• aspartic proteases;
  • drug discovery;
  • FRET;
  • inhibitors;
  • peptides


The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease β-secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax, KM, and kcat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.