Structure-Based Design of High-Affinity and Selective Peptidomimetic Hepsin Inhibitors

In many solid tumors, increased upregulation of transmembrane serine proteases (TTSPs) leads to an overactivation of growth factors, which promotes tumor progression. Here, we have used a combinatorial methodology to develop high-affinity tetrapeptidic inhibitors. A previous virtual screening of 8000 peptide combinations against the crystal structure of the TTSP hepsin identified a series of recognition sequences, customized for the non-prime substrate binding (P) sites of this serine protease. A combination of the top recognition sequences with an electrophilic warhead resulted in highly potent inhibitors with good selectivity against coagulation proteases factor Xa and thrombin. Structure–activity relationships of two selected compounds were further elucidated by investigation of their stability in biological fluids as well as the influence of the warhead and truncated inhibitors on the inhibitory potency. Overall, this methodology yielded compounds as selective inhibitors for potential cancer drug development, where hepsin is overexpressed.