Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
EMBO Mol Med ; 15(1): e14850, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36515561

RESUMO

High-throughput (HT) screening drug discovery, during which thousands or millions of compounds are screened, remains the key methodology for identifying active chemical matter in early drug discovery pipelines. Recent technological developments in mass spectrometry (MS) and automation have revolutionized the application of MS for use in HT screens. These methods allow the targeting of unlabelled biomolecules in HT assays, thereby expanding the breadth of targets for which HT assays can be developed compared to traditional approaches. Moreover, these label-free MS assays are often cheaper, faster, and more physiologically relevant than competing assay technologies. In this review, we will describe current MS techniques used in drug discovery and explain their advantages and disadvantages. We will highlight the power of mass spectrometry in label-free in vitro assays, and its application for setting up multiplexed cellular phenotypic assays, providing an exciting new tool for screening compounds in cell lines, and even primary cells. Finally, we will give an outlook on how technological advances will increase the future use and the capabilities of mass spectrometry in drug discovery.


Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Descoberta de Drogas/métodos , Espectrometria de Massas , Ensaios de Triagem em Larga Escala/métodos
2.
SLAS Discov ; 28(1): 3-11, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36414185

RESUMO

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.


Assuntos
Aminopeptidases , Ensaios de Triagem em Larga Escala , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Reprodutibilidade dos Testes , Ensaios de Triagem em Larga Escala/métodos , Peptídeos
3.
Sci Rep ; 12(1): 3114, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35210470

RESUMO

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.


Assuntos
SARS-CoV-2
4.
Anal Chem ; 92(20): 13847-13854, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32970424

RESUMO

Drug discovery usually begins with a high-throughput screen (HTS) of thousands to millions of molecules to identify starting points for medicinal chemistry. Conventional HTS platforms require expensive reagents and typically have complex assay formats. HTS platforms based on radioactivity are expensive, both in terms of reagent cost and disposal. Furthermore, nonspecific interferences common to these technologies result in an extensive attrition of hits during validation experiments. Mass spectrometry (MS) is a highly selective, label-free technology that can quantify multiple analytes in a single experiment. However, most commercial MS platforms typically involve a separation or cleanup prior to analysis and are too slow for large-scale screening campaigns. Recently, an MS platform (AMI-MS) was introduced that uses acoustically generated droplets to deliver analyte molecules directly from microtiter plates into the mass spectrometer at subsecond per well sampling rates. Here, we demonstrate the application of AMI-MS by developing an HTS-compatible assay that measures the inhibition of histone acetyltransferase activity. Real-time kinetic measurements from a single well were used to determine enzyme Km and Vmax values. We compare the AMI-MS readout with conventional platforms in single-shot screening and multipoint profiling modes. The AMI-MS assay identified 86% of hits previously identified, with a pIC50 ≥ 5.0, in a scintillation proximity assay (SPA) HTS at a lower hit rate and with a significantly reduced cost per well compared to the SPA-based readout. Furthermore, pIC50s, as measured by AMI-MS, showed a good correlation with values generated by RapidFire-MS. AMI-MS has the potential to provide significant improvements to high-throughput bioassays.


Assuntos
Inibidores Enzimáticos/análise , Ensaios de Triagem em Larga Escala , Espectrometria de Massas/métodos , Acústica , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Cinética
5.
J Med Chem ; 63(11): 5816-5840, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32410449

RESUMO

Non-BET bromodomain-containing proteins have become attractive targets for the development of novel therapeutics targeting epigenetic pathways. To help facilitate the target validation of this class of proteins, structurally diverse small-molecule ligands and methodologies to produce selective inhibitors in a predictable fashion are in high demand. Herein, we report the development and application of atypical acetyl-lysine (KAc) methyl mimetics to take advantage of the differential stability of conserved water molecules in the bromodomain binding site. Discovery of the n-butyl group as an atypical KAc methyl mimetic allowed generation of 31 (GSK6776) as a soluble, permeable, and selective BRD7/9 inhibitor from a pyridazinone template. The n-butyl group was then used to enhance the bromodomain selectivity of an existing BRD9 inhibitor and to transform pan-bromodomain inhibitors into BRD7/9 selective compounds. Finally, a solvent-exposed vector was defined from the pyridazinone template to enable bifunctional molecule synthesis, and affinity enrichment chemoproteomic experiments were used to confirm several of the endogenous protein partners of BRD7 and BRD9, which form part of the chromatin remodeling PBAF and BAF complexes, respectively.


