s8a.m1.p41 De Novo Design and Protein X-ray Analysis of a Protein Kinase Inhibitor

In pharmaceutical research, lead structures are still mainly identified by screening. When the threedimensional structure of the target protein is known, however, this information can be used to design, de novo, molecules fitting the protein binding site. The molecules designed by molecular modeling can then be synthesized or retrieved from a database if they have already been made. A biological activity in the micromolar range or below warrants consideration as a potential lead structure. Following this approach, we have identified PKF049365 as a representative of a new class of inhibitors of the cell cycle kinases CDK1/2. The three-dimensional structure of the CDK2/PKF049-365 complex was subsequently determined by protein crystallography and refined to 1.53Å resolution. The X-ray analysis shows that PKF049-365 binds to the adenine pocket of the ATP binding site and interacts with the hinge region of the kinase molecule, as designed by molecular modeling. However, two overlapping and hence mutually exclusive binding modes are detected, which are characterized by a different pattern of hydrogen-bonded interactions with the main chain of the hinge region of the kinase. In the first binding mode, which corresponds exactly to the prediction of modeling, N2 of the pyrazole ring of PKF049-365 receives an H-bond from the amide nitrogen of Leu 83 and N1 donates an H-bond to the carbonyl oxygen of the same residue In the second binding mode, N2 of the pyrazole ring donates a H-bond to the carbonyl oxygen of Glu 81, and N1 receives an H-bond from the amide nitrogen of Leu 83. Therefore, the observed binding modes involve two distinct tautomeric forms of PKF049-365, differing by the presence of a proton either on N1 or N2 of the pyrazole ring of the inhibitor. These two alternate binding modes appear to be equally populated. Furthermore, switching from one to the other binding mode only requires a small translation/rotation of the inhibitor molecule within the same plane and does not induce any significant structural rearrangement of the kinase.