Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Nat Chem Biol ; 17(3): 280-290, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33462494

RESUMO

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.


Assuntos
Proteínas de Ciclo Celular/genética , Epiderme/efeitos dos fármacos , Reepitelização/efeitos dos fármacos , Úlcera Cutânea/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/genética , Ferimentos não Penetrantes/tratamento farmacológico , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Reepitelização/genética , Úlcera Cutânea/genética , Úlcera Cutânea/metabolismo , Úlcera Cutânea/patologia , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ferimentos não Penetrantes/genética , Ferimentos não Penetrantes/metabolismo , Ferimentos não Penetrantes/patologia
2.
Biochim Biophys Acta ; 1804(3): 445-53, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19922818

RESUMO

As a drug used to treat imatinib-resistant and -intolerant, chronic and advanced phase chronic myelogenous leukaemia, nilotinib is well characterised as a potent inhibitor of the Abl tyrosine kinase activity of wild-type and imatinib-resistant mutant forms of BCR-Abl. Here we review the profile of nilotinib as a protein kinase inhibitor. Although an ATP-competitive inhibitor of Abl, nilotinib binds to a catalytically inactive conformation (DFG-out) of the activation loop. As a consequence of this, nilotinib exhibits time-dependent inhibition of Abl kinase in enzymatic assays, which can be extrapolated to other targets to explain differences between biochemical activity and cellular assays. Although these differences confound assessment of kinase selectivity, as assessed using a combination of protein binding and transphosphorylation assays, together with cellular autophosporylation and proliferation assays, well established kinase targets of nilotinib in rank order of inhibitory potency are DDR-1>DDR-2>BCR-Abl (Abl)>PDGFRalpha/beta>KIT>CSF-1R. In addition nilotinib has now been found to bind to both MAPK11 (p38beta) and MAPK12 (p38alpha), as well as with very high affinity to ZAK kinase. Although neither enzymatic nor cellular data are yet available to substantiate the drug as an inhibitor of ZAK phosphorylation, modeling predicts that it binds in an ATP-competitive fashion.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Pirimidinas/química , Trifosfato de Adenosina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/uso terapêutico
3.
Bioorg Med Chem Lett ; 21(23): 7030-3, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22004721

RESUMO

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the k(cat)/K(M) ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR.


Assuntos
Sondas Moleculares/química , Peptídeos/química , Receptor IGF Tipo 1/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Sondas Moleculares/genética , Dados de Sequência Molecular , Peptídeos/genética , Fosforilação , Ligação Proteica , Receptor de Insulina/química , Receptor de Insulina/genética , Especificidade por Substrato
6.
Eur J Med Chem ; 187: 112004, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31881458

RESUMO

Protein phosphorylation by kinases is of critical importance for the regulation of many cellular functions. When kinases are deregulated numerous biological processes are affected, which may cause a variety of diseases. Therefore, kinase inhibition plays an important role for therapeutic intervention. A number of kinase inhibitors have been approved as drugs, initially in oncology where promiscuous (multi-kinase) inhibitors were most efficacious. Exploring kinase inhibitor selectivity and promiscuity for therapy is among the most challenging aspects of kinase drug discovery. Herein, we thoroughly analyze a kinase profiling experiment in which 637 designated inhibitors of p38α MAP kinase (p38α) were tested against a panel of 60 kinases distributed across the human kinome. In this experiment, only 19% of the inhibitors were found to be promiscuous when the median p38α inhibition level was applied as an activity threshold. Promiscuous inhibitors had a median value of two targets per compound, and many of these inhibitors were only active against the p38α and closely related JNK3 enzymes. Promiscuity cliffs were identified and analyzed in a network representation revealing structural modifications that were implicated in triggering compound promiscuity. Taken together, the findings revealed a high degree of selectivity of designated p38α directed inhibitors although they target the ATP binding site that is largely conserved across the human kinome.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
7.
Anal Chem ; 81(1): 408-19, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19067573

