RESUMEN
Mulberry extract from Fructus Mori contains an anthocyanin pigment and has been widely used as a food additive in China and other Eastern Asian countries. Only few research has been done on toxicological profiling of mulberry extract for its safety evaluation; however, the data is inconclusive. In the current study, mulberry extract of 4200, 1400, or 466 mg/kg were orally administrated to Sprague Dawley rats for 90 consecutive days followed by a recovery period of 28 days. No abnormalities were detected in body weights, food intake, ophthalmological, hematological, coagulation, clinical chemistry, and organ weights parameters. Discoloration of urine (red, purple, and brown) and feces (black), along with bedding material (purple) were observed in the 4200 mg/kg group. Further, microscopic examination revealed brown granules in the renal tubular cells for rats in 4200 and 1400 mg/kg groups. Since these changes were associated with excretory effect of the extract, the No Observed Adverse Effect Level was determined to be 4200 mg/kg, which was equivalent to the 1058.5 mg/kg of anthocyanin.
Asunto(s)
Morus , Animales , Nivel sin Efectos Adversos Observados , Extractos Vegetales/toxicidad , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad AgudaRESUMEN
Previous studies have shown that conflict monitoring is integrated with negative emotions. However, the idea that conflict resolution facilitates positive stimuli processing has not reached a consistent conclusion. We suggested that conflict resolution was integrated with positive emotions. The present study used ERPs, took the flanker task as primes, set different durations (i.e., 600 ms and 1200 ms) between the resolution of conflicts and the presentation of targets, and chose affective words as targets to investigate the affective effect of cognitive conflict during the resolution stage. Participants' task was to react to the flanker task and then evaluate the valence of the target words. The findings of experiment1 (600 ms) and experiment2 (1200 ms) were consistent. Behavioral results showed that the conflict effect was significant, and the positive signal effect of conflict resolution was found. In ERPs results, the enhanced N2 amplitudes for incongruent primes showed a significant conflict effect. The enhanced conflict SP amplitudes for incongruent primes reflected conflict resolution. As expected, the enhanced N400 amplitudes for positive targets after incongruent primes indicated that conflict resolution facilitated positive stimuli processing. Time-frequency analyses showed that incongruent primes elicited larger theta (4-8 Hz) power than congruent primes over the frontal areas. More importantly, we found that theta (4-8 Hz) power for positive targets after incongruent primes was lower than those after congruent primes over the central areas. These findings suggested that conflict resolution facilitated positive stimulus processing, and this positive effect was a carry-over effect, which indicated that conflict resolution was integrated with positive emotions.
Asunto(s)
Electroencefalografía , Potenciales Evocados , Conflicto Psicológico , Emociones , Femenino , Humanos , Masculino , Negociación , Tiempo de ReacciónRESUMEN
RUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET-proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network-based Cellular Signatures 1000-connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1.
Asunto(s)
Antineoplásicos/farmacología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Leucemia Mieloide Aguda/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Técnicas de Silenciamiento del Gen , Mutación de Línea Germinal , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , RatonesRESUMEN
Anti-leukemic effect of BET/BRD4 (BETP) protein inhibition has been largely attributed to transcriptional downregulation of cellular anabolic/anti-apoptotic processes but its effect on bone marrow microenvironment, a sanctuary favoring persistence of leukemia stem/progenitor cells, is unexplored. Sustained degradation of BETP with small-molecule BET proteolysis-targeting chimera (PROTAC), ARV-825, resulted in marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interaction, in AML cells. Abrogation of surface CXCR4 expression impaired SDF-1α directed migration and was mediated through transcriptional down-regulation of PIM1 kinase that in turn phosphorylates CXCR4 and facilitates its surface localization. Down-regulation of CD44/CD44v8-10 impaired cystine uptake, lowered intracellular reduced glutathione and increased oxidative stress. More importantly, BETP degradation markedly decreased CD34+CD38-CD90-CD45RA+ leukemic stem cell population and alone or in combination with Cytarabine, prolonged survival in mouse model of human leukemia including AML-PDX. Gene expression profiling and single cell proteomics confirmed down regulation of the gene signatures associated with 'stemness' in AML and Wnt/ß-catenin, Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell intrinsic signaling, stromal interactions and metabolism in AML.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/antagonistas & inhibidores , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Azepinas/farmacología , Médula Ósea/metabolismo , Línea Celular Tumoral , Movimiento Celular , Quimiocina CXCL12/metabolismo , Cisteína/química , Perfilación de la Expresión Génica , Glutatión/química , Células HL-60 , Humanos , Receptores de Hialuranos/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Trasplante de Neoplasias , Estrés Oxidativo , Fosforilación , Receptores CXCR4/metabolismo , Células THP-1 , Talidomida/análogos & derivados , Talidomida/farmacología , Antígenos Thy-1/metabolismo , Células U937RESUMEN
Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear ß-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of ß-catenin or treatment with BC2059 that disrupts binding of ß-catenin to TBL1X (TBL1) depleted nuclear ß-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of ß-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of ß-catenin and BETP antagonists against post-MPN sAML BPCs.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Sinergismo Farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , beta Catenina/antagonistas & inhibidores , Acetanilidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Núcleo Celular/patología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Leucemia Mieloide Aguda/complicaciones , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trastornos Mieloproliferativos/complicaciones , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Nitrilos , Pirazoles/farmacología , Pirimidinas , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Although the number of proteins effectively targeted for posttranslational degradation by PROTAC has grown steadily, the number of E3 ligases successfully exploited to accomplish this has been limited to the few for which small-molecule ligands have been discovered. Although the E3 ligase MDM2 is bound by the nutlin class of small-molecule ligands, there are few nutlin-based PROTAC. Because a nutlin-based PROTAC should both knockdown its target protein and upregulate the tumor suppressor p53, we examined the ability of such a PROTAC to decrease cancer cell viability. A nutlin-based, BRD4-degrading PROTAC, A1874, was able to degrade its target protein by 98% with nanomolar potency. Given the complementary ability of A1874 to stabilize p53, we discovered that the nutlin-based PROTAC was more effective in inhibiting proliferation of many cancer cell lines with wild-type p53 than was a corresponding VHL-utilizing PROTAC with similar potency and efficacy to degrade BRD4. This is the first report of a PROTAC in which the E3 ligase ligand and targeting warhead combine to exert a synergistic antiproliferative effect. Our study highlights the untapped potential that may be unlocked by expanding the repertoire of E3 ligases that can be recruited by PROTAC. SIGNIFICANCE: These findings present the first BRD4-targeting MDM2-based PROTAC that possesses potent, distinct, and synergistic biological activities associated with both ends of this heterobifunctional molecule.
Asunto(s)
Proliferación Celular , Sinergismo Farmacológico , Neoplasias , Proteínas Nucleares , Péptidos , Proteínas Proto-Oncogénicas c-mdm2 , Factores de Transcripción , Proteína p53 Supresora de Tumor , Humanos , Proteínas de Ciclo Celular , Fibrinolíticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Péptidos/farmacología , Estabilidad Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.
Asunto(s)
Antineoplásicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Proteínas/metabolismo , Secuencias de Aminoácidos/efectos de los fármacos , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Proteínas Nucleares/metabolismo , Dominios Proteicos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis/efectos de los fármacos , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Polarización de Fluorescencia , Genes ras , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Estructura Molecular , Mutación , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Relación Estructura-Actividad , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.
Asunto(s)
Inhibidores Enzimáticos , Proteolisis , Proteínas Tirosina Quinasas Receptoras , Ubiquitina-Proteína Ligasas , Humanos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ligandos , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.
Asunto(s)
Antineoplásicos/administración & dosificación , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Humanos , Masculino , Ratones , Proteínas Nucleares/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Proteínas de Unión al ARN/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt's lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest antiproliferative activity, and lack of apoptotic induction. To address these limitations we designed ARV-825, a hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small-molecule BRD4 inhibitors, resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small-molecule inhibitors.
Asunto(s)
Azepinas/farmacología , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/metabolismo , Transducción de Señal/efectos de los fármacos , Talidomida/análogos & derivados , Factores de Transcripción/metabolismo , Acetanilidas/toxicidad , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/efectos de los fármacos , Azepinas/química , Azepinas/toxicidad , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/toxicidad , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Talidomida/química , Talidomida/farmacología , Factores de Transcripción/antagonistas & inhibidores , Triazoles/toxicidad , Ubiquitina-Proteína LigasasRESUMEN
High throughput screening (HTS) of our chemical library identified 3-alkylamino-2-aryl-5H-imidazo[1,2,b]pyrazol-7-carbonitrile 1 as a potent antagonist of the LPA1 receptor (LPA1R). Further evaluation of this class of compounds indicated that LPA1R antagonist activity originated from the degradation of the parent molecule in DMSO during the assay conditions. Here, we describe the isolation and characterization of the degradation products and their LPA1R antagonist activity. We further profiled these novel non-carboxylic acid LPA1R antagonists and demonstrated their inhibition of LPA-induced proliferation and contraction of normal human lung fibroblasts (NHLF).
