RESUMO
Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli- or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
Assuntos
Ácidos Cetoglutáricos/química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/biossíntese , Animais , Células CHO , Carbono/química , Dióxido de Carbono/química , Cromatografia Líquida , Colágeno/química , Cricetulus , Meios de Cultivo Condicionados/química , Glicosilação , Humanos , Luciferases/química , Lisina/química , Proteínas Recombinantes/biossíntese , Ácido Succínico/química , Espectrometria de Massas em TandemRESUMO
eEF-2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6-diamino-4-(2-fluorophenyl)-4H-thiopyran-3,5-dicarbonitrile (DFTD) as an inhibitor of eEF-2K. Here we describe its mechanism of action against eEF-2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile-containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF-2K with a two-step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF-2K.
Assuntos
Quinase do Fator 2 de Elongação/antagonistas & inibidores , Nitrilas/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nitrilas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
PERK, as one of the principle unfolded protein response signal transducers, is believed to be associated with many human diseases, such as cancer and type-II diabetes. There has been increasing effort to discover potent PERK inhibitors due to its potential therapeutic interest. In this study, a computer-based virtual screening approach is employed to discover novel PERK inhibitors, followed by experimental validation. Using a focused library, we show that a consensus approach, combining pharmacophore modeling and docking, can be more cost-effective than using either approach alone. It is also demonstrated that the conformational flexibility near the active site is an important consideration in structure-based docking and can be addressed by using molecular dynamics. The consensus approach has further been applied to screen the ZINC lead-like database, resulting in the identification of 10 active compounds, two of which show IC50 values that are less than 10 µM in a dose-response assay.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , eIF-2 Quinase/antagonistas & inibidores , Animais , Domínio Catalítico , Bases de Dados de Produtos Farmacêuticos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/metabolismo , Reprodutibilidade dos Testes , Interface Usuário-Computador , eIF-2 Quinase/química , eIF-2 Quinase/metabolismoRESUMO
Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site and one or more substrate-binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer the γ phosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time-dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Measuring protein kinase activity using fluorescent peptides Alternate Protocol: Measuring protein kinase activity using a fluorescence plate reader Support Protocol: Labeling peptides with sox fluorophore Basic Protocol 2: Measuring time-dependent ATP-competitive inhibition of protein kinases using fluorescent peptides.
Assuntos
Peptídeos , Proteínas Quinases , Fosforilação , Corantes Fluorescentes , Trifosfato de AdenosinaRESUMO
Evidence that elongation factor 2 kinase (eEF-2K) has potential as a target for anticancer therapy and possibly for the treatment of depression is emerging. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC(50) of 60 nM, its mechanism of action was not established. Using the same kinetic assay, the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC(50) of 18 ± 0.25 µM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation, we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions, NH125 exhibits a similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1% Triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is a nonspecific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a manner similar to that of eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment, its ability to inhibit eEF2 phosphorylation was assessed in MDA-MB-231 breast cancer, A549 lung cancer, and HEK-293T cell lines using a Western blot approach. No sign of a decrease in the level of eEF2 phosphorylation was observed up to 12 h following addition of NH125 to the media. Furthermore, contrary to the previously reported literatures, NH125 induced the phosphorylation of eEF-2.
Assuntos
Quinase do Fator 2 de Elongação/antagonistas & inibidores , Imidazóis/farmacologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Artefatos , Linhagem Celular , Relação Dose-Resposta a Droga , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Células HEK293 , Humanos , Imidazóis/administração & dosagem , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Serina/genética , Treonina/genética , Sequência de Aminoácidos , Sítios de Ligação , Quinase do Fator 2 de Elongação/genética , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Treonina/metabolismoRESUMO
The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the suboptimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and noncanonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the noncanonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteína Quinase 1 Ativada por Mitógeno/química , Sítio Alostérico , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Estatísticos , Conformação Molecular , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , RatosRESUMO
The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 µM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 µM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.
Assuntos
Cátions Bivalentes/metabolismo , Citoplasma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Proteínas Reguladoras de Apoptose , Ativação Enzimática , Humanos , Luz , Espalhamento de Radiação , UltracentrifugaçãoRESUMO
The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of â¼85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.
Assuntos
Clonagem Molecular/métodos , Quinase do Fator 2 de Elongação/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Plasmídeos/genética , Biossíntese de Proteínas/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/isolamento & purificação , Endopeptidases/metabolismo , Escherichia coli , Histidina/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Fosforilação , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tiorredoxinas/metabolismo , Transformação BacterianaRESUMO
Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z' (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.
Assuntos
Frutose-Bifosfato Aldolase/isolamento & purificação , Ensaios de Triagem em Larga Escala , Análise Custo-Benefício , Frutose/genética , Frutose/metabolismo , Frutose-Bifosfato Aldolase/genética , Glicólise/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , LuminescênciaRESUMO
Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.
Assuntos
Inibidores Enzimáticos/farmacologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios de Triagem em Larga Escala/métodosRESUMO
Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.
Assuntos
Proteínas de Transporte de Cátions/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/enzimologia , GTP Fosfo-Hidrolases/isolamento & purificação , Ensaios de Triagem em Larga Escala , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Colorimetria/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Luminescência , Modelos Moleculares , Domínios Proteicos/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The use of AlphaScreen® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Thus, we describe the optimization of an AlphaScreen detection platform by systematically reducing the Alpha reagents down to 2.5 µg/ml beads, without compromising assay integrity. We demonstrate that reducing bead concentration reduces detection costs substantially while yielding robust statistics. We believe this simple new protocol will enhance the future utilization of AlphaScreen technology in high-throughput screening.
