RESUMEN
Targeted immune agonist (TIA) comprising a TLR7 agonist conjugated to tumor-targeting antibodies have been shown to induce potent anti-tumor responses in various preclinical models. However, the clinical proof-of-concept of a TIA has been hampered by systemic dose-limiting immune-related toxicities, including rapid induction of anti-drug antibodies in patients. We have developed ELISPOT-based assay to measure activation of antibody-secreting cells (ASCs), intended to simulate the interaction between TIA and peripheral B cells as a tool to pre-clinically de-risk tumor target-independent peripheral B-cell activation by TIA. This method has proven to be robust and has fast turn-around time to evaluate the induction of spontaneous B-cell activation by TIA in a tumor target- and FcγR-independent manner. This novel ASC assay platform may serve as a preclinical tool to de-risk TIAs that can potentially induce immune-related adverse effects in the clinic.
Asunto(s)
Neoplasias , Receptor Toll-Like 7 , Humanos , Adyuvantes Inmunológicos , Anticuerpos , Activación de LinfocitosRESUMEN
Peggy Sue is a capillary-based western/immunoassay platform that can separate and characterize proteins by size or charge. A quick and automated immunogenicity assay was developed on Peggy Sue based on charge separation and compared with a traditional bridging method using preclinical samples from non-human primate studies. The results generated with the Peggy Sue assay were comparable to those of the bridging assays. The Peggy Sue platform has several advantages, including time efficiency, low sample consumption, and easy automation. The platform is especially ideal for further characterization of anti-drug antibody (ADA) specificity against complex biologics such as bispecific or multi-specific biotherapeutics as it is easy to conduct domain specificity assessment of observed ADA responses. Our evaluation suggests that the Peggy Sue platform is a promising tool for preclinical ADA analysis.
Asunto(s)
Anticuerpos/aislamiento & purificación , Productos Biológicos/uso terapéutico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Inmunoensayo/métodos , Anticuerpos de Dominio Único/aislamiento & purificación , Animales , Automatización , Camélidos del Nuevo Mundo , Electroforesis Capilar , Humanos , Macaca mulattaRESUMEN
PURPOSE: Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 (89Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells. PROCEDURES: Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis. RESULTS: 89Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation. CONCLUSIONS: 89Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02760225.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Imagen Molecular/métodos , Neoplasias/diagnóstico por imagen , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/farmacocinética , Femenino , Inmunoterapia/métodos , Macaca fascicularis , Masculino , Modelos Animales , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Radioisótopos , Distribución Tisular , CirconioRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2020.573405.].
RESUMEN
The molecule "T cell immunoreceptor with immunoglobulin and ITIM domain," or TIGIT, has recently received much attention as a promising target in the treatment of various malignancies. In spite of the quick progression of anti-TIGIT antibodies into clinical testing both as monotherapy and in combination with programmed cell death-1 (PD-1)-directed immune checkpoint blockade, the molecular mechanism behind the observed therapeutic benefits remains poorly understood. Here we demonstrate, using mouse tumor models, that TIGIT blocking antibodies with functional Fc binding potential induce effective anti-tumor response. Our observations reveal that the anti-TIGIT therapeutic effect is not achieved by depletion of intratumoral regulatory T cells (Treg) or any cell population expressing TIGIT, but instead is mediated by possible "reverse activating signals" through FcγRs on myeloid cells, inducing expression of various mediators such as cytokines and chemokines. Furthermore, we discovered an induction of a robust and persistent granzyme B and perforin response, distinct from a predominantly interferon-γ (IFN-γ)-driven anti-PD-1 blockade. Our observations, for the first time, provide the basis for a mechanistic hypothesis involving the requirement of a functional Fc domain of anti-TIGIT monoclonal antibodies, of which various isotypes are currently under intense clinical investigation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/farmacología , Células Mieloides/efectos de los fármacos , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Granzimas/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Microambiente TumoralRESUMEN
Assessment of the factors that regulate antibody exposure-response relationships in the relevant animal models is critical for the design of successful translational strategies from discovery to the clinic. Depending on the specific clinical indication, preclinical development paradigms may require that the efficacy or dosing-related attributes for the existing antibody be assessed in various species when cross-reactivity of the lead antibody to the intended species is justified. Additionally, with the success of monoclonal antibodies for management of various human conditions, a parallel interest in therapeutic use of these novel modalities in various veterinary species has followed. The protective role of neonatal Fc receptor (FcRn) in regulation of IgG homeostasis and clearance is now well recognized and the "nonspecific clearance" of antibodies through bone marrow-derived phagocytic and vascular endothelial cells (via lysosomal processes) is modulated by interactions with FcRn receptors. In this study, we have attempted to examine the PK properties of human IgG antibodies in dog and monkey. These studies establish a translational framework for evaluation of IgG antibody PK properties across species.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Inmunoglobulina G/farmacología , Administración Intravenosa , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Macaca fascicularis , Macaca mulatta , Ratones , Modelos Animales , Virus Sincitiales Respiratorios/inmunología , Especificidad de la EspecieRESUMEN
LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.
