Neutrophils are directly responsible for destroying invading pathogens via reactive oxygen species, antimicrobial peptides, and neutrophil serine proteases (NSPs). Imbalance between NSP activity and endogenous protease inhibitors is associated with chronic inflammatory disorders, and engineered inhibitors of NSPs are a potential therapeutic pathway. In this study we characterized the extended substrate specificity (P4-P1) of the NSP cathepsin G using a peptide substrate library. Substituting preferred cathepsin G substrate sequences into sunflower trypsin inhibitor-1 (SFTI-1) produced a potent cathepsin G inhibitor (K = 0.89 nM). Cathepsin G's P2' preference was determined by screening against a P2' diverse SFTI-based library, and the most preferred residue at P2' was combined in SFTI-1 with a preferred substrate sequence (P4-P2) and a nonproteinogenic P1 residue (4-guanidyl-l-phenylalanine) to produce a potent (K = 1.6 nM) and the most selective (≥360-fold) engineered cathepsin G inhibitor reported to date. This compound is a promising lead for further development of cathepsin G inhibitors targeting chronic inflammatory disorders.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with similar sequence and structure to a larger family of serine protease inhibitors, the Bowman-Birk inhibitors. Here, we focus on the P5′ residue in the Bowman-Birk inhibitory loop and produce a library of SFTIvariants to characterize the P5′ specificity of 11 different proteases. We identify seven amino acids that are generally preferred by these enzymes and also correlate with P5′ sequence diversity in naturally occurring Bowman-Birk inhibitors. Additionally, we show that several enzymes have divergent specificities that can be harnessed in engineering studies. By optimizing the P5′ residue, we improve the potency or selectivity of existing inhibitors for kallikrein-related peptidase 5 and show that a variant with substitutions at seven of the scaffold's fourteen residues retains a similar structure to SFTI-1. These findings provide new insights into P5′ specificity requirements for the Bowman-Birk inhibitory loop.

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 = 30 ± 0.8 pM) but shows 165,000-fold weaker activity against mesotrypsin (K = 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.

Neutrophils produce at least four serine proteases that are packaged within azurophilic granules. These enzymes contribute to antimicrobial defense and inflammation but can be destructive if their activities are not properly regulated. Accordingly, they represent therapeutic targets for several diseases, including chronic obstructive pulmonary disease, cystic fibrosis, and rheumatoid arthritis. In this study, we focused on proteinase 3 (PR3), a neutrophil protease with elastase-like specificity, and engineered potent PR3 inhibitors based on the cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1). We used an iterative optimization approach to screen targeted substitutions at the P1, P2, P2′, and P4 positions of SFTI-1, and generated several new inhibitors with K i values in the low nanomolar range. These SFTI-variants show high stability in human serum and are attractive leads for further optimization.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with similar sequence and structure to a larger family of serine protease inhibitors, the Bowman-Birk inhibitors.Here, we focus on the P5′ residue in the Bowman-Birk inhibitory loop and produce a library of SFTIvariants to characterize the P5′ specificity of 11 different proteases. We identify seven amino acids that are generally preferred by these enzymes and also correlate with P5′ sequence diversity in naturally occurring Bowman-Birk inhibitors. Additionally, we show that several enzymes have divergent specificities that can be harnessed in engineering studies. By optimizing the P5′ residue, we improve the potency or selectivity of existing inhibitors for kallikrein-related peptidase 5 and show that a variant with substitutions at seven of the scaffold's fourteen residues retains a similar structure to SFTI-1. These findings provide new insights into P5′ specificity requirements for the Bowman-Birk inhibitory loop.