The 3/4- and 3/6-Subfamily Variants of α-Conotoxins GI and MI Exhibit Potent Inhibitory Activity against Muscular Nicotinic Acetylcholine Receptors

Abstract

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.

Marine natural products

“…656 Synthesised 3/4- and 3/6-, but not 3/7-, loop-size variants of α-conotoxins GI and MI are potent inhibitors of muscular nAChR's. 657 Both the α4/7-conotoxin CIC (venom of C. catus ) and an N -terminus truncated mutant selectively inhibit α3β2 and α6/α3β2β3 nAChR, suggesting the N -terminal tail can accommodate structural changes without any effect on observed receptor activity. 658 α4/7-Conotoxin LvIF, a 16-residue synthesised peptide based upon genomic DNA clone data derived from C. lividis also exhibits potent inhibition of rα3β2 and rα6/α3β2β3 nAChR's.…”

A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABABR-Coupled N-Type Calcium Channel

“…According to the potency, the disulfide bond connectivity of several AuIB variants folded by one-step oxidation was determined by comparison with peptide folding products of known disulfide connectivity, as described previously [32,33]. Briefly, linear AuIB variants containing an acetamidomethyl (Acm)-protecting group at the C1-C3 or C1-C4 positions were synthesized and then folded by incubation in 0.1 M NH 4 HCO 3 (pH 8.0) at room temperature for 24-72 h. The folded products were further oxidized with an iodine mixture (30% CH 3 CN, 2% TFA, 68% H 2 O) for 10 min to form disulfide bond connectivity "C1-C3, C2-C4" or "C1-C4, C2-C3," and then co-analyzed with a one-step folding product using HPLC.…”