Amino Acid Scanning at P5′ within the Bowman–Birk Inhibitory Loop Reveals Specificity Trends for Diverse Serine Proteases

Abstract

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.

Sunflower Trypsin Inhibitor‐1 (SFTI‐1): Sowing Seeds in the Fields of Chemistry and Biology

“…Initially, this approach was applied to generate a 12‐member inhibitor library to screen the P1 specificity of trypsin, chymotrypsin, and neutrophil elastase, together with a 21‐member library to screen the P4′‐position [27a] . In recent studies, SFTI‐based libraries were produced to screen preferred residues at P2′ and P5′ using an engineered variant 34 that has multi‐target activity [43b, 46] . Each inhibitor library contained more than 20 variants and was screened against at least 10 separate proteases, with the specificity profiles for each target identifying useful substitutions for inhibitor design [23, 39, 43b, 46] .…”

Ranacyclin-NF, a Novel Bowman–Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus

“…Taken together, the different trypsin inhibitory activities of RNF and ranacyclin-T could be generally explained by three points: (1) different secondary structures; (2) different cationicities; and (3) different residues at the P5 position. In a previous study, amino acid scanning at the P5 position within the BBIs inhibitory loop, revealed that the residue Gln (Q) was the optimal residue at the P5 position with a relatively higher inhibitory activity in both bovine beta-trypsin and human cationic trypsin inhibitory studies [4]. Therefore, residues at the P5 position might not be the main reason for induction of different activities.…”

“…To construct rational explanations for the different trypsin inhibitory activities between ranacyclin-T and RNF and to further study structure and trypsin inhibitory activity relationships, we compared some structural parameters of these two peptides (Table 3). Compared with RNF, ranacyclin-T contained a larger net positive charge and a different residue (Lys) at the P5′ position-a position thought to play important role in the trypsin inhibitory activity of naturally-occurring BBIs [4]. Analysis of predicted secondary structures also revealed that ranacyclin-T may adopt a beta-sheet structure which was different for RNF3L ( Figure 7).…”

“…Although most current serine protease-targeted drugs are small molecules, there is an increasing attention shift to polypeptide inhibitors as these more complex molecules offer an achievable approach for optimising the affinity and specificity of inhibitors. Inhibitor scaffolds are normally contained in natural proteins or peptides which have been optimised and improved through a process of parallel evolution for activities against endogenous targets [4].…”

A Chameleonic Macrocyclic Peptide with Drug Delivery Applications

“…The grafting concept has been applied to a number of macrocycles, including both SFTI-1 and the larger cyclotides, and used in disease models for cancer 20,21 , multiple sclerosis 22 , or to improve proangiogenic activity 23 . Rather than replacing the entire bioactivity with a new segment, the inherent protease inhibitory activity of SFTI-1 can also be tuned to specifically target different proteases 24,25 .…”