Abstract
Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes - such as protein kinases - adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand K d, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.