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1.
Chem Biol ; 21(6): 754-65, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24836908

RESUMO

Protein kinases play important regulatory roles in cells and organisms. Therefore, they are subject to specific and tight mechanisms of regulation that ultimately converge on the catalytic domain and allow the kinases to be activated or inhibited only upon the appropriate stimuli. AGC protein kinases have a pocket in the catalytic domain, the PDK1-interacting fragment (PIF)-pocket, which is a key mediator of the activation. We show here that helix αC within the PIF-pocket of atypical protein kinase C (aPKC) is the target of the interaction with its inhibitory N-terminal domains. We also provide structural evidence that the small compound PS315 is an allosteric inhibitor that binds to the PIF-pocket of aPKC. PS315 exploits the physiological dynamics of helix αC for its binding and allosteric inhibition. The results will support research on allosteric mechanisms and selective drug development efforts against PKC isoforms.


Assuntos
Compostos de Bifenilo/farmacologia , Cinamatos/farmacologia , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Regulação Alostérica/efeitos dos fármacos , Compostos de Bifenilo/química , Cinamatos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína Quinase C/antagonistas & inibidores , Estrutura Terciária de Proteína/efeitos dos fármacos , Relação Estrutura-Atividade
2.
Chem Biol ; 19(9): 1152-63, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22999883

RESUMO

The PIF-pocket of AGC protein kinases participates in the physiologic mechanism of regulation by acting as a docking site for substrates and as a switch for the transduction of the conformational changes needed for activation or inhibition. We describe the effects of compounds that bind to the PIF-pocket of PDK1. In vitro, PS210 is a potent activator of PDK1, and the crystal structure of the PDK1-ATP-PS210 complex shows that PS210 stimulates the closure of the kinase domain. However, in cells, the prodrug of PS210 (PS423) acts as a substrate-selective inhibitor of PDK1, inhibiting the phosphorylation and activation of S6K, which requires docking to the PIF-pocket, but not affecting PKB/Akt. This work describes a tool to study the dynamics of PDK1 activity and a potential approach for drug discovery.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Chalconas/farmacologia , Ácidos Dicarboxílicos/farmacologia , Pró-Fármacos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular , Chalconas/química , Ácidos Dicarboxílicos/química , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Pró-Fármacos/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Relação Estrutura-Atividade , Especificidade por Substrato
3.
J Med Chem ; 54(19): 6714-23, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21863889

RESUMO

Protein kinase inhibitors with an allosteric mode of action are expected to reach, in many cases, higher selectivity for the target enzyme than ATP-competitive compounds. Therefore, basic research is aiming at identifying and establishing novel sites on the catalytic domain of protein kinases which might be targeted by allosteric inhibitors. We previously published the first structure-activity relationships (SARs) for allosteric activators of protein kinase PDK1. Here, we present the design, synthesis, and SAR data on a series of novel compounds, 4-benzimidazolyl-3-phenylbutanoic acids, that inhibit the atypical protein kinace C (PKC) ζ via binding to the PIF-pocket. Key positions were identified in the compounds that can be modified to increase potency and selectivity. Some congeners showed a high selectivity toward PKCζ, lacking inhibition of the most closely related isoform, PKCι, and of further AGC kinases. Furthermore, evidence is provided that these compounds are also active toward cellular PKCζ without loss of potency compared to the cell-free assay.


Assuntos
Butiratos/síntese química , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Regulação Alostérica , Sítios de Ligação , Butiratos/química , Butiratos/farmacologia , Sistema Livre de Células , Humanos , NF-kappa B/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Células U937
4.
Nat Chem Biol ; 5(10): 758-64, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19718043

RESUMO

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.


Assuntos
Fosfopeptídeos/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Regulação Alostérica , Sítio Alostérico , Linhagem Celular , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Peso Molecular , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato
5.
J Med Chem ; 52(15): 4683-93, 2009 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-19606904

RESUMO

The modulation of protein kinase activities by low molecular weight compounds is a major goal of current pharmaceutical developments. In this line, important efforts are directed to the development of drugs targeting the conserved ATP binding site. However, there is very little experience on targeting allosteric, regulatory sites, different from the ATP binding site, in protein kinases. Here we describe the synthesis, cell-free activation potency, and calorimetric binding analysis of 3,5-diphenylpent-2-enoic acids and derivatives as allosteric modulators of the phosphoinositide-dependent kinase-1 (PDK1) catalytic activity. Our SAR results combined with thermodynamic binding analyses revealed both favorable binding enthalpy and entropy and confirmed the PIF-binding pocket of PDK1 as a druggable site. In conclusion, we defined the minimal structural requirements for compounds to bind to the PIF-binding pocket and to act as allosteric modulators and identified two new lead structures (12Z and 13Z) with predominating binding enthalpy.


Assuntos
Ácidos Graxos Monoinsaturados/síntese química , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Termodinâmica , Sítio Alostérico , Calorimetria , Entropia , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Relação Estrutura-Atividade
6.
EMBO J ; 25(23): 5469-80, 2006 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-17110931

RESUMO

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositide-dependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.


Assuntos
Acetatos/farmacologia , Desenho de Fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Acetatos/química , Regulação Alostérica , Motivos de Aminoácidos , Sítios de Ligação , Células Cultivadas , Ativação Enzimática , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Mutação , Fosfopeptídeos/farmacologia , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética
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