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

• Published in: Biochimica et biophysica acta. Proteins and proteomics Vol. 1868; no. 8; p. 140427
• Main Authors: Nonga, Olivier Etebe, Lavogina, Darja, Ivan, Taavi, Viht, Kaido, Enkvist, Erki, Uri, Asko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2020
• Subjects: ATP-binding pocket; Bisubstrate inhibitor; cAMP-dependent protein kinase (PKA); Inhibitory antibody; Ligand-induced epitope restructuring; Time-gated luminescence; Ligand-induced epitope restructuring; Time-gated luminescence; cAMP-dependent protein kinase (PKA); Bisubstrate inhibitor; ATP-binding pocket; Inhibitory antibody
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2020.140427

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. •Inhibition of protein kinase A by monoclonal antibody mAb(D38C6).•Epitope restructuring leading to kinase dissociation from the complex with antibody.•Development of an immunoassay for the kinase with specificity and high sensitivity.•Immunoassay application to cell lysates and determination of cellular concentration.