Recognition-Domain Focused Chemosensors: Versatile and Efficient Reporters of Protein Kinase Activity

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• 作者：Elvedin Luković ; Juan A. Gonzá ; lez-Vera ; Barbara Imperiali
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：September 24, 2008
• 年：2008
• 卷：130
• 期：38
• 页码：12821-12827
• 全文大小：200K
• 年卷期：v.130,no.38(September 24, 2008)
• ISSN：1520-5126

文摘

Catalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation. Thus, assays that easily monitor kinase activity are critical in both academic and pharmaceutical settings. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a β-turn focused (BTF) design for the continuous assay of kinase activity in vitro and in cell lysates. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg²⁺ and undergoes chelation-enhanced fluorescence (CHEF). Although the design was applied successfully to the development of several kinase sensors, an intrinsic limitation was that only residues C- or N-terminal to the phosphorylated residue could be used to derive specificity for the target kinase. To address this limitation, a new, recognition-domain focused (RDF) strategy was developed that also relies on CHEF. In this approach, the requirement for the constrained β-turn motif is obviated by alkylation of a cysteine residue with a Sox-based derivative to afford an amino acid termed C-Sox. The RDF design allows inclusion of extended binding determinants to maximize recognition by the cognate kinase, which has now permitted the construction of chemosensors for a variety of representative Ser/Thr (PKC_α, PKC_βΙ, PKC_δ, Pim2, Akt1, MK2, and PKA) as well as receptor (IRK) and nonreceptor (Src, Abl) Tyr kinases with greatly enhanced selectivity. The new sensors have up to 28-fold improved catalytic efficiency and up to 66-fold lower K_M when compared to the corresponding BTF probes. The improved generality of the strategy is exemplified with the synthesis and analysis of Sox-based probes for PKC_βΙ and PKC_δ, which were previously unattainable using the BTF approach.