RESUMO

Protein kinases have emerged as a major drug target in the last years. Since more than 500 kinases are encoded in the human genome, cross-reactivity of a majority of kinase inhibitors causes problems. Tools are required for a rapid classification of inhibitors according to their affinity for a certain target to refine the search for new, more specific lead compounds. Mass spectrometry (MS) is increasingly used in pharmaceutical research and drug discovery to investigate protein-ligand interactions and determination of binding affinities. We present a comparison of different existing nanoelectrospray-MS based methods to quantify binding affinities and qualitatively rank, by competitive experiments, the affinity of several clinical inhibitors. We also present a new competitive method which is derived from our previous work for quantitative assessment of binding strengths (Wortmann et al., J. Mass Spectrom. 2008, 43(5), 600-608). The human kinases studied for this purpose were p38alpha (MAPK14) and LCK (lymphocyte specific kinase), and their interaction with 17 known small molecule kinase inhibitors was probed. Moreover, we present a new method to differentiate type I from type II inhibitors (Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2(7), 358-364) based on a kinetic experiment with direct MS read-out of the noncovalent complex between the human kinase and the inhibitor. This method was successfully applied to p38alpha binding to BIRB796, as well as to a BIRB796 analogue. Quantitative determination of the binding strength is also described. The results of our competitive experiments for the affinity classification of different inhibitors, as well as the results for the kinetic study, are in good agreement with IC(50) measurements and data found in the literature.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Ligação Competitiva , Humanos , Concentração Inibidora 50 , Cinética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Quinase 14 Ativada por Mitógeno/química , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Proteômica/instrumentação , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/instrumentação
8.
Cancer Res ; 67(17): 8325-34, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17804748

RESUMO

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Compostos Aza/química , Compostos Aza/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Células Cultivadas , Cristalografia por Raios X , Ciclina A/química , Ciclina A/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Haematologica ; 93(5): 653-61, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18367480

RESUMO

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.


Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Leucemia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/farmacologia , Anilidas/química , Animais , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mesilato de Imatinib , Camundongos , Transplante de Neoplasias , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Pirimidinas/química
10.
J Med Chem ; 61(18): 8120-8135, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30137981

RESUMO

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.


Assuntos
Descoberta de Drogas , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Regulação Alostérica , Animais , Cães , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação , Niacinamida/química , Niacinamida/farmacologia , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/química , Pirazóis/química , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Methods Mol Biol ; 1439: 143-57, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27316993

RESUMO

Biochemical selectivity profiling is an integral part of early drug development. Typically compounds from optimization phase are regularly tested for off-target activities within or across target families. This article presents workflow and critical aspects of biochemical protein kinase profiling based on microfluidic mobility shift assays.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Técnicas Analíticas Microfluídicas/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaio de Desvio de Mobilidade Eletroforética/instrumentação , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/instrumentação
12.
ACS Chem Biol ; 11(12): 3338-3346, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27712055

RESUMO

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Descoberta de Drogas , Células HEK293 , Células HT29 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK
13.
ACS Chem Biol ; 10(9): 2116-25, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26158339

RESUMO

Kinases can switch between active and inactive conformations of the ATP/Mg(2+) binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in-out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target.


Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Ciclinas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos
14.
Clin Cancer Res ; 20(7): 1834-45, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24474669

RESUMO

PURPOSE: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies. EXPERIMENTAL DESIGN: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context. RESULTS: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. CONCLUSIONS: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies.


Assuntos
Neoplasias Hematológicas/terapia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas/genética , Animais , Linhagem Celular Tumoral , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Eur J Med Chem ; 57: 1-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23041456

RESUMO

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia. Although "traditional" kinase inhibitors typically act by preventing the interaction between the kinase and ATP, thus stopping substrate phosphorylation, an alternative approach consists in disrupting the protein-protein interaction between the kinase and its downstream partners. In order to facilitate the identification of potential chemical starting points for substrate-site inhibition approaches, we desired to investigate the application of Substrate Activity Screening to kinases. We herein report a proof-of-concept study demonstrating, on a model tyrosine kinase, that the key requirements of this methodology can be met. Namely, using peptides as model substrates, we show that a simple ADP-accumulation assay can be used to monitor substrate efficiency and that efficiency can be optimized in a modular manner. More importantly, we demonstrate that structure-efficiency relationships translate into structure-activity relationships upon conversion of the substrates into inhibitors.


