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Mar Drugs
2021 Dec 14;1912:. doi: 10.3390/md19120705.
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The 3/4- and 3/6-Subfamily Variants of α-Conotoxins GI and MI Exhibit Potent Inhibitory Activity against Muscular Nicotinic Acetylcholine Receptors.
Ma X, Huang Q, Yu S, Xu S, Huang Y, Zhao Z, Xiao X, Dai Q.
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α-Conotoxins GI and MI belong to the 3/5 subfamily of α-conotoxins and potently inhibit muscular nicotinic acetylcholine receptors (nAChRs). To date, no 3/4- or 3/6-subfamily α-conotoxins have been reported to inhibit muscular nAChRs. In the present study, a series of new 3/4-, 3/6-, and 3/7-subfamily GI and MI variants were synthesized and functionally characterized by modifications of loop2. The results show that the 3/4-subfamily GI variant GI[∆8G]-II and the 3/6-subfamily variants GI[+13A], GI[+13R], and GI[+13K] displayed potent inhibition of muscular nAChRs expressed in Xenopus oocytes, with an IC50 of 45.4-73.4 nM, similar to or slightly lower than that of wild-type GI (42.0 nM). The toxicity of these GI variants in mice appeared to be about a half to a quarter of that of wild-type GI. At the same time, the 3/7-subfamily GI variants showed significantly lower in vitro potency and toxicity. On the other hand, similar to the 3/6-subfamily GI variants, the 3/6-subfamily MI variants MI[+14R] and MI[+14K] were also active after the addition of a basic amino acid, Arg or Lys, in loop2, but the activity was not maintained for the 3/4-subfamily MI variant MI[∆9G]. Interestingly, the disulfide bond connectivity "C1-C4, C2-C3" in the 3/4-subfamily variant GI[∆8G]-II was significantly more potent than the "C1-C3, C2-C4" connectivity found in wild-type GI and MI, suggesting that disulfide bond connectivity is easily affected in the rigid 3/4-subfamily α-conotoxins and that the disulfide bonds significantly impact the variants' function. This work is the first to demonstrate that 3/4- and 3/6-subfamily α-conotoxins potently inhibit muscular nAChRs, expanding our knowledge of α-conotoxins and providing new motifs for their further modifications.
Figure 1. HPLC analyses of one-step folding products of linear GI variants. (A) GI[ΔG8]-II. (B) GI[+13K]. (C) GI[+13K, +14A]. (D) GI[+13G, +14G].Traces from bottom to top: linear peptide (a), folded products (b), purified products (c). Samples were applied to an Agilent Eclipse Plus C18 column (5 μm, 4.6 mm×250 mm) and eluted with a linear gradient of 5–10% B for 0–1 min; 10–50% B (B is acetonitrile containing 0.1% trifluoroacetic acid (TFA)) for 1–25 min. Absorbance was monitored at 214 nm. Flow rate was 1.0 mL/min.
Figure 2. HPLC analyses of the folded products of linear GI and MI variants with Acm modification. (A) Linear GI[Δ8G]-I with Acm modifications at Cys1 and Cys3. (B) Linear GI[Δ8G]-II with Acm modifications at Cys1 and Cys4. (C) Linear GI[+13K] with Acm modifications at Cys1 and Cys3. (D) Linear GI[+13G, +14G] with Acm modifications at Cys1 and Cys3. (E) Linear MI[Δ9G]-I with Acm modifications at Cys1 and Cys3. (F) Linear MI[Δ9G]-II with Acm modifications at Cys1 and Cys4. Traces from bottom to top: linear peptide (a), primary oxidized product (b), secondary oxidized product (c), and co-elution of the two-step folding product and one-step natural-air folding oxidation product (d). HPLC analysis conditions are the same as in Figure 1.
Figure 3. Circular dichroism spectra of α-conotoxins and their variants in 0.01 M PBS (pH = 7.2). (A) GI mutants. (B) MI mutants. n = 3.
Figure 4. Concentration-dependent response curves of the GI and MI variants interacting with rat muscular nAChRs. (A) GI variants; (B) MI variants. The error bars for the data denote the SEM. Five to seven oocytes were used for each determination. The IC50 values and 95% confidence intervals are summarized in Table 1.
Figure 5. Kinetic analyses of GI and MI variants interacting with Xenopus oocyte-expressed rat muscular nAChRs. (A) GI, (B) GI[∆8G], (C) GI[+13A] (D) MI, (E) MI[+14R]. The toxins were applied as described in Materials and Methods, and the data were fit to a single exponential equation. The error bars denote the SEM of the data obtained using four to seven oocytes for each determination. The kinetic data are summarized in Table 2.
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