Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (<b>4</b>) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was >40 fold more potent (<i>K</i> _i = 2.3 μM) than <b>4</b>. Linear and macrocyclic peptides exhibited unexpected differences in structure-activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of <b>31</b> in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of <b>31</b> suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors.