Rational drug-design approach supported with thermodynamic studies — a peptide leader for the efficient bi-substrate inhibitor of protein kinase CK2

Abstract

Numerous inhibitors of protein kinases act on the basis of competition, targeting the ATP binding site. In this work, we present a procedure of rational design of a bi-substrate inhibitor, complemented with biophysical assays. The inhibitors of this type are commonly engineered by combining ligands carrying an ATP-like part with a peptide or peptide-mimicking fragment that determines specificity. Approach presented in this paper led to generation of a specific system for independent screening for efficient ligands and peptides, by means of thermodynamic measurements, that assessed the ability of the identified ligand and peptide to combine into a bi-substrate inhibitor. The catalytic subunit of human protein kinase CK2 was used as the model target. Peptide sequence was optimized using peptide libraries [KGDE]-[DE]-[ST]-[DE]3–4-NH2, originated from the consensus CK2 sequence. We identified KESEEE-NH2 peptide as the most promising one, whose binding affinity is substantially higher than that of the reference RRRDDDSDDD peptide. We assessed its potency to form an efficient bisubstrate inhibitor using tetrabromobenzotriazole (TBBt) as the model ATP-competitive inhibitor. The formation of ternary complex was monitored using Differential Scanning Fluorimetry (DSF), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC).