RESUMO
The development of a fluorescent LCK inhibitor that exhibits favourable solvatochromic properties upon binding the kinase is described. Fluorescent properties were realised through the inclusion of a prodan-derived fluorophore into the pharmacophore of an ATP-competitive kinase inhibitor. Fluorescence titration experiments demonstrate the solvatochromic properties of the inhibitor, in which dramatic increase in emission intensity and hypsochromic shift in emission maxima are clearly observed upon binding LCK. Microscopy experiments in cellular contexts together with flow cytometry show that the fluorescence intensity of the inhibitor correlates with the LCK concentration. Furthermore, multiphoton microscopy experiments demonstrate both the rapid cellular uptake of the inhibitor and that the two-photon cross section of the inhibitor is amenable for excitation at 700â nm.
Assuntos
2-Naftilamina/análogos & derivados , Corantes Fluorescentes/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , 2-Naftilamina/química , Trifosfato de Adenosina/metabolismo , Ligação Competitiva , Citometria de Fluxo , Humanos , Células Jurkat , Microscopia de Fluorescência por Excitação Multifotônica , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/químicaRESUMO
Fluorescence microscopy studies using 4-morpholinoscriptaid (4MS) demonstrated rapid cellular uptake of this scriptaid analogue into the cytoplasm but no nuclear penetration. As 4MS and scriptaid have the same in vitro activity against HDACs and KASUMI-1 cells; 4MS exemplifies a rational approach to subtly modify 'profluorogenic' substrates for intracellular studies.