Asunto(s)
Descubrimiento de Drogas , Pirazoles/farmacología , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Pulmón/citología , Lisofosfolípidos/antagonistas & inhibidores , Lisofosfolípidos/farmacología , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Receptores del Ácido Lisofosfatídico/metabolismo , Relación Estructura-ActividadRESUMEN
The beneficial effects of thyroid hormone (TH) on lipid levels are primarily due to its action at the thyroid hormone receptor ß (THR-ß) in the liver, while adverse effects, including cardiac effects, are mediated by thyroid hormone receptor α (THR-α). A pyridazinone series has been identified that is significantly more THR-ß selective than earlier analogues. Optimization of this series by the addition of a cyanoazauracil substituent improved both the potency and selectivity and led to MGL-3196 (53), which is 28-fold selective for THR-ß over THR-α in a functional assay. Compound 53 showed outstanding safety in a rat heart model and was efficacious in a preclinical model at doses that showed no impact on the central thyroid axis. In reported studies in healthy volunteers, 53 exhibited an excellent safety profile and decreased LDL cholesterol (LDL-C) and triglycerides (TG) at once daily oral doses of 50 mg or higher given for 2 weeks.
Asunto(s)
Descubrimiento de Drogas , Dislipidemias/tratamiento farmacológico , Piridazinas/síntesis química , Receptores beta de Hormona Tiroidea/agonistas , Uracilo/análogos & derivados , Animales , Densidad Ósea/efectos de los fármacos , Ensayos Clínicos como Asunto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Piridazinas/metabolismo , Piridazinas/farmacología , Piridazinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Uracilo/síntesis química , Uracilo/metabolismo , Uracilo/farmacología , Uracilo/uso terapéuticoRESUMEN
Lysophosphatidic acid (LPA) is a class of bioactive phospholipid that displays a wide range of cellular effects via LPA receptors, of which six have been identified (LPAR1-6). In serum and plasma, LPA production occurs mainly by the hydrolysis of lysophosphatidylcholine by the phospholipase D activity of autotaxin (ATX). The involvement of the LPA pathway in driving chronic wound-healing conditions, such as idiopathic pulmonary fibrosis, has suggested targets in this pathway could provide potential therapeutic approaches. Mice with LPAR1 knockout or tissue-specific ATX deletion have demonstrated reduced lung fibrosis following bleomycin challenge. Therefore, strategies aimed at antagonizing LPA receptors or inhibiting ATX have gained considerable attention. This Review will summarize the current status of identifying small-molecule modulators of the LPA pathway. The therapeutic utility of LPA modulators for the treatment of fibrotic diseases will soon be revealed as clinical trials are already in progress in this area.
Asunto(s)
Lisofosfolípidos/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Ácidos Fosforosos/química , Ácidos Fosforosos/uso terapéutico , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Benzimidazole and indane are the two key fragments in our potent and selective MCH-1 receptor (MCHR1) antagonists. To identify novel linkers connecting the two fragments, we investigated diamino-cycloalkane-derived analogs and discovered highly potent antagonists with cis-1,4-diaminocyclohexane as a unique spacer in this chemical class. Structural overlay suggested that cis-1-substituted-4-aminocyclohexane functions as a bioisostere of 4-substituted-piperidine and that the active conformation adopts a U-shaped orientation.
Asunto(s)
Ciclohexanos/química , Indanos/química , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Animales , Bencimidazoles/química , Semivida , Indanos/metabolismo , Indanos/farmacocinética , Isomerismo , Ratones , Unión Proteica , Ratas , Receptores de la Hormona Hipofisaria/metabolismoRESUMEN
Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen polymer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen levels and decreased muscle glucose uptake is the major defect in whole body glucose disposal. Therefore, activating GS may provide a potential approach for the treatment of type 2 diabetes. In order to identify non-carboxylic acids GS activators, we designed and synthesized a series of 2-N-alkyl- and 2-N-aryl-indazolone derivatives and studied their activity in activating human GS.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa/antagonistas & inhibidores , Indazoles/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/síntesis química , Glucógeno Sintasa/metabolismo , Indazoles/administración & dosificación , Indazoles/síntesis química , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Relación Estructura-ActividadRESUMEN
To resolve the metabolite redox cycling associated with our earlier clinical compound 2, we carried out lead optimization of lead molecule 1. Compound 4 showed improved lipophilic ligand efficiency and demonstrated robust glucose lowering in diet-induced obese mice without a liability in predictive preclinical drug safety studies. Thus, it was selected as a clinical candidate and further studied in type 2 diabetic patients. Clinical data suggests no evidence of metabolite cycling, which is consistent with the preclinical profiling of metabolism.
RESUMEN
Lysophosphatidic acid (LPA) is a class of bioactive lyso-phospholipid that mediates most of its biological effects through a family of G protein-coupled receptors of which six have been identified. The role of the LPA pathway in driving chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) has gained considerable academic and industry attention. Modulation of the pulmonary artery endothelial barrier function by the LPA1 receptor has been shown to drive pulmonary fibrosis in murine models of disease. The purpose of this study was (i) to assess the effect of LPA on the barrier function of human pulmonary arterial (HPAEC) and microvascular (HMVEC) endothelial cells and (ii) to identify the LPA receptor subtype(s) responsible for changes in human pulmonary endothelial cell permeability using LPA receptor antagonists and siRNA technology. Analysis of the LPA receptor subtype expression demonstrated predominant expression of LPA2 and LPA6 receptor subtypes in both HPAECs and HMVECs. HPAECs also exhibit low expression of LPA1, LPA3, and LPA4 receptor subtypes. Treatment of cells with increasing concentrations of LPA caused loss of barrier function in HPAECs but not HMVECs, despite both cell types exhibiting very similar LPA receptor expression profiles. The LPA-mediated loss of barrier function in HPAECs appears to be independent of the LPA1 receptor and likely to be mediated via the LPA6 receptor although we cannot exclude an additional role for the LPA2 and LPA4 receptors in mediating these effects. These results suggest cell-specific mechanisms exist in human pulmonary endothelial cells to permit regulation of barrier function downstream of LPA receptors. More importantly, our data indicate that selective LPA1 receptor antagonism may be insufficient for therapeutic use in pulmonary diseases where impaired endothelial barrier function is related to disease initiation and progression.
Asunto(s)
Células Endoteliales/citología , Pulmón/citología , Lisofosfolípidos/metabolismo , Arterias/patología , Calcio/química , Cartilla de ADN/genética , Relación Dosis-Respuesta a Droga , Endotelio/citología , Humanos , Fibrosis Pulmonar Idiopática/patología , Microcirculación , Microscopía Fluorescente/métodos , Permeabilidad , Reacción en Cadena de la Polimerasa/métodos , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Factores de TiempoRESUMEN
Lysophosphatidic acid is a class of bioactive phospholipid that mediates most of its biological effects through LPA receptors, of which six isoforms have been identified. The recent results from LPA1 knockout mice suggested that blocking LPA1 signaling could provide a potential novel approach for the treatment of idiopathic pulmonary fibrosis. Here, we report the design and synthesis of pyrazole- and triazole-derived carbamates as LPA1-selective and LPA1/3 dual antagonists. In particular, compound 2, the most selective LPA1 antagonist reported, inhibited proliferation and contraction of normal human lung fibroblasts (NHLF) following LPA stimulation. Oral dosing of compound 2 to mice resulted in a dose-dependent reduction of plasma histamine levels in a murine LPA challenge model. Furthermore, we applied our novel antagonists as chemistry probes and investigated the contribution of LPA1/2/3 in mediating the pro-fibrotic responses. Our results suggest LPA1 as the major receptor subtype mediating LPA-induced proliferation and contraction of NHLF.
Asunto(s)
Descubrimiento de Drogas , Pulmón/efectos de los fármacos , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Administración Oral , Animales , Fibroblastos/efectos de los fármacos , Humanos , Pulmón/citología , Espectroscopía de Resonancia Magnética , Ratones , Pirazoles/química , Pirazoles/farmacología , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
Through high throughput screening and subsequent hit identification and optimization, we synthesized a series of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as the first reported potent and reversible GFAT inhibitors. SAR studies of this class of compounds indicated significant impact on GFAT inhibition potency by substitutions on the A-ring and C-ring. The ketone group was found to be necessary for high potency. Compound 28 (RO0509347) demonstrated potent GFAT inhibition (IC(50)=1µM) with a desirable pharmacokinetic profile in rats, and showed significant efficacy in reducing the glucose excursion in an OGTT test in ob/ob mice.