Assuntos
Proteínas Cromossômicas não Histona/antagonistas & inibidores , Lisina/química , Piridazinas/química , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Humanos , Ligantes , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Piridazinas/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
6.
Bioorg Med Chem ; 27(8): 1456-1478, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30858025

RESUMO

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Quinolinas/química , Quinolinas/farmacologia , Animais , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Quinolinas/farmacocinética
7.
SLAS Discov ; 23(9): 881-897, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29874524

RESUMO

There has been much debate around the success rates of various screening strategies to identify starting points for drug discovery. Although high-throughput target-based and phenotypic screening has been the focus of this debate, techniques such as fragment screening, virtual screening, and DNA-encoded library screening are also increasingly reported as a source of new chemical equity. Here, we provide examples in which integration of more than one screening approach has improved the campaign outcome and discuss how strengths and weaknesses of various methods can be used to build a complementary toolbox of approaches, giving researchers the greatest probability of successfully identifying leads. Among others, we highlight case studies for receptor-interacting serine/threonine-protein kinase 1 and the bromo- and extra-terminal domain family of bromodomains. In each example, the unique insight or chemistries individual approaches provided are described, emphasizing the synergy of information obtained from the various tactics employed and the particular question each tactic was employed to answer. We conclude with a short prospective discussing how screening strategies are evolving, what this screening toolbox might look like in the future, how to maximize success through integration of multiple tactics, and scenarios that drive selection of one combination of tactics over another.


Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Animais , Descoberta de Drogas/normas , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Ensaios de Triagem em Larga Escala , Humanos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
8.
SLAS Discov ; 22(10): 1262-1269, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-27932699

RESUMO

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) offers a label-free alternative for the screening of biochemical targets in both 1536- and 6144-assay formats, as well as potentially providing increased sensitivity, reproducibility, and the simultaneous detection of multiple assay components within a specified m/z range. Ion suppression effects are one of the principal limitations reported for MS analysis. Within MALDI-MS screening, it has been identified that certain biochemical components incorporated into the assay (e.g., the buffers used to preserve the physiological conditions of the enzyme, salts, and other additives) induce suppression of the analyte ion signals monitored. This poorly understood phenomenon of ion suppression is a key reason the screening community has been reluctant to shift their investigations toward MS methods with reduced sample cleanup. Using acetylcholine as an assay substrate mimic, we have generated robust data to quantify the degree to which the most highly used components (base buffers, additional components, detergents, cell culture media, and other additives) within current screening assays are compatible with MALDI-MS. Here, the most suitable buffers and components, along with their identified optimal concentrations in terms of limiting ion suppression effects, are proposed for use in screening assays measured by MALDI-MS.


Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetilcolina/análise , Soluções Tampão , Processamento de Imagem Assistida por Computador , Processamento de Sinais Assistido por Computador , Software
9.
J Med Chem ; 59(4): 1357-69, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26771107

RESUMO

Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 µM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).


Assuntos
Inibidores Enzimáticos/química , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Piridinas/química , Aminação , Linhagem Celular , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Piridinas/farmacocinética , Piridinas/farmacologia
10.
J Med Chem ; 59(4): 1370-87, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26771203

RESUMO

Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Pirimidinonas/química , Pirimidinonas/farmacologia , Linhagem Celular , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Inibidores Enzimáticos/farmacocinética , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Pirimidinonas/farmacocinética , Relação Estrutura-Atividade
11.
J Biomol Screen ; 21(2): 145-55, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26403521

RESUMO

LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson's disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.


Assuntos
Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular , DNA Complementar/genética , GTP Fosfo-Hidrolases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Espectrometria de Massas/métodos , Mutação/genética , Fosforilação/genética , Estrutura Terciária de Proteína/genética , Células Sf9
12.
J Med Chem ; 59(4): 1425-39, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25856009

RESUMO

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain "reader" modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.


Assuntos
Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fatores de Transcrição/química
13.
J Biomol Screen ; 21(2): 176-86, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26428484

RESUMO

Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography-coupled MS, an analysis time of 8-10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetilcolinesterase/química , Cromatografia Líquida/métodos , Histona Desmetilases/química , Peptídeos/química
15.
J Biomol Screen ; 19(2): 278-86, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23896685

RESUMO

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.


Assuntos
Ensaios de Triagem em Larga Escala , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espectrometria de Massas/métodos , Epigenômica , Humanos , Especificidade por Substrato
16.
Nature ; 488(7411): 404-8, 2012 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-22842901

RESUMO

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Sequência de Aminoácidos , Animais , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Evolução Molecular , Histonas/química , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/classificação , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Metilação/efeitos dos fármacos , Camundongos , Modelos Moleculares , Especificidade por Substrato , Fator de Necrose Tumoral alfa/biossíntese
17.
J Biomol Screen ; 17(5): 641-50, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22337655

RESUMO

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.


Assuntos
Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Oxirredutases Intramoleculares/antagonistas & inibidores , Espectrometria de Massas/métodos , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Corantes Fluorescentes/metabolismo , Humanos , Concentração Inibidora 50 , Oxirredutases Intramoleculares/metabolismo , Prostaglandina-E Sintases
18.
J Biomol Screen ; 17(1): 108-20, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22223398

RESUMO

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.


Assuntos
Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Especificidade de Anticorpos , Linhagem Celular , Histonas/imunologia , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Permeabilidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas
19.
J Biomol Screen ; 17(1): 39-48, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21859681

RESUMO

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espectrometria de Massas/métodos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Epigênese Genética/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Cinética , Lisina/metabolismo , Oxiquinolina/metabolismo , Oxiquinolina/farmacologia , Peptídeos/metabolismo , Piridinas/metabolismo , Piridinas/farmacologia , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA