RESUMO
We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Interleucina-17/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Ligação Competitiva , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Estrutura Molecular , Ratos , Ratos Wistar , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.