A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa

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.

Transcriptomic Analysis of Four Cerianthid (Cnidaria, Ceriantharia) Venoms

“…Because ShK-scaffolds are abundant in sea anemone venom peptides, characterizing the venoms from sea anemones (and cnidarians in general) could yield additional candidates for novel therapeutic compounds [ 30 , 34 , 35 ]. Kunitz-domain containing serine inhibitors, also found in sea anemone venoms, can also be used as potential therapeutic resources [ 25 , 36 ]. These cnidarian-derived neuropeptide inhibitors have potential applications as analgesics, antiepileptics, and other neuroprotective drugs [ 37 ].…”

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity

“…Serine proteases and their inhibitors are involved in a wide variety of cellular processes (e.g. coagulation, inflammation) and therefore, are molecules of interest when targeting therapeutic applications [ 1 , 2 ]. A commonly studied family of serine protease inhibitors is the one containing a Kunitz type motif.…”

Characterising Functional Venom Profiles of Anthozoans and Medusozoans within Their Ecological Context

“…Therefore, MSI offers the opportunity to analyse peptide mixtures and evaluate peptide localisation for any species. This approach has been applied to the imaging of venom toxins from sea anemones, snakes and centipedes to date [16,25,[140][141][142][143][144]. Identification of venom components directly from MSI spectra, however, remains non-trivial.…”

“…MSI of toxins is therefore most informative when analysed in light of a venom peptidome obtained by more "traditional" venomic approaches, such as combined transcriptomic and venom proteomic analyses [25]. It has also contributed to understanding the variable tissue expression patterns of toxins within order Actiniaria, having been employed to visualise both widely distributed and highly localised toxins in A. tenebrosa [16,25,140]. Taking into account the presence of enzymes within sea anemone venom, the use of MSI as a novel assay to investigate the regulation of enzyme activity also confers considerable utility [144].…”