DSPE-PEG Modification of α-Conotoxin TxID

Abstract

In order to improve stability of a peptide marine drug lead, α-conotoxin TxID, we synthesized and modified TxID at the N-terminal with DSPE-PEG-NHS by a nucleophilic substitution reaction to prepare the DSPE-PEG-TxID for the first time. The reaction conditions, including solvent, ratio, pH, and reaction time, were optimized systematically and the optimal one was reacted in dimethyl formamide at pH 8.2 with triethylamine at room temperature for 120 h. The in vitro stabilities in serum, simulated gastric juice, and intestinal fluid were tested, and improved dramatically compared with TxID. The PEG-modified peptide was functionally tested on α3β4 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus laevis oocytes. The DSPE-PEG-TxID showed an obvious inhibition effect on α3β4 nAChR. All in all, the PEG modification of TxID was improved in stability, resistance to enzymatic degradation, and may prolong the half-life in vivo, which may pave the way for the future application in smoking cessation and drug rehabilitation, as well as small cell lung cancer.

Keywords: PEG modification; stability; α-conotoxin TxID; α3β4 nAChR.

Figure 1

(A) Sequence and disulfide bond connection of TxID, # represents a C-terminal amide; (B) RP-UPLC chromatogram of TxID; (C) ESI-MS data of TxID.

Figure 2

(A) Synthesis scheme of DSPE-PEG-TxID; (B) MADLI-TOF spectrum of DSPE-PEG-TxID; (C) MADLI-TOF spectrum of DSPE-PEG-NHS.

Figure 3

Chromatograms of reaction optimization under various conditions, the changes in solvent (A), ratio of polypeptide to PEG (B), pH (C) and reaction time (D) (see Table 2).

Figure 4

α-Conotoxin TxID and DSPE-PEG-TxID were tested on neuronal rat α3β4 nAChR subtype expressed in Xenopus laevis oocytes. The representative current traces showing the inhibition of rat α3β4 ACh-evoked currents by TxID (A) and DSPE-PEG-TxID (B).

Figure 5

Stability of TxID and DSPE-PEG-TxID in serum (A), simulated gastric fluid (SGF) (B), and simulated intestinal fluid (SIF) (C). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the TxID.