or selectivity. A gateway strategy designed to provide general synthetic access to natural and non-natural daphnanes is described and utilized in the synthesis of two novel members of this class. In this study, a commercially available tartrate derivative was elaborated through a key late-stage diversification intermediate into B-ring yuanhuapin analogues to initiate exploration of the structure-function relationships of this class. Protein kinase C was identified as a cellular target for these agents, and their activity against human lung and leukaemia cell lines was evaluated. The natural product and a novel non-natural analogue exhibited significant potency, but the epimeric epoxide was essentially inactive.
Asunto(s)
División Celular , Diterpenos/síntesis química , Proteína Quinasa C/metabolismo , Diterpenos/metabolismoRESUMEN
A new enantioselective synthesis of the idolizidine alkaloid (-)-swainsonine 1 in 40% overall yield starting from the known oxazolidinone 6 is described. Throughout the synthesis, the high efficiency of metal-catalyzed reactions is illustrated. The key step is a new ruthenium-catalyzed metathesis rearrangement reaction. In this ring-closing/ring-opening tandem process, stereocenters are transferred from a ring to the olefinic side chain of the formed heterocycle. The metathesis precursor was obtained by palladium-catalyzed desymmetrization of cyclopentenediol. The synthesis was completed by functionalization of the terminal double bond, cyclization of the second ring, and diastereoselective dihydroxylation.