Consistent with our previous report, the undecorated parent homodimeric CC-Di peptide did not show a response in the BID/MCL-1 FA competition assay, whereas increasing the number of grafted hot-spot residues led to increased inhibition (CC-Di_S < CC-Di_E1 < CC-Di_E2, Fig. ). Similarly, the 1:1 mixture of CC-Di-A and CC-Di-B_S showed inhibition (IC50 = 46 ± 5 µM, Fig. ). None of these coiled-coil assemblies acted as BID/BCL-xL inhibitors (Fig. ). Next we determined the binding affinities of the coiled-coil sequences with modified a and d positions that change the oligomerization state of the peptide scaffold to explore multivalent binding. The monomeric control peptide, Mono_E1, was comparable in potency to NOXA-B and CC-Di_E1 (IC50 = 0.20 ± 0.05 µM, Fig. ). This result may seem surprising. However, MCL-1 is known to recognize its ligands through a bind-and-fold mechanism. Thus, the high alanine content in the sequence favors adoption of the MCL-1 bound conformation, and the sequence contains eight hot-spot residues. Trimeric CC-Tri_E1 and tetrameric CC-Tet_E1 scaffolds acted as BID/MCL-1 inhibitors with IC50 values of 68 ± 16 µM and 7.4 ± 0.8 µM, respectively (Fig. ). Thus, compared to the dimer CC-Di_E1 (IC50 = 0.8 ± 0.1 µM), grafting the E1 sequence into higher-order trimeric and tetrameric coiled coils resulted in a significant decrease in potency; as the valency of binding sites increased, potency decreased. This behavior may reflect stepwise decreases in the accessible surface areas of the binding site with increasing oligomerization state (Table -2). In other words, higher-order coiled-coil assemblies occlude the grafted binding motifs, and, thus, coiled-coil oligomerization begins to compete with MCL-1 binding. This effect will be compounded as increasing numbers of target proteins are recruited to the scaffolds; i.e, bound proteins will increasingly crowd the scaffold surface and further occlude the binding sites.