Inhibitors and fluorescent probes for protein kinase PKAcß and its S54L mutant, identified in a patient with cortisol producing adenoma.

Recently, a mutation was discovered in the gene PRKACB encoding the catalytic subunit ß of PKA (PKAcß) from a patient with severe Cushing's syndrome. This mutation, S54L, leads to a structural change in the glycine-rich loop of the protein. In the present study, an inhibitor with six-fold selectivity toward S54L-PKAcß mutant over the wild-type enzyme was constructed. Moreover, we developed a fluorescent assay allowing to determine side by side the affinity of commercially available PKA inhibitors, newly synthesized compounds, and fluorescent probes toward PKAcß and S54L-PKAcß.

Discovery of strong inhibitory properties of a monoclonal antibody of PKA and use of the antibody and a competitive photoluminescent orthosteric probe for analysis of the protein kinase.

We show that the antibody, clone mAb(D38C6), of the α isoform of the catalytic subunit of PKA (PKAcα) inhibits the kinase-catalyzed phosphorylation with low-nanomolar inhibitory potency (Ki = 2.4 nM). This property of the antibody was established by its capacity to displace a synthetic small-molecule active site-binding (orthosteric) photoluminescent ARC-Lum(Fluo) probe from the complex with PKAcα. Likely, the competitiveness of association of the two binders with the protein is coming from two excluding conformations of PKAcα to which the binders bind. mAb(D38C6) possesses a linear peptide epitope and it binds to the disordered C-tail of unliganded inactive conformer of PKAcα. ARC-Lum(Fluo) probes bind to the ordered and active conformation of PKAcα with Phe327 residue from the C-tail taking part in the formation of the active core. Consecutive application of these competitive PKAcα binders was used to develop an immunoassay allowing the determination of PKAcα concentration in complex biological solutions. At first, PKAcα was captured from the solution by the isoform-specific antibody and thereafter a high-affinity ARC-Lum(Fluo) probe was used to displace PKAcα from the binary complex. The developed immunoassay could be used for quantification of small amounts (starting from 93 pg, 2.3 fmol) of PKAcα in cell lysates.