Asunto(s)
Anticuerpos Bloqueadores/análisis , Antígenos CD/inmunología , Ingeniería Celular , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/inmunología , Anticuerpos Bloqueadores/inmunología , Antígenos CD/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Células Jurkat , Receptor de Muerte Celular Programada 1/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Linfocitos T/inmunología , Células Tumorales Cultivadas , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.
Asunto(s)
Anticuerpos Monoclonales/sangre , Bioensayo/métodos , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/química , Macaca mulatta , Proteolisis , Tripsina/químicaRESUMEN
Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by â¼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diseño de Fármacos , Hipoglucemiantes/uso terapéutico , Insulina Regular Humana/análogos & derivados , Lectinas Tipo C/agonistas , Lectinas de Unión a Manosa/agonistas , Receptor de Insulina/agonistas , Receptores de Superficie Celular/agonistas , Animales , Animales Endogámicos , Unión Competitiva , Células CHO , Cricetulus , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Hiperglucemia/prevención & control , Hipoglucemia/inducido químicamente , Hipoglucemia/prevención & control , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/farmacocinética , Insulina Regular Humana/efectos adversos , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/uso terapéutico , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Tasa de Depuración Metabólica , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Porcinos , Porcinos EnanosRESUMEN
Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acute-phase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1ß-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1ß exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC50 values for IL-1ß-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1ß interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1ß antibody. Unlike IL-1ß, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.
Asunto(s)
Proteínas Portadoras/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Interleucinas/metabolismo , Macrófagos del Hígado/metabolismo , Células 3T3 , Adulto , Animales , Transporte Biológico/fisiología , Proteína C-Reactiva/metabolismo , Línea Celular , Técnicas de Cocultivo/métodos , Citocromo P-450 CYP3A/metabolismo , Regulación hacia Abajo/fisiología , Glicoproteínas/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteína Amiloide A Sérica/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antitoxinas/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Clostridioides difficile/inmunología , Enterocolitis Seudomembranosa/prevención & control , Enterotoxinas/inmunología , Animales , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Antitoxinas/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/inmunología , Ratones , Ratones Endogámicos C57BL , Mutación , Receptores de IgG/inmunología , Recurrencia , Células VeroRESUMEN
Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy, and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Análisis Químico de la Sangre/instrumentación , Péptidos/análisis , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/sangre , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Semivida , Inmunoglobulina G/metabolismo , Inmunoprecipitación , Marcaje Isotópico , Macaca fascicularis , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.
Asunto(s)
Desoxicitidina/análogos & derivados , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Línea Celular Tumoral , Desoxicitidina/química , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ribonucleósido Difosfato Reductasa , Relación Estructura-Actividad , GemcitabinaRESUMEN
Drug design efforts in the emerging 2-aminothiazole-4-carboxamide class of CHK1 inhibitors have uncovered specific combinations of key substructures within the molecule; resulting in significant improvements in cell-based activity while retaining a greater than one hundred-fold selectivity against CDK2. The X-ray crystal structure of a complex between compound 39 and the CHK1 protein detailing a 'U-shaped' topology and key interactions with the protein surface at the ATP site is also reported.
Asunto(s)
Amidas/química , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Tiazoles/química , Amidas/síntesis química , Amidas/metabolismo , Sitios de Unión , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.
Asunto(s)
Interferón gamma/metabolismo , Interleucina-10/metabolismo , Neoplasias Experimentales/inmunología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Granzimas/metabolismo , Humanos , Interferón gamma/genética , Interleucina-10/genética , Interleucina-10/inmunología , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Perforina/metabolismo , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Bazo/metabolismo , Trasplante Heterólogo , Carga Tumoral , Escape del TumorRESUMEN
Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Óxidos N-Cíclicos , Quinasas Ciclina-Dependientes/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Histonas/metabolismo , Humanos , Immunoblotting , Indolizinas , Ratones , Ratones Desnudos , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirazoles/química , Compuestos de Piridinio/administración & dosificación , Compuestos de Piridinio/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/química , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Pirazoles/química , Pirimidinas/química , Sitios de Unión , Dominio Catalítico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
The synthesis and hit-to-lead SAR development of a pyrazolo[1,5-a]pyrimidine hit 4 is described leading to a series of potent, selective CHK1 inhibitors such as compound 17r. In the Letter, the further utility of the pyrazolo[1,5-a]pyrimidine template for the development of potent, selective kinase inhibitors is detailed.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Pirazoles/química , Pirimidinas/química , Sitios de Unión , Dominio Catalítico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
A novel series of CHK1 inhibitors based on thienopyridine template has been designed and synthesized. These inhibitors maintain critical hydrogen bonding with the hinge and conserved water in the ATP binding site. Several compounds show single digit nanomolar CHK1 activities. Compound 70 shows excellent enzymatic activity of 1 nM.