Structural Insight Into Protein Kinase D Small Molecule Inhibition

Protein kinase D (PKD) is a family of serine/threonine kinases that has emerged as a novel therapeutic target in multiple diseases; therefore, the development of small molecule inhibitors that abrogate PKD’s function has been an area of intense investigation. However, these efforts have not yet resulted in a PKD therapeutic that is available for clinical applications. Access to structural information can greatly accelerate this drug discovery process and allow future initiatives to benefit from structure-based drug design. Therefore, we employed both computational and experimental methods to investigate the important characteristics of PKD small molecule inhibitors that allow for excellent potency and selectivity. We found that type I ATP-competitive PKD inhibitors bind to the hinge region and position a hydrogen bond acceptor near the charged side chain of Lys612. An additional moiety (typically a basic amino group) capable of developing a critical salt bridge with Glu710 was shown to be important for improved affinity. Further analysis from inhibitor selectivity profiles suggested strategic functionalization in pocket II could potentially reduce kinase off-target effects. From our experimental investigation of CRT0066101 inhibition, we developed a chemical genetic tool kit for evaluating PKD function. Mutating the gatekeeper residue, Met659, to phenylalanine or tyrosine dramatically sensitized PKD to CRT0066101 inhibition. Conversely, mutating hinge residue, G664, to valine or isoleucine conferred resistance. Taken together, our study provides key insights into the chemical features associated with PKD inhibition, which will aid in the future development of a novel PKD therapeutic