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Mar Drugs
2024 Aug 29;229:. doi: 10.3390/md22090390.
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Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor.
Yu J
,
Xie J
,
Ma Y
,
Wei P
,
Zhang P
,
Tang Z
,
Zhu X
,
Zhangsun D
,
Luo S
.
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α-Conotoxins are disulfide-rich peptides obtained from the venom of cone snails, which are considered potential molecular probes and drug leads for nAChR-related disorders. However, low specificity towards different nAChR subtypes restricts the further application of many α-conotoxins. In this work, a series of loop1 amino acid-substituted mutants of α-conotoxin RegIIA were synthesized, whose potency and selectivity were evaluated by an electrophysiological approach. The results showed that loop1 alanine scanning mutants [H5A]RegIIA and [P6A]RegIIA blocked rα7 nAChR with IC50s of 446 nM and 459 nM, respectively, while their inhibition against rα3β2 and rα3β4 subtypes was negligible, indicating the importance of the fifth and sixth amino acid residues for RegIIA's potency and selectivity. Then, second-generation mutants were designed and synthesized, among which the analogues [H5V]RegIIA and [H5S]RegIIA showed significantly improved selectivity and comparable potency towards rα7 nAChR compared with the native RegIIA. Overall, these findings provide deep insights into the structure-activity relationship of RegIIA, as well as revealing a unique perspective for the further modification and optimization of α-conotoxins and other active peptides.
2022GXNSFBA035662 the Guangxi Natural Science Foundation, 82104059 the National Natural Science Foundation of China, 2023GXNSFAA026039 the Guangxi Natural Science Foundation, 2024GXNSFBA010423 the Guangxi Natural Science Foundation, D20010 the 111 Project, GUIKE AD22035948 the Guangxi Science and Technology Base and Talents Fund, 2022YFE0132700 the Major Intergovernmental Joint Research Project of the National Key R & D Program of China
Figure 1. Sequences of the native RegIIA and its analogues. Conserved cysteine residues are marked in blue. Disulfide connectivity (I–III, II–IV) is indicated by lines connecting the Cys residues. The amino acids substituted in mutants are marked in red. # indicates the C-terminal amide. O, hydroxyproline (Hyp).
Figure 2. Relative current amplitude (%) of RegIIA and its first-generation mutants at a concentration of 10 μM on rα3β2 (A), rα3β4 (B), and rα7 (C). The inhibition of 10 μM [H5A]RegIIA and [P6A]RegIIA (both marked in red) were less than 50% on rα3β2 and rα3β4 subtypes, which were comparable with that of the native RegIIA on rα7 nAChR. All data were conducted at a holding potential of −70 mV and analyzed from 6–8 separate oocytes.
Figure 3. α-Conotoxin RegIIA (A), as well as its analogs [H5A]RegIIA (B) and [P6A]RegIIA (C), differentially blocking the current mediated by the rα3β2, rα3β4, and rα7 nAChR subtypes. Superimposed representative ACh-evoked currents were recorded from Xenopus laevis oocytes expressing rα3β2, rα3β4, and rα7 nAChRs in the absence (black trace) and presence (red trace) of incubation with 10 μM RegIIA or its analogs.
Figure 4. Concentration–response curves for the inhibition of the rα3β2 (A), rα3β4 (B), and rα7 nAChR (C) by RegIIA and its first-generation mutants. The concentration–response curves of [H5A]RegIIA and [P6A]RegIIA towards rα7 nAChR are shifted to the right compared to the native RegIIA, while their inhibition on the rα3β2 and rα3β4 subtypes were not obvious. All data points represent mean ± SEM values from 6–8 separate oocytes.
Figure 5. Concentration–response curves for the inhibition of the rα3β4 (A) and rα7 nAChR (B) by RegIIA’s second-generation mutants. The second-generation mutants showed substantial loss in potency towards all three nAChR subtypes, except for [H5S]RegIIA and [H5V]RegIIA, which exhibited only a ~2-fold potency decrease towards rα7 nAChR and >56-fold potency decrease towards rα3β2 and rα3β4 nAChRs, suggesting a >24-fold increase in selectivity compared with the native RegIIA. All data points represent mean ± SEM values from 6–8 oocytes.
Figure 6. Interactions between RegIIA and its mutants with the extracellular domains (ECDs) of rα3β2, rα3β4, and rα7 nAChRs. (A) The overall structure of RegIIA binding to the ECD of the rα3β2 nAChR. (B) The overall structure of RegIIA binding to the ECD of the rα3β4 nAChR. (C) The overall structure of RegIIA binding to the ECD of the rα7 nAChR. (D) Detailed interactions of RegIIA with the rα3β2 nAChR, displaying all hydrogen bonds. Disulfide bonds between the side chains of C2–C8 and C3–C16 are shown. (E) Detailed interactions of RegIIA with the α3β4 nAChR, showing all hydrogen bonds. Disulfide bonds between the side chains of C2–C8 and C3–C16 are indicated. (F) Detailed interactions of RegIIA with the α7 nAChR, illustrating all hydrogen bonds. Disulfide bonds between the side chains of C2–C8 and C3–C16 are displayed. (G) Detailed interactions of RegIIA H5 and P6 with the α3β2 nAChR. Hydrogen bonds are formed between the side chain of H5 of RegIIA and the side chains of K142 and Y187 of the α3 subunit. (H) Detailed interactions of RegIIA H5 and P6 with the α3β4 nAChR. Hydrogen bonds are formed between the side chain of H5 of RegIIA and the oxygen of the main chain of Q195 of the α3 subunit. (I) Detailed interactions of H5 and P6 of RegIIA with the α7 nAChR. No hydrogen bonds are formed between H5 and the surrounding amino acids. (J) Interactions of the [H5A]RegIIA with the α7 nAChR, displaying all hydrogen bonds. Disulfide bonds between the side chains of C2–C8 and C3–C16 are shown. (K) Interactions of the [P6A]RegIIA with the α7 nAChR, illustrating all hydrogen bonds. Disulfide bonds between the side chains of C2–C8 and C3–C16 are indicated. Hydrogen bonds are represented by black dashed lines, with the cutoff set at 3.5 Å. Blue, Nitrogen; Red, Oxygen; Yellow, Sulfur.
Figure 7. CD spectra of native RegIIA and its mutants. All CD spectra of examined peptides showed negative peaks at 208 nm and 222 nm, indicating the existence of α-helices, while the mutations H5A, H5V, and H5S made the most changes in secondary structure.
Figure 8. Serum stability of RegIIA and its analogues. The Stability of [H5S]RegIIA and [H5V]RegIIA vs. RegIIA were assayed in human serum. All peptides were dissolved in 100% human serum AB type and incubated at 37 °C. As the results indicated, mutations H5S and H5V led to significant improvements in stability after 48 h incubation in human serum. ** p < 0.01, * p < 0.05 (two-way ANOVA). All results are expressed as means ± SEM (n = 3).
Figure S3: Amino acid sequence alignment of the N-terminal extracellular domains of rat and human α7, as well as α3, β2, and β4 nAChR subunits
Figure S4: Interactions between RegIIA and its mutants with the ECDs of hα3β2, hα3β4, and hα7 nAChRs; additional figures illustrating the RP-HPLC and ESI-MS analysis of RegIIA and its mutants (Figures S1 and S2).
