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.

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure–activity relationship (SAR) between GI and mouse α1β1δε nAChRs. The Pro⁵, Gly⁸, Arg⁹, and Tyr¹¹ were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1β1δε nAChR. On the contrary, substituting Asn⁴, His¹⁰ and Ser¹² with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1β1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1β1δε nAChR, and Tyr¹¹ of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu⁹³, Tyr⁹⁵ and Leu¹⁰³. Our research elucidates the interaction of GI and mouse α1β1δε nAChR in detail that will help to develop the novel analogues of GI.

α-Conotoxin TxID was discovered from Conus textile by gene cloning, which has 4/6 inter-cysteine loop spacing and selectively inhibits α3β4 nicotinic acetylcholine receptor (nAChR) subtype. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID using a combination of electrophysiological recordings and molecular modelling. The results showed most TxID analogues had substantially decreased activities on α3β4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on α3β4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at α3β4 nAChR with an IC₅₀ of 69 nM, which only exhibited 3.8-fold less compared with TxID. Molecular dynamics simulations were performed to expound the decrease in the affinity for α3β4 nAChR. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign conotoxins containing methionine residue.