D-Amino Acid Substitution of α-Conotoxin RgIA Identifies its Critical Residues and Improves the Enzymatic Stability.

Jie Ren,Yong Wu,Sulan Luo,Pan Xu,Shuai Dong,Rui Li,Dongting Zhangsun,Ying Fu,Xiaopeng Zhu

doi:10.3390/md17030142

Abstract

α-Conotoxin RgIA is a selective and potent competitive antagonist of rat α9α10 nicotinic acetylcholine receptors (nAChR), but it is much less potent towards human α9α10 nAChR. Furthermore, RgIA is susceptible to proteolytic degradation due to containing four arginine residues. These disadvantages greatly limit its use for clinical applications. The purpose of this research was to identify critical stereocenters of RgIA and discover more stable analogues, enhancing its bioavailability by using the d-amino acid scan method. The activity of each variant was investigated against rat and human α9α10 nAChRs, which were expressed in Xenopus oocytes. Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat α9α10 nAChR. Noticeably, analogue 13 retained full biological activity compared with RgIA. Meanwhile, two other analogues, 14 and 15, of which l-Args were substituted with d-Args, exhibited a significantly increased potency towards human α9α10 nAChR, although these analogues showed decreased activities against rat α9α10 nAChR. Additionally, these three analogues exhibited a high resistance against enzymatic degradation in human serum and simulated intestinal fluid (SIF). Collectively, our findings suggest that a d-amino acid scan is a useful strategy for investigating how the side-chain chirality of amino acids affects the structure and function of peptides and may facilitate the development of more stable analogues to increase therapeutic potential.

Highlights

Venoms from the Conus species comprise a mixture of peptides called conotoxins (CTxs) that target voltage- and ligand-gated ion channels with unparalleled selectivity and potency [1,2,3]
Ω-CTx MVIIA isolated from the venom of Conus magus targets a specific voltage-gated calcium channel subtype (Cav 2.2) and was approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) as a novel type of analgesic, whereas some other CTxs have entered clinical or preclinical trials [4,5,6]
We subsequently examined the stability of these peptides in simulated intestinal fluid (SIF)

Summary

Introduction

Venoms from the Conus species comprise a mixture of peptides called conotoxins (CTxs) that target voltage- and ligand-gated ion channels with unparalleled selectivity and potency [1,2,3]. These excellent properties provide potential for development as neuropharmacological probes or leads for drug development. Α-CTx RgIA is composed of 13 amino acids, belonging to the 3/4 subgroup, and was initially cloned from Conus regius [7] This peptide has an extremely high affinity and selectivity for the rat α9α10 nAChR, which is an available receptor for investigating neuropathic pain treatment [8,9,10].

Objectives

Methods

Conclusion