Assuntos
Ensaios de Triagem em Larga Escala/economia , Animais , Metabolismo dos Carboidratos , Análise Custo-Benefício , DNA/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Indicadores e Reagentes/economia , Medições Luminescentes/economia , Medições Luminescentes/métodos , Peptídeos/metabolismo , Proteínas/metabolismo , RNA/metabolismoRESUMO
A high rate of glycolysis, which supplies energy and materials for anabolism, is observed in a wide range of tumor cells, making it a potential pathway to control cancer growth. ALDOA is a multifunctional enzyme in the glycolytic pathway and also promotes HIF-1α, which is of importance in hypoxic solid tumors. The current method for assaying ALDOA activity involves monitoring the consumption of NADH in vitro using absorbance or intrinsic fluorescence via a coupled enzymatic reaction. Here, we report the development of a homogeneous biochemical assay that can overcome limitations of current methods, in particular for the application of high-throughput drug screening. The assay utilizes the commercially available Elite NADH Assay Kit, which incorporates an enzymatic reaction to measure the level of NADH using a fluorescent probe. Assay optimization and validation are discussed. Its feasibility for high-throughput screening (HTS) was demonstrated by screening 65,000 compounds for the identification of small molecules that inhibit ALDOA. Through a validation screen and dose-response evaluation, four inhibitors with IC50 below 10 µM were identified. In conclusion, we demonstrate that a traditional ALDOA assay can be transformed readily into a fluorescence-based assay utilizing a commercial NADH detection kit that is rapid, sensitive, inexpensive, and HTS friendly.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Corantes Fluorescentes , Glicólise/efeitos dos fármacos , Humanos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
Apyrase is a calcium-activated enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and Pi. It is currently used in studies involving cancer and platelet aggregation in humans, as well as herbicide resistance in plants. Inhibitors of apyrase are being investigated for their use to suppress tumors and combat herbicide resistance. Only a few inhibitors of apyrase have been reported, many of which were identified through automated screening using a 96-well plate format and colorimetric phosphate detection. However, these screens have had limitations, including large volumes, inconsistent reproducibility, high incidence of false hits, and lack of higher-throughput compatibility. A luciferin/luciferase-based detection system has been reported to examine potential inhibitors of apyrase; however, these reactions were performed in tubes with the assay completion in seconds, which necessitate the development of a high-throughput screening (HTS)-compatible format for screening. Therefore, a more cost-effective biochemical assay that improved the limitations of the previous assay formats using a commercially available luminescence-based detection system was developed. This new robust mix-and-read platform incorporates a low-volume luminescence-based protocol, formatted for use in 384-well microplates. This new format provides a simple and cost-effective method to screen for apyrase inhibitors and will facilitate larger HTS efforts to identify potent inhibitors of apyrase.
Assuntos
Apirase/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Luminescência , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , CinéticaRESUMO
Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.
RESUMO
Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.
Assuntos
Antineoplásicos/farmacologia , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Medições Luminescentes/métodos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Radiometria/métodos , Antineoplásicos/isolamento & purificação , Quinase do Fator 2 de Elongação/biossíntese , Quinase do Fator 2 de Elongação/isolamento & purificação , Raios gama , Humanos , Indicadores e Reagentes , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/isolamento & purificação , Neoplasias/enzimologia , Radioisótopos de Fósforo/análise , Inibidores de Proteínas Quinases/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Contagem de CintilaçãoRESUMO
We have recently demonstrated that the undifferentiated PSA-/lo prostate cancer (PCa) cell population harbors self-renewing long-term tumor-propagating cells that are refractory to castration, thus representing a therapeutic target. Our goals here are, by using the same lineage-tracing reporter system, to track the dynamic changes of PSA-/lo and PSA+ cells upon castration in vitro, investigate the molecular changes accompanying persistent castration, and develop large numbers of PSA-/lo PCa cells for drug screening. To these ends, we treated LNCaP cells infected with the PSAP-GFP reporter with three regimens of castration, i.e., CDSS, CDSS plus bicalutamide, and MDV3100 continuously for up to ~21 months. We observed that in the first ~7 months, castration led to time-dependent increases in PSA-/lo cells, loss of AR and PSA expression, increased expression of cancer stem cell markers, and many other molecular changes. Meanwhile, castrated LNCaP cells became resistant to high concentrations of MDV3100, chemotherapeutic drugs, and other agents. However, targeted and medium-throughput library screening identified several kinase (e.g., IGF-1R, AKT, PI3K/mTOR, Syk, GSK3) inhibitors as well as the BCL2 inhibitor that could effectively sensitize the LNCaP-CRPC cells to killing. Of interest, LNCaP cells castrated for >7 months showed evidence of cyclic changes in AR and the mTOR/AKT signaling pathways potentially involving epigenetic mechanisms. These observations indicate that castration elicits numerous molecular changes and leads to enrichment of PSA-/lo PCa cells. The ability to generate large numbers of PSA-/lo PCa cells should allow future high-throughput screening to identify novel therapeutics that specifically target this population.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Estudos Longitudinais , Masculino , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Células Tumorais CultivadasRESUMO
The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.
Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias/tratamento farmacológico , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/fisiologia , Humanos , Camundongos , Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.