RESUMEN
The human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R), a class B G-protein-coupled receptor (GPCR) identified almost 30 years ago, represents an important pharmacological target in the areas of neuroscience, oncology, and immunology. Despite interest in this target, only a very limited number of small molecule modulators have been reported for this receptor. We herein describe the results of a drug discovery program aiming for the identification of a potent and selective hPAC1-R antagonist. An initial high-throughput screening (HTS) screen of 3.05 million compounds originating from the Bayer screening library failed to identify any tractable hits. A second, completely revised screen using native human embryonic kidney (HEK)293 cells yielded a small number of hits exhibiting antagonistic properties (4.2 million compounds screened). BAY 2686013 (1) emerged as a promising compound showing selective antagonistic activity in the submicromolar potency range. In-depth characterization supported the hypothesis that BAY 2686013 blocks receptor activity in a noncompetitive manner. Preclinical, pharmacokinetic profiling indicates that BAY 2686013 is a valuable tool compound for better understanding the signaling and function of hPAC1-R. SIGNIFICANCE STATEMENT: Although the human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R) is of major significance as a therapeutic target with a well documented role in pain signaling, only a very limited number of small-molecule (SMOL) compounds are known to modulate its activity. We identified and thoroughly characterized a novel, potent, and selective SMOL antagonist of hPAC1-R (acting in an allosteric manner). These characteristics make BAY 2686013 an ideal tool for further studies.
Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Humanos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/químicaRESUMEN
Here we report on novel and potent pyridyl-cycloalkyl-carboxylic acid inhibitors of microsomal prostaglandin E synthase-1 (PTGES). PTGES produces, as part of the prostaglandin pathway, prostaglandin E2 which is a well-known driver for pain and inflammation. This fact together with the observed upregulation of PTGES during inflammation suggests that blockade of the enzyme might provide a beneficial treatment option for inflammation related conditions such as endometriosis. Compound 5a, a close analogue of the screening hit, potently inhibited PTGES in vitro, displayed excellent PK properties in vitro and in vivo and demonstrated efficacy in a CFA-induced pain model in mice and in a rat dyspareunia endometriosis model and was therefore selected for further studies.
Asunto(s)
Ácidos Carboxílicos/farmacología , Descubrimiento de Drogas , Endometriosis/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Prostaglandina-E Sintasas/antagonistas & inhibidores , Animales , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endometriosis/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Femenino , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Dolor/tratamiento farmacológico , Dolor/metabolismo , Prostaglandina-E Sintasas/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions. METHODS: The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models. RESULTS: AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice. CONCLUSIONS: These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Dioxigenasas , Neoplasias de Cabeza y Cuello , Neoplasias Pulmonares , Humanos , Ratones , Animales , Triptófano , Receptores de Hidrocarburo de Aril/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Leucocitos Mononucleares/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Quinurenina/metabolismo , Inmunoterapia , Factores Inmunológicos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Microambiente TumoralRESUMEN
PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Naftiridinas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up-regulated in response to physiological concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone-responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone-regulated sodium retention in the kidney. CCD cell lines stably overexpressing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC-mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.
Asunto(s)
Canales Epiteliales de Sodio/genética , Proteínas de la Membrana/genética , Receptores de Mineralocorticoides/fisiología , Aldosterona/farmacología , Animales , Línea Celular , Células Cultivadas , Canales Epiteliales de Sodio/efectos de los fármacos , Humanos , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/fisiología , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , Ratones , Regiones Promotoras Genéticas/fisiologíaRESUMEN
The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKε are noncanonical members of the inhibitor of the nuclear factor κB (IκB) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation. Herein, we describe our investigations on a family of benzimidazoles and the identification of the potent and highly selective TBK1/IKKε inhibitor BAY-985. BAY-985 inhibits the cellular phosphorylation of interferon regulatory factor 3 and displays antiproliferative efficacy in the melanoma cell line SK-MEL-2 but showed only weak antitumor activity in the SK-MEL-2 human melanoma xenograft model.
Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Fosforilación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identified Sum1 as an origin-binding protein that contributes to efficient replication initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1. RESULTS: In this study, we investigated how Sum1 affected replication initiation. We found that it functioned in initiation as a component of the Sum1/Rfm1/Hst1 complex, implying a role for histone deacetylation in origin activity. We identified several origins in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1Delta or hst1Delta caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was important for full initiation capacity of these origins. CONCLUSION: Taken together, our results demonstrate a role for histone deacetylation in origin activity and reveal a novel aspect of origin regulation by chromatin. These results suggest recruitment of the Sum1/Rfm1/Hst1 complex to a number of yeast origins, where Hst1 deacetylated H4 K5.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sirtuinas/genética , Acetilación , Animales , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Plásmidos/metabolismo , Proteínas Represoras , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuina 2 , Sirtuinas/metabolismoRESUMEN
MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Sistemas de Liberación de Medicamentos , Morfolinas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Células CACO-2 , Células Cultivadas , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Células HeLa , Hepatocitos/efectos de los fármacos , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Microsomas Hepáticos/efectos de los fármacos , Modelos Moleculares , Morfolinas/química , Neoplasias/fisiopatología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Pirimidinas/química , Pirimidinas/farmacología , RatasRESUMEN
Heterochromatinization at the silent mating-type loci HMR and HML in Saccharomyces cerevisiae is achieved by targeting the Sir complex to these regions via a set of anchor proteins that bind to the silencers. Here, we have identified a novel heterochromatin-targeting factor for HML, the protein Sum1, a repressor of meiotic genes during vegetative growth. Sum1 bound both in vitro and in vivo to HML via a functional element within the HML-E silencer, and sum1Delta caused HML derepression. Significantly, Sum1 was also required for origin activity of HML-E, demonstrating a role of Sum1 in replication initiation. In a genome-wide search for Sum1-regulated origins, we identified a set of autonomous replicative sequences (ARS elements) that bound both the origin recognition complex and Sum1. Full initiation activity of these origins required Sum1, and their origin activity was decreased upon removal of the Sum1-binding site. Thus, Sum1 constitutes a novel global regulator of replication initiation in yeast.