Previously we have shown that
Previously, we have shown that complexes of protein PKs with inhibitors that comprised sulfur or selenium atoms in aromatic structures were phosphorescent at room temperature, while the long-lifetime signal emitted by the free inhibitor was negligible.34, 35, 36 This phenomenon was caused by stabilization of the triplet excited state of the aromatic structure of the inhibitor inside the ATP-binding pocket of PK that slowed down competing non-radiative relaxation pathways (e.g., quenching by dissolved oxygen). Moreover, significant enhancement of kinase binding-responsive long lifetime photoluminescence was achieved when the inhibitor was covalently labeled with a bright fluorescent dye. This enhancement resulted from efficient radiationless intramolecular Förster-type resonant energy transfer (RET) from the donor phosphor in the excited triplet state to the non-excited acceptor fluorophore, leading it to the excited singlet state of the latter and emission of light.35, 38
Herein we present a new series of bisubstrate inhibitors incorporating either 4-(2-amino-1,3-thiazol-5-yl)benzoic UNC 3230 (ATB) moiety, a fragment that was previously introduced to inhibit CK2 by others39, 40, or 4-(2-amino-1,3-selenazol-5-yl)benzoic acid (ASB) moiety, the selenium-comprising counterpart of ATB. The invented conjugates have high affinity and selectivity in respect to CK2 and emit protein-responsive phosphorescence at room temperature that has not been previously described for thiazole- and selenazole-containing scaffolds. In addition, the inhibitors labeled with fluorescent dyes are tandem probes that detect the active forms of CK2α and CK2α’ in time-gated photoluminescence measurement mode, a technique otherwise used in the case of lanthanide-based long-lifetime labels. The developed probes have many potential applications in CK2-related studies such as screening of inhibitors, determination of binding affinity and kinetics of the inhibitors or quantifying the expression level of CK2 in cell lysates.
Results and discussion
Summary and conclusions For the construction of high-affinity sensors for CK2 determination based on measurement of protein binding-responsive long-lifetime photoluminescence, 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) were conjugated with an oligo-(l-aspartic acid) peptide for construction of sensitive CK2 sensors. Conjugation led to compounds ARC-1527 and ARC-1529, both of which revealed remarkable phosphorescence in complex with CK2 upon excitation with near-UV radiation while free compounds in buffer solution possessed no long-lifetime emission. Labeling of these compounds with the fluorescent dye 5-TAMRA gave probes ARC-1528 and ARC-1530 that possessed significantly higher long-lifetime (microsecond-scale) luminescence intensities in complex with CK2 than their unlabeled precursors. This strong signal enhancement resulted from efficient radiationless intramolecular Förster-type resonant energy transfer from the thiazole- or selenazole-comprising donor phosphor in the excited triplet state to the non-excited acceptor fluorophore 5-TAMRA, leading to the excited singlet state of the latter and tardy light emission at the emission spectral region 5-TAMRA dye. Very efficient energy transfer between adjacent luminophores well competes with various non-radiative decay pathways of the triplet state resulting in significant dye-mediated enhancement of phosphorescence signal. ARC-1528 and ARC-1530 can be used in simple mix-and-measure type binding assays with time-gated read-out to determine the concentration of the active form of CK2 (α and α’) and characterize non-labeled inhibitors in displacement assays. Compared to the previously reported benzoselenadiazole-based long-lifetime photoluminescence probe of CK2, disclosed by us four years ago, the ATB- and ASB-related probes have more than an order of magnitude higher affinity towards CK2 and the probe ARC-1530 possesses an order of magnitude stronger luminescence signal. ARC-1530 is thus the brightest highly selective probe that can be used to measure very low concentration of CK2. In our settings, isolated CK2α and CK2α’ could be detected at concentrations starting from 25 pM. In addition, the probes offer selectivity of detection both in the level of recognition of the target enzyme and in the level of appearance of the long-lifetime photoluminescence signal upon binding to the target enzyme. That makes these probes useful for determination of the concentration of the active form of CK2 in complex biological solutions such as cell lysates, as it was also demonstrated in the current report. Free ARC-1530 does not possess long-lifetime photoluminescence and therefore might be used in large excess. In displacement assay it opens the possibility to characterize very potent inhibitors whose differentiation would be impossible with other methods because of tight-binding conditions inherent to assays in use.41, 48 Moreover, monitoring the displacement of unlabeled inhibitors by the protein-responsive long-lifetime probes gives access to dissociation rate constants of those inhibitors.