We finally assessed if the increase in potency was gained at the expense of binding pocket specificity. We therefore tested TACC3518-532-L/R-Bph-I/fP in FA competition experiments against TPX2 and N-Myc (Fig. ). Similarly to TACC3, TPX21-43 has been shown to bind Aurora-A at the N-lobe of the protein. However, it binds to the opposing face, via two separate segments linked by a disordered region that can interact and insert into the W, F and Y pockets on Aurora-A (Fig. , TPX21-43; PDB: 1OL5). On the other hand, N-Myc has been shown to bind between the N-and C-lobes to a groove adjacent to the activation loop of the kinase (N-Myc61-89; PDB: 5G1X). In FA competition assays, the constrained peptidomimetics exhibited limited evidence of TPX2 displacement (IC50 >> 200 µM versus TPX27-43 IC50, = 20 ± 1 µM; see Fig. ) indicating promising specificity for the TACC3 binding site. More surprisingly, the constrained variants were observed to out-compete N-Myc (Fig. and Fig. ). The most potent constrained variant (TACC3518-532-L/R-Bph-I/fP IC50 = 28 ± 1 µM) was more potent than the control N-Myc sequence (N-Myc61-89, IC50 = 56 ± 5 µM). To assess if these observations could be the consequence of orthosteric competition for the N-Myc binding site we performed the reverse experiment where acetylated N-Myc61-89 was employed to displace fluorescentlylabeled TACC-3518-532-L/R-Bph-I/fP (Fig. ). This N-Myc sequence was also observed to outcompete the TACC3 peptide.