Mixed\nStereochemistry Macrocycle Acts as a Helix-Stabilizing\nPeptide N‑Cap

Interactions\nbetween proteins and α-helical peptides have\nbeen the focus of drug discovery campaigns. However, the large interfaces\nformed between multiple turns of an α-helix and a binding protein\nrepresent a significant challenge to inhibitor discovery. Modified\npeptides featuring helix-stabilizing macrocycles have shown promise\nas inhibitors of these interactions. Here, we tested the ability of\nN-terminal to side-chain thioether-cyclized peptides to inhibit the\nα-helix binding protein Mcl-1, by screening a trillion-scale\nlibrary. The enriched peptides were lariats featuring a small, four-amino-acid\nN-terminal macrocycle followed by a short linear sequence that resembled\nthe natural α-helical Mcl-1 ligands. These “Heliats”\n(helical lariats) bound Mcl-1 with tens of nM affinity, and inhibited\nthe interaction between Mcl-1 and a natural peptide ligand. Macrocyclization\nwas found to stabilize α-helical structures and significantly\ncontribute to affinity and potency. Yet, the 2nd and 3rd positions\nwithin the macrocycle were permissible to sequence variation, so that\na minimal macrocyclic motif, of an <i>N</i>-acetylated d-phenylalanine at the 1st position thioether connected to a\ncysteine at the 4th, could be grafted into a range of peptides and\nstabilize helical conformations. We found that d-stereochemistry\nis more helix-stabilizing than l- at the 1st position in\nthe motif, as the d-amino acid can utilize polyproline II\ntorsional angles that allow for more optimal intrachain hydrogen bonding.\nThis mixed stereochemistry macrocyclic N-cap is synthetically accessible,\nrequiring only minor modifications to standard solid-phase peptide\nsynthesis, and its compatibility with peptide screening can provide\nready access to helix-focused peptide libraries for <i>de novo</i> inhibitor discovery.