With two amino-acid modifications that enhanced binding potency of TACC3 to Aurora-A and two constrained backbones identified, we assessed the extent to which these modifications could be productively combined in a single compound (Fig. ). Four modified peptides were assessed: TACC3518-532-L/R-Bph-I and TACC3518-532-L/R-Bph-Br based on the L520C/R526C biphenylconstrained structure carrying either (4-I) 525 F or (4-Br) 525 F modifications, and the corresponding peptides: TACC3518-532-S/E-Bph-I and TACC3518-532-S/E-Bph-Br, based on the alternative S524C/E530C constrained template (Fig. ). All variants exhibited IC50 values of ~ 20-30 µM against the WT peptide (Fig. ). Next, we assessed the effect of incorporating the trans- ; Figs. ). These variants showed lower IC50 values in competition experiments than previous compounds, with all peptides exhibiting IC50 values in the range 10-22 ± 2 µM (the limit of sensitivity for the assay). To more accurately assess binding potencies to Aurora-A, we employed direct FA titrations of the protein in the presence of FAMlabelled peptides and assessed their corresponding dissociation constants, Kd. Twelve-fold improved Kd values were measured for the constrained peptides when compared to the linear sequences (Fig. ), with all variants showing low micromolar/high nanomolar affinities. MD simulations indicate that these combined modifications act as designed i.e. the halogenated phenylalanine better occupies its pocket and the trans-fluoroproline favors the exo-pucker conformation (Fig. ).