The structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C), a prototype for the protein kinase superfamily, laid the foundation for the development of targeted kinase inhibitors. Here we describe the structure and biophysical characterization of a PKA-C complex with BLU0588, a small PKA-selective inhibitor. The high-resolution crystal structure not only captures the inhibitor’s unusual T-shaped geometry, but also shows how the four rings of BLU0588 serve as surrogates for ATP’s adenosine and phosphate-organizing sites. Each site contains two subsites. BLU0588’s planar azaindole and pyridine rings, which are buried beneath the glycine-rich loop in a hydrophobic shell at the base of the active site cleft, fill the adenine and ribose subsites. In contrast, BLU0588’s indane and pyrrolidine rings fill the phosphate-organizing site. The indane ring occupies the α/β-phosphate organizing site while the pyrrolidine ring fills the Mg/γ-phosphate organizing site. The structure also shows how BLU0588 nucleates an open but stable conformation of the entire hydrophobic architecture of the N- and C-lobes. In addition to potently blocking phosphoryl transfer activity, BLU0588 also abolishes the synergistic high-affinity binding of the physiological pseudosubstrate inhibitor, protein kinase inhibitor. The residence time of BLU0588, measured by surface plasmon residence, contributes to its picomolar affinity and is distinct from H89, a commonly used but more promiscuous PKA inhibitor. These molecular insights provide a valuable framework for dissecting the organization of the active site cleft as well as different strategies for the rational design of more potent and selective kinase inhibitors in general.
Bruystens et al. (Thu,) studied this question.