RESUMO
Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.
Assuntos
Doenças Autoimunes , Janus Quinase 1 , Janus Quinase 3 , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Humanos , Isoenzimas , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Monitorização Imunológica/métodos , Inibidores de Proteínas Quinases/farmacologia , RatosRESUMO
8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Descoberta de Drogas , Imidazóis/farmacologia , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Artrite Reumatoide/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imidazóis/síntese química , Imidazóis/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Modelos Moleculares , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Regulador Transcricional ERG/antagonistas & inibidores , Regulador Transcricional ERG/metabolismoRESUMO
We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.
Assuntos
Ácidos Carboxílicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Humanos , RatosRESUMO
Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Descoberta de Drogas , Macaca mulatta , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyRESUMO
Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aß42 IC(50) in cell-based assays and reduced affinity for the hERG channel.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Transativadores/metabolismo , Triazóis/farmacologia , Amidas/química , Amidas/farmacologia , Peptídeos beta-Amiloides , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Lactamas , Relação Estrutura-Atividade , Regulador Transcricional ERG , Triazóis/químicaRESUMO
Synthesis, SAR, and evaluation of aryl triazoles as novel gamma secretase modulators (GSMs) are presented in this communication. Starting from the literature and in-house leads, we evaluated a range of five-membered heterocycles as replacements for olefins commonly found in non-acid GSMs. 1,2,3-C-aryl-triazoles were identified as suitable replacements which exhibited good modulation of γ-secretase activity, excellent pharmacokinetics and good central lowering of Aß42 in Sprague-Dawley rats.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Triazóis/síntese química , Triazóis/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Triazóis/metabolismoRESUMO
Fatty acid synthase (FAS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. To identify small-molecule inhibitors of FAS, the authors developed a bead-based scintillation proximity assay (SPA) to detect the fatty acid products of FAS enzymatic activity. This homogeneous SPA assay discriminates between a radiolabeled hydrophilic substrate of FAS (acetyl-coenzyme A) and the labeled lipophilic products of FAS (fatty acids), generating signal only when labeled fatty acids are present. The assay requires a single addition of unmodified polystyrene imaging SPA beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. High-potency FAS inhibitors were used to compare the sensitivity of the SPA bead assay with previously described assays that measure FAS reaction intermediates (CoA-SH and NADP+). The advantages and disadvantages of these different FAS assays in small-molecule inhibitor discovery are discussed.
Assuntos
Bioensaio/métodos , Ácido Graxo Sintases/metabolismo , Acetilcoenzima A/metabolismo , Linhagem Celular , Ácido Graxo Sintases/antagonistas & inibidores , Humanos , NADP/metabolismo , Palmitatos/metabolismo , Transdução de Sinais , Fatores de Tempo , TitulometriaRESUMO
c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. Using AlphaScreen, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z' statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen assay allows its adaptation to measure cellular activity of other kinases.
Assuntos
Bioensaio/métodos , Imageamento Tridimensional/métodos , Proteínas Proto-Oncogênicas c-met/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Humanos , Indicadores e Reagentes , Fosforilação , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Reprodutibilidade dos TestesRESUMO
PURPOSE: For many tumor cells, de novo lipogenesis is a requirement for growth and survival. A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. EXPERIMENTAL DESIGN: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. RESULTS: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1 and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. CONCLUSIONS: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.
Assuntos
Acetiltransferases/metabolismo , Neoplasias do Colo/enzimologia , Ácido Graxo Sintases/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Lipogênese , Transdução de Sinais , TransfecçãoRESUMO
The ubiquitin-proteasome pathway has a well-defined beginning and end. Target proteins are initially recognized by upstream components and tagged with polyubiquitin chains. The 26S proteasome then degrades these polyubiquitinated proteins. Until recently, it was not known what, if any, steps occurred between the initial polyubiquitination of target proteins and their final degradation. Several new papers investigating the function of the Cdc48-Ufd1-Npl4 complex indicate that there is indeed a middle to the ubiquitin-proteasome pathway. The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing. The elucidation of Cdc48, Ufd1 and Npl4 action not only provides long-sought functions for these specific proteins, but illuminates a poorly understood part of the ubiquitin-proteasome pathway.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases , Proteínas de Ciclo Celular/genética , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Mutação , Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Proteína com ValosinaRESUMO
The discovery of a potent selective low dose Janus kinase 1 (JAK1) inhibitor suitable for clinical evaluation is described. As part of an overall goal to minimize dose, we pursued a medicinal chemistry strategy focused on optimization of key parameters that influence dose size, including lowering human Clint and increasing intrinsic potency, bioavailability, and solubility. To impact these multiple parameters simultaneously, we used lipophilic ligand efficiency as a key metric to track changes in the physicochemical properties of our analogs, which led to improvements in overall compound quality. In parallel, structural information guided advancements in JAK1 selectivity by informing on new vector space, which enabled the discovery of a unique key amino acid difference between JAK1 (Glu966) and JAK2 (Asp939). This difference was exploited to consistently produce analogs with the best balance of JAK1 selectivity, efficacy, and projected human dose, ultimately culminating in the discovery of compound 28.
Assuntos
Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Animais , Cães , Descoberta de Drogas , Halogenação , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.
RESUMO
Recent studies have revealed that rabbit reticulocyte lysate (RRL) efficiently reconstitutes endoplasmic reticulum-associated degradation (ERAD) of mutant and misfolded membrane proteins. When supplemented with canine pancreas microsomal membranes, the RRL system faithfully carries out ER targeting, translocation, glycosylation, and membrane integration events and therefore provides a ready source of 35S-labeled protein with defined transmembrane topology. These substrates can be rapidly isolated in native ER membranes which, when incubated in RRL lacking exogenous hemin, are degraded in an ATP-dependent manner by the ubiquitin-proteasome pathway. Because the newly translated protein is the only source of radiolabel, degradation can be followed to its end state by conversion into trichloroacetic acid (TCA)-soluble peptide fragments. A particularly useful aspect of this system is that both membrane-associated and cytosolic components are amenable to biochemical and pharmacological manipulation. Here we describe techniques for preparing translation- and degradation-competent RRL, affinity depletion, identification of cytosolic factors involved in degrading the cystic fibrosis transmembrane conductance regulator (CFTR), and reconstitution of ERAD by add-back of purified recombinant proteins. These techniques provide a powerful tool for dissecting components involved in ubiquitination, degradation, and in particular, extraction of transmembrane ERAD substrates.
Assuntos
Sistema Livre de Células/enzimologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/enzimologia , Microssomos/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Reticulócitos/enzimologia , Animais , Cães , CoelhosRESUMO
Yeast biology has yielded major insights into fundamental cellular biology and has served as a remarkable platform for technical innovation. We review how these resources can be applied to the validation of mammalian or anti-fungal drug targets. These approaches range from elucidating synergistic interactions between drugs and targets to facile methods for tracking proteins in the cell or characterization of receptor biology. We also discuss web-based resources that integrate the extensive biochemical, cell biological, and genetic literature exploring the basic biology of these model eukaryotic cells.:
RESUMO
A series of 3-aryl-4-hydroxyquinolin-2(1H)-ones with fatty acid synthase inhibitory activity was prepared. Starting from a derivative with an IC(50) = 1.4 microM, SAR studies led to compounds with more than 70-fold increase in potency (IC(50) < 20 nM).