RESUMO
Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.
Assuntos
Autofagia/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Neurotoxinas , Doença de Parkinson/metabolismo , alfa-Sinucleína , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Química Encefálica , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Mitocôndrias , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidadeRESUMO
Because oxidative stress and mitochondrial dysfunction are well known contributors to Parkinson disease (PD), we set out to investigate the role mitochondrial JNK plays in the etiology of 6-hydroxydopamine-induced (6-OHDA) oxidative stress, mitochondrial dysfunction, and neurotoxicity in SHSY5Y cells and neuroprotection and motor behavioral protection in vivo. To do this, we utilized a cell-permeable peptide of the outer mitochondrial membrane protein, Sab (SH3BP5), as an inhibitor of JNK mitochondrial translocation. In vitro studies showed that 6-OHDA induced JNK translocation to the mitochondria and that inhibition of mitochondrial JNK signaling by Tat-Sab(KIM1) protected against 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and neurotoxicity. Administration of Tat-Sab(KIM1) via an intracerebral injection into the mid-forebrain bundle increased the number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta by 2-fold (p < 0.05) in animals lesioned with 6-OHDA, compared with animals treated only with 6-OHDA into the nigrostriatal pathway. In addition, Tat-Sab(KIM1) decreased the d-amphetamine-induced unilateral rotations associated with the lesion by 30% (p < 0.05). Steady-state brain levels of Tat-Sab(KIM1) at day 7 were 750 nm, which was â¼3.4-fold higher than the IC(50) for this peptide versus Sab protein. Collectively, these data suggest that 6-OHDA induced JNK translocation to the mitochondria and that blocking this translocation reduced oxidative stress, mitochondrial dysfunction, and neurotoxicity both in vitro and in vivo. Moreover, the data suggest that inhibitors that block association of JNKs with the mitochondria may be useful neuroprotective agents for the treatment of Parkinson disease.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/efeitos dos fármacos , Oxidopamina/toxicidade , Animais , Western Blotting , Morte Celular , Linhagem Celular , Dopamina/metabolismo , Inativação Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Mitocôndrias/metabolismo , Neurônios/citologia , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
To build upon recent findings that mitochondrial JNK signaling is inhibited by selectively blocking the interaction between JNK and Sab, we utilized a cell-permeable peptide to demonstrate that ischemia/reperfusion (I/R) injury could be protected in vivo and that JNK mitochondrial signaling was the mechanism by which reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cardiomyocyte cell death occur. We also demonstrated that 5 mg/kg SR-3306 (a selective JNK inhibitor) was able to protect against I/R injury, reducing infarct volume by 34% (p < 0.05) while also decreasing I/R-induced increases in the activity of creatine phosphokinase and creatine kinase-MB. TUNEL staining showed that the percent TUNEL positive nuclei in rat hearts increased 10-fold after I/R injury and that this was reduced 4-fold (p < 0.01) by SR-3306. These data suggest that blocking JNK mitochondrial translocation or JNK inhibition prevents ROS increases and mitochondrial dysfunction and may be an effective treatment for I/R-induced cardiomyocyte death.
Assuntos
MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Mitocôndrias Cardíacas/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Morte Celular , Linhagem Celular , Creatina Quinase/genética , Creatina Quinase/metabolismo , Creatina Quinase Forma MB/genética , Creatina Quinase Forma MB/metabolismo , Humanos , MAP Quinase Quinase 4/antagonistas & inibidores , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Proteínas Mitocondriais/genética , Proteínas Musculares/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
The design and synthesis of isoxazole 3 is described, a potent JNK inhibitor with two fold selectivity over p38. Optimization of this scaffold led to compounds 27 and 28 which showed greatly improved selectivity over p38 by maintaining the JNK3 potency of compound 3. Extensive SAR studies will be described as well as preliminary in vivo data of the two lead compounds.
