Cystine Knot Peptides with Tuneable Activity and Mechanism.

Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.

Binding Loop Substitutions in the Cyclic Peptide SFTI-1 Generate Potent and Selective Chymase Inhibitors.

Chymase is a serine protease that is predominantly expressed by mast cells and has key roles in immune defense and the cardiovascular system. This enzyme has also emerged as a therapeutic target for cardiovascular disease due to its ability to remodel cardiac tissue and generate angiotensin II. Here, we used the nature-derived cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1) as a template for designing novel chymase inhibitors. The key binding contacts of SFTI-1 were optimized by combining a peptide substrate library screen with structure-based design, which yielded several variants with potent activity. The lead variant was further modified by replacing the P1 Tyr residue with para-substituted Phe derivatives, generating new inhibitors with improved potency (Ki = 1.8 nM) and higher selectivity over closely related enzymes. Several variants were shown to block angiotensin I cleavage in vitro, highlighting their potential for further development and future evaluation as pharmaceutical leads.

Iterative Optimization of the Cyclic Peptide SFTI-1 Yields Potent Inhibitors of Neutrophil Proteinase 3.

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.

Amino Acid Scanning at P5' within the Bowman-Birk Inhibitory Loop Reveals Specificity Trends for Diverse Serine Proteases.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with a sequence and structure similar 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 SFTI variants 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 7 of the scaffold's 14 residues retains a similar structure to SFTI-1. These findings provide new insights into P5' specificity requirements for the Bowman-Birk inhibitory loop.

Characterising the Subsite Specificity of Urokinase-Type Plasminogen Activator and Tissue-Type Plasminogen Activator using a Sequence-Defined Peptide Aldehyde Library.

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1-P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (Ki =18 nm), but not for tPA. Additionally, synthetic peptide substrates corresponding to preferred inhibitor sequences were cleaved with high catalytic efficiency by uPA but not by tPA. These findings provide new insights into the binding specificity of uPA and tPA and the relative activity of tetrapeptide inhibitors and substrates against these enzymes.

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 (Ki = 30 ±â€¯0.8 pM) but shows 165,000-fold weaker activity against mesotrypsin (Ki = 4.96 ±â€¯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.

Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold.

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 (Ki = 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 (Ki = 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.