A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Xingchen Chen,Eivind A B Undheim,Bruno Madio,Peter J Prentis,Ana Pavasovic,Jonathan M Harris,Jessica Van Haeften,Darren Leahy,Joachim M Surm,Maria Brattsand,Perry Hartfield,Glenn F King,Brett R Hamilton,Chloé A Van Der Burg

doi:10.3390/md17120701

Abstract

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor’s stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Highlights

Serine proteases are pivotal regulators of multifarious cellular activities through the activation of protein precursors, such as prohormones, pro-enzymes [1], and protease-activated receptors [2].Serine proteases play important roles in disease progression [3] and cancer [4]
We previously reported on this phenomenon when carrying out substitutions within the sunflower trypsin inhibitor (SFTI-1) [6], a robust, 14-residue cyclized peptide stabilized by a bisecting disulfide bond and extensive internal H-bonds
We demonstrated that substitutions within the SFTI-1 binding loop could redirect its inhibitory selectivity and potency [6]

Summary

Introduction

Serine proteases are pivotal regulators of multifarious cellular activities through the activation of protein precursors, such as prohormones, pro-enzymes [1], and protease-activated receptors [2].Serine proteases play important roles in disease progression [3] and cancer [4]. The bioscaffolding approach has seen many successes with examples including ecallantide (Kalbitor® ), a variant of tissue factor pathway inhibitor-1 [8], which is being used clinically for the treatment of hereditary angioedema [9], and hirudin variants, such as lepirudin (Refludan® ), which is used for treatment of heparin-induced thrombocytopenia [10]. The success of these bioengineered protein-based drugs relies on both the potency and the selectivity of inhibition they show for their targets. The ability to selectively target a given protease is especially important for therapeutics as exemplified by the failure of early metalloprotease inhibitors undergoing development for use in anti-cancer therapies [11]

Methods

Results

Conclusion