Assuntos
Isoxazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Relação Dose-Resposta a Droga , Humanos , Isoxazóis/administração & dosagem , Isoxazóis/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Ratos , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
c-Jun N-terminal kinase (JNK) is a stress signal transducer linked to cell death, and survival. JNK1 has been implicated in obesity, glucose intolerance, and insulin resistance. In this study we report the kinetic mechanism for JNK1ß1 with transcription factors ATF2 and c-Jun along with interaction kinetics for these substrates. JNK1ß1 followed a random sequential mechanism forming a ternary complex between JNK-substrate-ATP. K(m) for ATF2 and c-Jun was 1.1 and 2.8 µM, respectively. Inhibition studies using adenosine 5'-(ß,γ-methylenetriphosphate) and a peptide derived from JNK interacting protein 1 (JIP1) supported the proposed kinetic mechanism. Biolayer interferometry studies showed that unphosphorylated JNK1ß1 bound to ATF2 with similar affinity as it did to c-Jun (K(D) = 2.60 ± 0.34 versus 1.00 ± 0.35 µM, respectively). The presence of ATP increased the affinity of unphosphorylated JNK1ß1 for ATF2 and c-Jun, to 0.80 ± 0.04 versus 0.65 ± 0.07 µM, respectively. Phosphorylation of JNK1ß1 decreased the affinity of the kinase for ATF2 to 11.0 ± 1.1 µM and for c-Jun to 17.0 ± 7.5 µM in the absence of ATP. The presence of ATP caused a shift in the K(D) of the active kinase for ATF2 to 1.70 ± 0.25 µM and for c-Jun of 3.50 ± 0.95 µM. These results are the first kinetic and biochemical characterization of JNK1ß1 and uncover some of the differences in the enzymatic activity of JNK1ß1 compared with other variants and suggest that ATP binding or JNK phosphorylation could induce changes in the interactions with substrates, activators, and regulatory proteins.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Processamento Alternativo/fisiologia , Morte Celular/fisiologia , Ativação Enzimática/fisiologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Cinética , Estresse Fisiológico/fisiologia , Especificidade por Substrato/fisiologiaRESUMO
Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2-associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45(-ve) /CD44(+ve) cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300-fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA-mediated knockdown of p53 in wild-type cells or exposure of p53(-/-) MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Oxigênio/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
SAR and lead optimization studies for Rock inhibitors based on amino acid-derived quinazolines are described. Studies demonstrated that these amino acid derived quinazolinones were mainly pan-Rock (I & II) inhibitors. While selectivity against other kinases could be achieved, selectivity for most of these compounds against PKA was not achieved. This is distinct from Rock inhibitors based on non-amino acid derived quinazolinones, where high selectivity against PKA could be obtained.(22) The inhibitors presented here in some cases possessed sub-nanomolar inhibition of Rock, nanomolar potency in ppMLC cell based assays, low to fair cytochrome P-450 inhibition, and good human microsomal stability.
Assuntos
Aminoácidos/química , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Quinazolinas/química , Quinases Associadas a rho/antagonistas & inibidores , Sítios de Ligação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Microssomos/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Quinazolinas/síntese química , Quinazolinas/metabolismo , Relação Estrutura-Atividade , Quinases Associadas a rho/metabolismoRESUMO
The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.
Assuntos
Fibroblastos/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Anisomicina/farmacologia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/citologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC(50)=40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/síntese química , Quinazolinas/farmacologia , Encéfalo/metabolismo , Concentração Inibidora 50 , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/química , Quinazolinas/farmacocinética , Relação Estrutura-AtividadeRESUMO
Therapeutic interventions with Rho kinase (ROCK) inhibitors may effectively treat several disorders such as hypertension, stroke, cancer, and glaucoma. Herein we disclose the optimization and biological evaluation of potent novel ROCK inhibitors based on substituted indole and 7-azaindole core scaffolds. Substitutions on the indole C3 position and on the indole NH and/or amide NH positions all yielded potent and selective ROCK inhibitors (25, 42, and 50). Improvement of aqueous solubility and tailoring of in vitro and in vivo DMPK properties could be achieved through these substitutions.
Assuntos
Descoberta de Drogas , Indóis/síntese química , Água/química , Quinases Associadas a rho/antagonistas & inibidores , Animais , Sítios de Ligação , Inibidores das Enzimas do Citocromo P-450 , Ativação Enzimática/efeitos dos fármacos , Humanos , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Ratos , SolubilidadeRESUMO
Rho kinase (ROCK) inhibitors are potential therapeutic agents to treat disorders such as hypertension, multiple sclerosis, cancers, and glaucoma. Here, we disclose the synthesis, optimization, biological evaluation of potent indole and 7-azaindole based ROCK inhibitors that have high potency on ROCK (IC(50)=1 nM) with 740-fold selectivity over PKA (47). Moreover, 47 showed very good DMPK properties making it a good candidate for further development. Finally, docking studies with a homology model of ROCK-II were performed to rationalize the binding mode of these compounds and showed the compounds bound in both orientations to take advantage to H-bonds with Lys-121 of ROCK-II.
