Stapling Cysteine[2,4] Disulfide Bond of α-Conotoxin LsIA and Its Potential in Target Delivery

Abstract

α-Conotoxins, as selective nAChR antagonists, can be valuable tools for targeted drug delivery and fluorescent labeling, while conotoxin-drug or conotoxin-fluorescent conjugates through the disulfide bond are rarely reported. Herein, we demonstrate the [2,4] disulfide bond of α-conotoxin as a feasible new chemical modification site. In this study, analogs of the α-conotoxin LsIA cysteine[2,4] were synthesized by stapling with five linkers, and their inhibitory activities against human α7 and rat α3β2 nAChRs were maintained. To further apply this method in targeted delivery, the alkynylbenzyl bromide linker was synthesized and conjugated with Coumarin 120 (AMC) and Camptothecin (CPT) by copper-catalyzed click chemistry, and then stapled between cysteine[2,4] of the LsIA to construct a fluorescent probe and two peptide-drug conjugates. The maximum emission wavelength of the LsIA fluorescent probe was 402.2 nm, which was essentially unchanged compared with AMC. The cytotoxic activity of the LsIA peptide-drug conjugates on human A549 was maintained in vitro. The results demonstrate that the stapling of cysteine[2,4] with alkynylbenzyl bromide is a simple and feasible strategy for the exploitation and utilization of the α-conotoxin LsIA.

Keywords: disulfide bond stapling; fluorescent probe; peptide-drug conjugates; target delivery; α-conotoxin.

Scheme 1

Synthetic routes of α-Conotoxin LsIA, LsIA[2,4]-1, 2, 3, 4, 5, and LsIA[1,3]-1 (# signifies C-term amidation). (A) Oxidative folding process of LsIA. (B) Cysteine[2,4] linker stapling of LsIA. (C) Cysteine[1,3] linker stapling of LsIA. (D) Structures of the linkers. Reagents and conditions: (a) K₃[Fe(CN)₆], Tris-HCl, MeCN, ultrapure water, rt, and 45 min; (b) I₂, TFA, MeCN, ultrapure water, rt, 10 min, and vitamin C; (c) Chemical scaffolds 1–5, NH₄HCO₃ buffer, MeCN, rt, and 65 min. (E) Illustration of the chemical scaffolds 1, 2, 3, 4, and 5 after reaction with the sulfhydryl groups of cysteines.

Figure 1

HPLC and ESI-MS profiles of linear LsIA (A), LsIA (B), and its analogs (C–H). The chromatographic conditions were 10% to 55% buffer B in buffer A with a flow rate of 1 mL/min at UV-214 nm over 20 min, and the column temperature was 40 °C. A = 0.1% trifluoroacetic acid in H₂O and B = 0.1% trifluoroacetic acid in acetonitrile. AU, absorbance units.

Figure 2

Concentration–response curves of LsIA analogs on human α7 and rat α3β2 nAChRs. Each data point was presented as the mean ± SEM of 3–10 separated oocytes.

Figure 3

Circular dichroism (CD) spectra of LsIA and its analogs.

Scheme 2

Synthetic route of LsIA[2,4]-6, 7, 8, and 9. Reagents and conditions: (a) CuSO₄·5H₂O, sodium ascorbate, MeCN-H₂O, rt, and overnight; (b) NH₄HCO₃ buffer, MeCN, rt, and 65 min; (c) I_2, TFA, MeCN, H₂O, rt, and 10 min.

Figure 4

HPLC and ESI-MS profiles of LsIA[2,4]-6 (A), LsIA[2,4]-7 (B), LsIA[2,4]-8 (C), and LsIA[2,4]-9 (D). The chromatographic conditions were 10% to 55% buffer B in buffer A with a flow rate of 1 mL/min at UV-214 nm over 20 min, and the column temperature was 40 °C. A = 0.1% trifluoroacetic acid in H₂O and B = 0.1% trifluoroacetic acid in acetonitrile. AU, absorbance units.

Figure 5

Fluorescence emission spectra of AMC (red), LsIA[2,4]-6 (grey), and LsIA[2,4]-7 (blue). Concentration: 100 μM, excitation at 340 nm.

Figure 6

CCK8 experiment to evaluate the cytotoxicity of LsIA, CPT, LsIA[2,4]-8, and LsIA[2,4]-9 at 2.5 nM, 25 nM, 250 nM, 2.5 μM, and 25 μM. Each data point represents mean ± SEM (n = 5).