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

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 (PKCα, PKCβΙ, PKCδ, 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 PKCβΙ and PKCδ, which were previously unattainable using the BTF approach.