Assuntos
Descoberta de Drogas , Indóis/síntese química , Quinases Associadas a rho/antagonistas & inibidores , Sítios de Ligação , Inibidores das Enzimas do Citocromo P-450 , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Estrutura MolecularRESUMO
Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hypertension, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on 4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase A (PKA).
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinonas/síntese química , Quinazolinonas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Quinazolinonas/química , Relação Estrutura-AtividadeRESUMO
Rho Kinase (ROCK) is a serine/threonine kinase whose inhibition could prove beneficial in numerous therapeutic areas. We have developed a promising class of ATP-competitive inhibitors based upon a benzimidazole scaffold, which show excellent potency toward ROCK (IC(50)<10nM). This report details the optimization of selectivity for ROCK over other related kinases such as Protein kinase A (PKA).
Assuntos
Benzimidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Inibidores de Proteínas Quinases/químicaRESUMO
A series of benzothiazole derivatives as ROCK inhibitors have been discovered. Compounds with good biochemical and cellular potency, and sufficient kinase selectivity have been identified.
Assuntos
Benzotiazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Benzotiazóis/síntese química , Benzotiazóis/química , Desenho de Fármacos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.
Assuntos
Amidas/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Piperazinas/síntese química , Amidas/farmacologia , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-AtividadeRESUMO
Here, we describe the first example of a cell-based myosin light chain phosphorylation assay in 96-well format that allows for the rapid screening of novel Rho-kinase inhibitors. We obtained IC(50) values for the prototypic Rho-kinase inhibitors Y-27632 (1.2+/-0.05microM) and Fasudil (3.7+/-1.2microM) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC(50) value of 77+/-30nM. Data derived from a set of 21 novel Rho-kinase inhibitors correlate with those generated by a well-established cell-based phenotypic Rho-kinase inhibition assay (R(2)=0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC(50) values and to screen a larger set of small molecule Rho-kinase inhibitors and suggests that this approach can be broadly applied to other cell lines and signaling pathways.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Cadeias Leves de Miosina/análise , Fosfoproteínas/análise , Inibidores de Proteínas Quinases/isolamento & purificação , Quinases Associadas a rho/antagonistas & inibidores , Amidas/farmacologia , Animais , Linhagem Celular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologiaRESUMO
Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Medições Luminescentes/métodos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Sequência de Bases , Primers do DNA/genética , Humanos , Técnicas In Vitro , Luciferases , Miniaturização , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Quinases Associadas a rho/genéticaRESUMO
c-jun-N-terminal kinase 1alpha1 (JNK1alpha1) is a serine/threonine kinase of the mitogen-activated protein (MAP) kinase family that phosphorylates protein transcription factors after activation by a variety of environmental stressors. In this study, the kinetic mechanism for JNK1alpha1 phosphorylation of activating transcription factor 2 (ATF2) was determined utilizing steady-state kinetics in the presence and absence of both ATF2 and ATP competitive inhibitors. Data from initial velocity studies were consistent with a sequential mechanism for JNK1alpha1. AMP-PCP exhibited competitive inhibition versus ATP and pure noncompetitive inhibition versus ATF2. JIP-1 peptide (RPKRPTTLNLF) was competitive versus ATF2 and mixed noncompetitive versus ATP. These data suggest that JNK1alpha1 proceeded via a random sequential kinetic mechanism with non-interacting ATF2 and ATP substrate sites.
Assuntos
Fator 2 Ativador da Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal/química , Trifosfato de Adenosina/química , Proteína Quinase 8 Ativada por Mitógeno/química , Modelos Químicos , Fragmentos de Peptídeos/química , Simulação por Computador , Ativação EnzimáticaRESUMO
Inhibitors of c-jun-N-Terminal Kinase (JNK) have many potential therapeutic indications ranging from neurodegenerative disease, to metabolic disorders, inflammation, cardiovascular disease, and cancer. This overview will highlight biological inhibitors such as JNK-interacting protein (JIP) as well as small molecule inhibitors from various structural classes including, aminopyrimidines and indazoles.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Piridinas/química , Piridinas/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benzoxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective inhibitors of ROCK-II with excellent potency in both enzyme and cell-based assays were obtained. These inhibitors possess good microsomal stability, low cytochrome P-450 inhibitions and good oral bioavailability.