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Mar Drugs
2019 Feb 28;173:. doi: 10.3390/md17030142.
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d-Amino Acid Substitution of α-Conotoxin RgIA Identifies its Critical Residues and Improves the Enzymatic Stability.
Ren J
,
Zhu X
,
Xu P
,
Li R
,
Fu Y
,
Dong S
,
Zhangsun D
,
Wu Y
,
Luo S
.
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α-Conotoxin RgIA is a selective and potent competitive antagonist of rat α9α10 nicotinic acetylcholine receptors (nAChR), but it is much less potent towards human α9α10 nAChR. Furthermore, RgIA is susceptible to proteolytic degradation due to containing four arginine residues. These disadvantages greatly limit its use for clinical applications. The purpose of this research was to identify critical stereocenters of RgIA and discover more stable analogues, enhancing its bioavailability by using the d-amino acid scan method. The activity of each variant was investigated against rat and human α9α10 nAChRs, which were expressed in Xenopus oocytes. Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat α9α10 nAChR. Noticeably, analogue 13 retained full biological activity compared with RgIA. Meanwhile, two other analogues, 14 and 15, of which l-Args were substituted with d-Args, exhibited a significantly increased potency towards human α9α10 nAChR, although these analogues showed decreased activities against rat α9α10 nAChR. Additionally, these three analogues exhibited a high resistance against enzymatic degradation in human serum and simulated intestinal fluid (SIF). Collectively, our findings suggest that a d-amino acid scan is a useful strategy for investigating how the side-chain chirality of amino acids affects the structure and function of peptides and may facilitate the development of more stable analogues to increase therapeutic potential.
81872794, 81660585 and 31860246 National Natural Science Foundation of China, 81420108028 Major International Joint Research Project of National Natural Science Foundation of China, IRT_15R15 Changjiang Scholars and Innovative Research Team in University Grant
Figure 1. Sequence and structure of RgIA and its d-amino acid scan analogues. (A) The sequence of RgIA. Disulfide connectivity of CysI-CysIII and CysII-CysIV is labeled with black lines. Amino acids in loop I and loop II are marked in blue and purple, respectively. (B) NMR structure of RgIA (PDB ID 2JUT) [30]. (C) d-amino acid scan analogues of RgIA. The amino acids indicated by red lower case letters are d-amino acids. a Compound number. The # indicates a C-terminal amide.
Figure 2. Analytical RP-HPLC and ESI-MS analysis of RgIA and 13. The peptides were determined on a Vydac C18 column (4.6 à 250 mm, 5 μm) with a flow rate of 1 mL/min. The gradient of RP-HPLC was 10% buffer B ramping linearly to 40% over 20 min, where buffer A is 0.65% trifluoroacetic acid (TFA) in water, and buffer B is 0.5% TFA and 90% acetonitrile in water. UV detection was performed at 214 nm. (A) HPLC chromatogram of RgIA with a retention time of 13.64 min. (B)ESI-MS profile of RgIA with a mass of 1571.13 Da. (C) HPLC chromatogram of peptide 13 with a retention time of 14.36 min. (D) ESI-MS profile for peptide 13 with a mass of 1571.13 Da.
Figure 3. RgIA and its D-scan substitution were tested on rat and human α9α10nAChR. (A) ACh-evoked current inhibition of rat α9α10 nAChR by D-scan analogues (open bars) versus native RgIA (filled bar) at 100 nM; (B) ACh-evoked current inhibition of human α9α10 nAChR by RgIA (filled bar) and its analogues (open bars). Analogues incorporating higher or similar inhibition activity compared with wild-type peptide l (blue bars). The difference between the relative current amplitude of RgIA and each mutant was evaluated using one-way analysis of variance (ANOVA) followed by Dunnettâs t test; and the P values are indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Error bars represent the mean ± SEM (n = 3â11).
Figure 4. Concentration-response curves of RgIA and its D-amino acid substitutions for inhibition of α9α10 nAChR. (A) RgIA and its mutants were applied to rat α9α10 nAChR; (B) Peptides were tested on human α9α10 nAChR. Values are mean ± SEM from 5â11 separate oocytes.
Figure 5. Relative stability of RgIA, 13, 14, and 15. (A) Stability of RgIA, and peptides 13, 14, and 15 in human serum. (B) Stability of RgIA, 13, 14, and 15 in the SIF. Error bars represent the mean ± SEM (n = 3).
Figure 6. Representative CD spectra of wild-type RgIA and its variants. (A) The spectra of the native RgIA and peptide 13; (B) The spectra of the native RgIA, and peptides 14 and 15.
Figure 7. Molecular models of the interactions between wildtype RgIA, peptide 13, and rat α9α10 nAChR. The α9 subunit is drawn in green, the α10 subunit is in cyan, and the peptides are in white. (A) RgIA bound with the rat α9(+)α10(â) interface, where all arginine residues in RgIA are labeled. (B) The molecular model is shown between peptide 13 and α9(+)α10(â) interface, and amino acids around 4 à radius of the r-13 are labeled. (C) RgIA bound with rat α10(+)α9(â) interface, where all arginine residues in RgIA are labeled. (D) The molecular model is shown between peptide 13 and α10(+)α9(â) interface. All images were produced by PyMOL.
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