Asn85 N-Myc /Gly89 N-Myc , and Glu86 N-Myc /Ser90 N-Myc , could be explored. These pairs of residues were replaced with cysteines, and constrained (mal), oxidized (ox), or reduced (red). The sequences and key CD spectral data for these 6 new variants are shown in Table . Using competition FA, both new maleimide constrained variants showed improved IC50 potencies compared to the unmodified peptide (N-Myc73-94 IC50 = 199 ± 13 μM; Table , Fig. ). Indeed, maleimide constrained N-Myc73-94-N85C/G89C-mal (IC50 = 49 ± 5 μM) was found to exhibit 4-fold improvement (ΔΔG ~ 3.5 kJ mol -1 relative to N-Myc73-94) in potency to disrupt N-Myc/Aurora-A compared to the WT peptide. The inhibitory potency exhibited by the constrained N-Myc73-94-N85C/G89C-mal variant was found to be in the same range as that observed for the longer WT sequence (N-Myc61-89, IC50 = 42 ± 4 μM; Table ), despite lacking the N-terminal turn region and being ~25% shorter. The second variant, N-Myc73-94-E86C/S90C-mal (IC50= 111 ± 18 μM) was also found to be 2-fold more potent than the control peptide. For the most potent sequence (N-Myc73-94-N85C/G89C-mal), a number of features should be noted: (i) Gly89 N-Myc is replaced with cysteine to introduce the mal constraint and glycine is known to have low helix propensity; Ser90 N-Myc has the potential to act as a helix-capping residue before Pro91 N-Myc which likely interrupts the helical structure. Finally, based on the Aurora-A/N-Myc crystal structure, a mal constraint introduced at Asn85 N-Myc and Gly89 N-Myc is unlikely to be completely solvent-exposed and may form direct contacts with the surface of Aurora-A surface. Overall, these results highlight a crucial role in selecting an appropriate sequence length to allow identification of optimal sites for constraining peptide ligands. .