Three N-methylated SIM variants with ΔΔG values close to that of the parent SIM peptide (Me-Ile2708, Me-Ile2712, Me-Lys2717) and two variants with positive ΔΔG values (Me-Val2710 and Me-Lys216) were selected (Fig. ) to interrogate the determinants of binding affinity, relative to the parent SIM peptide. Using one-dimensional 1 H NMR experiments, we ruled out changes in the population of the cis isomer at the N-methylated amide bond and changes in peptide oligomeric state as drivers of the observed changes in potency (Figs. S2).