Structure and function of benzoy Iurea-derived A-helix mimetics targeting the Bcl-x_L/bak binding interface

The Bel-x_LBak protein-protein interaction has emerged as an important target for cancer therapy due to its role in apopto-sis. Inhibition of this interaction by small-molecule antagonists induces apoptosis in unhealthy cells. Bak, a pro-apoptotic Bcl-2 protein, projects four hydrophobic side chains (V74, L78, 181, and 185), corresponding to the i, i+4, i+7, and i+11 positions of an α-helix, into a hydrophobic cleft on Bcl-x_L. Herein, we present a novel family of rationally designed α-helix mimetics with improved solubility and synthetic feasibility based on a benzoylurea scaffold. These benzoylurea derivatives favor a linear conformation stabilized by an intramolecular hydrogen bond, and are able to mimic the spatial projection of the i, i+4, and i+7 residues of and α-helix. The binding of the benzoylurea derivatives to Bcl-x_Lwas assessed using fluorescence polarization competition assays, isothermal titration calorimetry, and "N-HSQC experiments. These experiments showed that these agents bind to and disrupt Bcl-x_L with low micromolar inhibition and dissociation constants, with ¹⁵N-HSQC experiments confirming binding to the hydrophobic pocket of Bcl-x_L normally occupied by the Bak helix.