Stability and Safety of Inhibitor Cystine Knot Peptide, GTx1-15, from the Tarantula Spider Grammostola rosea.

Tadashi Kimura

doi:10.3390/toxins13090621

Abstract

Inhibitor cystine knot (ICK) peptides are knotted peptides with three intramolecular disulfide bonds that affect several types of ion channels. Some are proteolytically stable and are promising scaffolds for drug development. GTx1-15 is an ICK peptide that inhibits the voltage-dependent calcium channel Cav3.1 and the voltage-dependent sodium channels Nav1.3 and Nav1.7. As a model molecule to develop an ICK peptide drug, we investigated several important pharmaceutical characteristics of GTx1-15. The stability of GTx1-15 in rat and human blood plasma was examined, and no GTx1-15 degradation was observed in either rat or human blood plasma for 24 h in vitro. GTx1-15 in blood circulation was detected for several hours after intravenous and intramuscular administration, indicating high stability in plasma. The thermal stability of GTx1-15 as examined by high thermal incubation and protein thermal shift assays indicated that GTx1-15 possesses high heat stability. The cytotoxicity and immunogenicity of GTx1-15 were examined using the human monocytic leukemia cell line THP-1. GTx1-15 showed no cytotoxicity or immunogenicity even at high concentrations. These results indicate that GTx1-15 itself is suitable for peptide drug development and as a peptide library scaffold.

Highlights

Ion channels play essential roles in a wide range of biological processes and have become attractive pharmaceutical targets related to various conditions such as hypertension, diabetes, pain and cancer [2,3,4,5].Bioactive peptides from various venomous animals serve as a basis for peptide drug development; examples include ziconotide from cone snails for pain control [6] and exendin-4 from the Gila monster lizard for diabetes treatment [7]
GTx1-15 concentrations in circulation gradually decreased and kept dropping within 4 and 8 h dose-dependently, meaning that GTx1-15 is stable in blood circulation, which is supported by a previous report showing that GTx1-15 has proteolytic resistance, i.e., GTx1-15 is not degraded by trypsin, chymotrypsin, pepsin or elastase [22]
In the PERISS method, a target protein and a peptide library are coexpressed in the E. coli inner-membrane and periplasmic space, respectively [34]

Summary

Introduction

Bioactive peptides from various venomous animals serve as a basis for peptide drug development; examples include ziconotide from cone snails for pain control [6] and exendin-4 from the Gila monster lizard for diabetes treatment [7]. Spider venoms contain various types of bioactive peptides that have attracted attention as peptide drug candidates [8,9,10,11,12,13,14]. Short peptides cannot form structural motifs and are likely to be readily degraded by proteases. Known as cystine-knot mini-proteins, are characteristic structural motifs due to multiple covalent bonds formed between side chain thiols of cysteine residues that are spatially distant from each other [15]. The knotted rigid structure is a common feature of knottins and is thought to endow them with unusual proteolytic, thermal and chemical stability [16,17]

Methods

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Discussion

Conclusion