Engineering potent mesotrypsin inhibitors based on the plant-derived cyclic peptide, sunflower trypsin inhibitor-1

Plants produce a diverse range of peptides and proteins that inhibit the activity of different serine proteases. The value of these inhibitors not only stems from their native role(s) in planta, but they are also regarded as promising templates for inhibitor engineering. Interest in this field has grown rapidly in recent years, particularly for therapeutic applications. The serine protease mesotrypsin has been implicated in several cancers, but is a challenging target for inhibitor engineering as a number of serine protease inhibitors that typically display broad-range activity show limited activity against mesotrypsin. In this study, we use a cyclic peptide isolated from sunflower seeds, sunflower trypsin inhibitor-1 (SFTI-1), as a scaffold for engineering potent mesotrypsin inhibitors. SFTI-1 comprises 14-amino acids and is a potent inhibitor of human cationic trypsin (K_i = 30 ± 0.8 pM) but shows 165,000-fold weaker activity against mesotrypsin (K_i = 4.96 ± 0.2 μM). Using an inhibitor library based on SFTI-1, we show that the inhibitor's P2' residue (Ile) is a key contributor to SFTI-1's limited activity against mesotrypsin. Substituting P2' Ile with chemically diverse amino acids, including non-canonical aromatic residues, produced new inhibitor variants that maintained a similar structure to SFTI-1 and showed marked improvements in activity (exceeding 100-fold). An assessment of the activity of the new inhibitors against closely-related trypsin paralogs revealed that the improved activity against mesotrypsin was accompanied by a loss in activity against off-target proteases, such that several engineered variants showed comparable activity against mesotrypsin and human cationic trypsin. Together, these findings identify potent mesotrypsin inhibitors that are suitable for further optimisation studies and demonstrate the potential gains in activity and selectivity that can be achieved by optimising the P2' residue, particularly for engineered SFTI-based inhibitors.