Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Bioorg Med Chem ; 26(14): 4014-4024, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-29941193

RESUMO

As a member of the Wee-kinase family protein kinase PKMYT1 is involved in G2/M checkpoint regulation of the cell cycle. Recently, a peptide microarray approach led to the identification of a small peptide; EFS247-259 as substrate of PKMYT1, which allowed for subsequent development of an activity assay. The developed activity assay was used to characterize the PKMYT1 catalyzed phosphorylation of EFS247-259. For the first time kinetic parameters for PKMYT1, namely Km, Km, ATP and vmax were determined. The optimized assay was used to screen the published protein kinase inhibitor sets (PKIS I and II), two sets of small molecule ATP-competitive kinase inhibitors reported by GlaxoSmithKline. We identified ten inhibitors, providing different scaffolds. The inhibitors were further characterized by using binding assay, activity and functional assay. In addition, docking studies were carried out in order to rationalize the observed biological activities. The derived results provide the basis for further chemical optimization of PKMYT1 inhibitors and for further analysis of PKMYT1 as target for anti-cancer therapy.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células HT29 , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade
2.
Molecules ; 22(12)2017 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-29168755

RESUMO

In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transition.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas de Membrana/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Família Multigênica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade
3.
Bioorg Med Chem ; 23(15): 4936-4942, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26059593

RESUMO

Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Polarização de Fluorescência , Corantes Fluorescentes/química , Humanos , Peptídeos/síntese química , Peptídeos/química , Fosforilação , Análise Serial de Proteínas , Especificidade por Substrato , Fatores de Transcrição/química
4.
J Enzyme Inhib Med Chem ; 30(3): 514-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24939100

RESUMO

Previously, a glycoglycerolipid isolated from marine algae was reported to be a potent and selective inhibitor of the human Myt1 kinase, an enzyme involved in cell cycle regulation with great potential as an anti-cancer target. Based on that report, a lot of research effort has been invested by several working groups to synthesize and derivatize this compound. However, reliable assay data confirming the inhibitory potential and the mechanism of action of these glycoglycerolipids are missing so far. Here, based on experimental data and theoretical considerations, we show that the aforesaid glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6'-amino-6'-deoxy-α-d-glucosyl)-sn-glycerol is not an inhibitor of the human Myt1 kinase.


Assuntos
Glicolipídeos/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores
5.
J Chem Inf Model ; 54(3): 881-93, 2014 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-24490903

RESUMO

Identification of compounds that can bind to a target protein with high affinity is a nontrivial task in structure-based drug design. Several approaches ranging from simple scoring methods to more computationally demanding methods are usually applied for this purpose. In the current work, we used ligand docking in combination with QM/MM-GBSA, MM-GBSA, and MM-PBSA rescoring to discriminate between active and inactive Myt1 kinase inhibitors. Results show that QM/MM-GBSA rescoring performs better than normal docking scores or MM-GBSA rescoring in classifying active and inactive inhibitors. We also applied QM/MM-GBSA rescoring to estimate the binding affinities of compounds from different virtual screening runs. To prove our approach and to confirm its predictive power, a few compounds which were predicted to be active were purchased and experimentally tested. Among the five selected compounds, three showed significant inhibition of recombinant Myt1. PD-173952, which yielded a favorable QM/MM-GBSA binding free energy, showed a K(i) value of 8.1 nM. In addition, two compounds, PD-180970 and saracatinib, showed inhibition at the low micromolar level. Thus, the developed protocol might be useful for further virtual screening experiments to better discriminate between active and inactive compounds and to further optimize the identified hits.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Compostos Aza/química , Compostos Aza/farmacologia , Benzodioxóis/química , Benzodioxóis/farmacologia , Desenho de Fármacos , Humanos , Ligantes , Proteínas de Membrana/metabolismo , Morfolinas/química , Morfolinas/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Piridonas/química , Piridonas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia
6.
Assay Drug Dev Technol ; 12(2): 136-44, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24229357

RESUMO

Abstract The human Myt1 kinase is a regulator of Cdk1/CycB and, hence, important for the G2/M transition in the cell cycle. It may act as a target for drug development, but suitable assay systems for assessing potential inhibitors are lacking so far. Herein, we describe the rational development of a fluorescence anisotropy-based kinase binding assay. A suitable fluoroprobe based on the tyrosine kinase inhibitor dasatinib was synthesized and tested with Myt1 and several other kinases for control purposes. The probe acted as expected in terms of specificity and reversibility, and a Myt1 assay was set up. Notwithstanding the moderate Kd of the starting compound dasatinib before chemical modification, satisfying Z' factors >0.5 were achieved. A validation with known kinase inhibitors demonstrated the applicability of the assay and led to a reliable ranking of the tested active compounds.


Assuntos
Cristalografia por Raios X/métodos , Polarização de Fluorescência/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Dasatinibe , Relação Dose-Resposta a Droga , Humanos , Ligação Proteica/fisiologia , Pirimidinas/química , Pirimidinas/metabolismo , Tiazóis/química , Tiazóis/metabolismo
7.
Eur J Med Chem ; 61: 41-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22770610

RESUMO

In the human cell cycle, the Myt1 kinase is a crucial regulator of the G2/M transition. Because this membrane-associated kinase is hard to obtain and assay, there is a distinct lack of data so far. Here we report the derivatization of a glycoglycerolipid which was shown previously to be active in a Myt1 activity assay. These compounds were tested in a binding assay together with a set of common kinase inhibitors against a full-length Myt1 expressed in a human cell line. Dasatinib exhibited nanomolar affinity whereas broad coverage inhibitors such as sunitinib and staurosporine derivatives did not show any effect. We also carried out docking studies for the most potent compounds allowing further insights into the inhibitor interaction of this kinase. The glycoglycerolipids showed no significant effects in the binding assay, endorsing the idea of a mechanism of action distant from the active site.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Glicolipídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Relação Dose-Resposta a Droga , Glicolipídeos/síntese química , Glicolipídeos/química , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade
8.
Bioorg Med Chem Lett ; 22(2): 1219-23, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22189141

RESUMO

The human Myt1 kinase (PKMYT1) is an important regulator of the G2/M transition in the cell cycle. Presently, limited knowledge about its substrate recognition is available. Here, various potential substrates were investigated by different antibody based techniques including fluorescence polarization immunoassays and immunoblotting. Regarding both Thr and Tyr kinase activity, only protein substrates were found to be phosphorylated by Myt1, whereas any tested peptide was not recognized. In silico molecular dynamics studies were used to compare the stability of the Myt1 peptide complex with Wee1 peptide complex and support the biochemical findings. Furthermore, a Myt1 kinase binding assay suggests Myt1 being insensitive to staurosporine.


Assuntos
Proteínas de Membrana/química , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Anticorpos Monoclonais/imunologia , Imunoensaio de Fluorescência por Polarização , Células HEK293 , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...