High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

“…In recent years, the bisubstrate approach to the design of PK inhibitors has made remarkable progress: improvements in synthetic procedures, sub-nanomolar affinities of lead compounds, and a large variety of applications in novel in vitro and ex vivo assays. [18,19,67] The therapeutic benefit of a PK inhibitor is the protection of the critical protein from over phosphorylation, and the restoration of a healthy phosphorylation balance for the given protein. Due to the overlapping consensus sequences of PKs, the phosphorylation at a particular amino acid residue of the www.chemmedchem.org target protein (including physiologically important targets CREB [69] and Tau [70] protein phosphorylation) can be catalyzed by several PKs.…”

“…[17] During the displacement studies, the binding properties of the bisubstrate inhibitor (usually labeled with a fluorescent tag) to the PK are measured at different concentrations of the substances interacting either with the ATP-or protein/peptidebinding site of the PK. [18,19] This kind of assay does not provide information on the effect of the inhibitor on the catalytic properties of the enzyme, but demonstrates whether the binding of ATP or the peptide substrate would be disturbed in the presence of the inhibitor. The greatest advantage of displacement assays, compared with kinetic assays, is the possibility to use the protein/peptide binding site-targeted inhibitors instead of the corresponding weakly binding substrates.…”

“…[19] In this assay, the change in fluorescence anisotropy of a solution is measured as a response to binding of the free fluorescent probe to the PK (e.g., fluorescent probe AdcAhx-(d-Arg) 6 …”

“…Indeed, in several works where kinetic assays were performed in parallel with binding assays, the bisubstrate inhibitors that were competitive only against ATP in kinetic studies could be displaced from the kinase complex with either ATP or a protein/peptide substrate-competitive inhibitor in binding studies. [18,19,24] Bisubstrate inhibitor-PK co-crystals structures Protein X-ray crystallography is a powerful technique for the determination of static high-resolution structures of proteins in complex with bound ligands. [25,26] In the case of an enzymebisubstrate inhibitor complex, the crystal structure also provides the most definitive evidence for true bisubstrate binding as it reveals whether the inhibitor occupies both substratebinding sites of the enzyme.…”

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2β interaction

“…We obtained a Z' of 0.72 for 48 positive control wells and 48 negative control wells in three separate experiments (Fig. 4A), which is in the range of reported FA-based kinase assays [37,74,75] and proves the chosen assay conditions (0.1 lM CF-Ahx-Pc and 3 lM CK2a 1-335 ) to be suitable for the screening of compound libraries.…”