As an incomplete kinase, 26,27 control of Aurora-A localization and activation is achieved through interaction with its intrinsically disordered clients. Indeed, TACC3 behaves as an allosteric activator of Aurora-A, whereby the protein binds to and activates the kinase, then in turn is phosphorylated, and recruited to microtubules. Such client/Aurora-A interactions cover a significant surface area on the kinase and so delineating the minimal structural determinants and changes needed to promote kinase activity is challenging. Similarly, orthosteric inhibitors of Aurora-A PPIs that simultaneously inactivate the kinase are unlikely to be differentiated from active site kinase inhibitors. We therefore assessed the effect of the peptidomimetics on Aurora-A kinase activity by performing Aurora-A autophosphorylation experiments in vitro (Fig. ). When unphosphorylated Aurora-A was incubated in the presence of the constrained peptides and ATP, they were found to stimulate Aurora-A kinase activity more effectively when compared to the WT unconstrained control peptide, with the effect being qualitatively more pronounced for the L/R constrained variants. ADP-Glo assays provided further quantitative data on the phosphorylation activity of the protein (10 nM) in the presence of excess substrate (kemptide; 100 µM) at increasing concentrations of the linear and constrained peptides (Fig. ). These experiments illustrate that both linear WT variants are relatively poor activators of the kinase, whereas the constrained peptidomimetics stimulate Aurora-A and promote substrate phosphorylation in a dose-dependent manner by up to 160-190%.