Assuntos
Peptídeos/química , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/química , Trifosfato de Adenosina/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Peptídeos/antagonistas & inibidores , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade , Especificidade por Substrato
16.
ACS Med Chem Lett ; 2(1): 22-7, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-24900250

RESUMO

The natural product L-783277 is a resorcylic lactone type covalent kinase inhibitor. We have prepared the 5'-deoxy analogue of L-783277 (1) in a stereoselective fashion. Remarkably, this analogue retains almost the full kinase inhibitory potential of natural L-783277, with low nanomolar IC50 values against the most sensitive kinases, and it exhibits essentially the same selectivity profile (within the panel of 39 kinases investigated). In contrast, removal of both the 4'- and the 5'-hydroxyl groups leads to a more significant reduction in kinase inhibitory activity and so does a change in the geometry of the C7'-C8' double bond in 1 from Z to E. These findings offer new perspectives for the design of second generation resorcylic lactone-based kinase inhibitors.

17.
J Biomol Screen ; 16(1): 65-72, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21148014

RESUMO

We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.


Assuntos
Corantes Fluorescentes/química , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Espectrometria de Fluorescência/métodos , Sequência de Aminoácidos , Bioensaio , Humanos , Concentração Inibidora 50 , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Especificidade por Substrato , Tirosina/metabolismo
18.
J Med Chem ; 54(20): 7066-83, 2011 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-21936542

RESUMO

A novel series of N-aryl-N'-pyrimidin-4-yl ureas has been optimized to afford potent and selective inhibitors of the fibroblast growth factor receptor tyrosine kinases 1, 2, and 3 by rationally designing the substitution pattern of the aryl ring. On the basis of its in vitro profile, compound 1h (NVP-BGJ398) was selected for in vivo evaluation and showed significant antitumor activity in RT112 bladder cancer xenografts models overexpressing wild-type FGFR3. These results support the potential therapeutic use of 1h as a new anticancer agent.


Assuntos
Antineoplásicos/síntese química , Compostos de Fenilureia/síntese química , Pirimidinas/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacocinética , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Compostos de Fenilureia/farmacocinética , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Ratos Wistar , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Relação Estrutura-Atividade , Transplante Heterólogo , Neoplasias da Bexiga Urinária
19.
Cancer Res ; 71(15): 5255-64, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21697284

RESUMO

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.


Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Mutação de Sentido Incorreto , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Quinolinas/farmacologia , Receptores de Fatores de Crescimento/antagonistas & inibidores , Substituição de Aminoácidos , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Animais , Antineoplásicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cristalografia por Raios X , Análise Mutacional de DNA , DNA de Neoplasias/genética , Ativação Enzimática/genética , Humanos , Camundongos , Modelos Moleculares , Mutagênese , Neoplasias/tratamento farmacológico , Neoplasias/genética , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Pirazóis/metabolismo , Pirazóis/farmacologia , Quinolinas/metabolismo , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/genética , Tirosina/metabolismo
20.
Mol Cancer Ther ; 9(7): 1945-55, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20587663

RESUMO

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.


Assuntos
Proliferação de Células/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Policitemia/prevenção & controle , Quinoxalinas/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Eritropoese/efeitos dos fármacos , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Mutação , Fosforilação/efeitos dos fármacos , Policitemia/metabolismo , Policitemia/patologia , Estrutura Terciária de Proteína , Quinoxalinas/química , Ratos , Fator de Transcrição STAT5/metabolismo , Esplenomegalia/metabolismo , Esplenomegalia/patologia , Esplenomegalia/prevenção & controle
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA