Fragment-Linking Approach Using ¹⁹F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of β‑Secretase

Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with ¹⁹F NMR-based methods has been shown to provide several advantages over ¹H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of ¹⁹F-based fragment screening by detailing the identification of a second-site fragment through ¹⁹F NMR screening that binds to a specific pocket of the aspartic acid protease, β-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.