Exploiting stereo-electronic effects (i.e. gauche effect; Fig. ) to favor the 528 Pro exopucker conformation observed in the TACC3/Aurora-A structure was more profitable. Incorporation of fluorine has proven effective in controlling the prolyl-ring preference, with trans-(4-F)Pro favoring the C γ -exo-pucker conformation at a 6:1 ratio, and cis-(4-F)Pro biasing the equilibrium to the opposite endo-conformer in an estimated 20:1 ratio. When TACC3522-536 variants based on both possible (4-F)Pro isomers (i.e. cis or trans-fluoroproline) were tested using competition FA assays (Fig. ), we observed that the exo-pucker stabilizing trans-isomer had ~3-fold increased TACC3/Aurora-A inhibitory potency (TACC3522-536-trans-(4-F)528P IC50 = 60 ± 5µM vs TACC3522-536 IC50 = 163 ± 13 µM; Fig. ). In contrast, incorporation of the endo-pucker stabilizing cis-(4-F)Pro residue resulted in the opposing effect, reducing the inhibitory potency of the peptide to IC50 ≥ 200 µM. Overall, these data (reinforced with MD simulations on the trans-(4-F)Pro variant Fig. -12 for further discussion) demonstrate that the TACC3/Aurora-A interaction is sensitive to the conformation of Pro528TACC3 and that inhibitory potency can be tuned using synthetic variation.