Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Mar Drugs
2019 Feb 28;173:. doi: 10.3390/md17030142.
Show Gene links
Show Anatomy links
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.
???displayArticle.abstract???
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 × 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 Dunnett’s 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 = 3–11).
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 5–11 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(–) interface, where all arginine residues in RgIA are labeled. (B) The molecular model is shown between peptide 13 and α9(+)α10(–) interface, and amino acids around 4 Å radius of the r-13 are labeled. (C) RgIA bound with rat α10(+)α9(–) interface, where all arginine residues in RgIA are labeled. (D) The molecular model is shown between peptide 13 and α10(+)α9(–) interface. All images were produced by PyMOL.
Adams,
Analgesic conotoxins: block and G protein-coupled receptor modulation of N-type (Ca(V) 2.2) calcium channels.
2012, Pubmed
Adams,
Analgesic conotoxins: block and G protein-coupled receptor modulation of N-type (Ca(V) 2.2) calcium channels.
2012,
Pubmed Agersø,
Pharmacokinetics and renal excretion of desmopressin after intravenous administration to healthy subjects and renally impaired patients.
2004,
Pubmed Akondi,
Discovery, synthesis, and structure-activity relationships of conotoxins.
2014,
Pubmed Anil,
Exploiting the right side of the Ramachandran plot: substitution of glycines by D-alanine can significantly increase protein stability.
2004,
Pubmed Azam,
Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA.
2012,
Pubmed
,
Xenbase Azam,
Molecular interaction of α-conotoxin RgIA with the rat α9α10 nicotinic acetylcholine receptor.
2015,
Pubmed Chhabra,
Dicarba analogues of α-conotoxin RgIA. Structure, stability, and activity at potential pain targets.
2014,
Pubmed Christensen,
RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.
2017,
Pubmed
,
Xenbase Clark,
The three-dimensional structure of the analgesic alpha-conotoxin, RgIA.
2008,
Pubmed Di Cesare Mannelli,
α-conotoxin RgIA protects against the development of nerve injury-induced chronic pain and prevents both neuronal and glial derangement.
2014,
Pubmed Ellison,
Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR.
2006,
Pubmed
,
Xenbase Ellison,
Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10 nAChR: structure and identification of key receptor-binding residues.
2008,
Pubmed Everhart,
Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors.
2004,
Pubmed
,
Xenbase Gao,
Cone Snails: A Big Store of Conotoxins for Novel Drug Discovery.
2017,
Pubmed Grieco,
D-Amino acid scan of gamma-melanocyte-stimulating hormone: importance of Trp(8) on human MC3 receptor selectivity.
2000,
Pubmed Grishin,
Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3β4 nicotinic acetylcholine receptors.
2013,
Pubmed
,
Xenbase Halai,
Effects of cyclization on stability, structure, and activity of α-conotoxin RgIA at the α9α10 nicotinic acetylcholine receptor and GABA(B) receptor.
2011,
Pubmed
,
Xenbase Halai,
Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor.
2009,
Pubmed
,
Xenbase Hone,
Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3β2β3 and α3β2 nicotinic acetylcholine receptors.
2013,
Pubmed
,
Xenbase Hruby,
Designing peptide receptor agonists and antagonists.
2002,
Pubmed Jamieson,
Peptide scanning for studying structure-activity relationships in drug discovery.
2013,
Pubmed Jia,
D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP.
2017,
Pubmed Kompella,
Alanine scan of α-conotoxin RegIIA reveals a selective α3β4 nicotinic acetylcholine receptor antagonist.
2015,
Pubmed
,
Xenbase Lewis,
Conus venom peptide pharmacology.
2012,
Pubmed Lindorff-Larsen,
Improved side-chain torsion potentials for the Amber ff99SB protein force field.
2010,
Pubmed Luo,
Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist.
2015,
Pubmed
,
Xenbase McIntosh,
Alpha9 nicotinic acetylcholine receptors and the treatment of pain.
2009,
Pubmed
,
Xenbase Mohammadi,
Conotoxin Interactions with α9α10-nAChRs: Is the α9α10-Nicotinic Acetylcholine Receptor an Important Therapeutic Target for Pain Management?
2015,
Pubmed Morales-Perez,
X-ray structure of the human α4β2 nicotinic receptor.
2016,
Pubmed Nielsen,
Effects of chirality at Tyr13 on the structure-activity relationships of omega-conotoxins from Conus magus.
1999,
Pubmed Peeters,
D-amino acid and alanine scans of the bioactive portion of porcine motilin.
1992,
Pubmed Pérez,
Molecular modeling of the alpha9alpha10 nicotinic acetylcholine receptor subtype.
2009,
Pubmed Pronk,
GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit.
2013,
Pubmed Romero,
Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain.
2017,
Pubmed Roth,
Structure-activity studies on neuropeptide S: identification of the amino acid residues crucial for receptor activation.
2006,
Pubmed Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed Simon,
d-Amino Acid Scan of Two Small Proteins.
2016,
Pubmed Terlau,
Conus venoms: a rich source of novel ion channel-targeted peptides.
2004,
Pubmed Tugyi,
Partial D-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide.
2005,
Pubmed Vetter,
Therapeutic potential of cone snail venom peptides (conopeptides).
2012,
Pubmed Vincler,
Targeting the alpha9alpha10 nicotinic acetylcholine receptor to treat severe pain.
2007,
Pubmed Vincler,
Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors.
2006,
Pubmed Wu,
α-Conotoxin [S9A]TxID Potently Discriminates between α3β4 and α6/α3β4 Nicotinic Acetylcholine Receptors.
2017,
Pubmed
,
Xenbase Yu,
Single Amino Acid Substitution in α-Conotoxin TxID Reveals a Specific α3β4 Nicotinic Acetylcholine Receptor Antagonist.
2018,
Pubmed Yu,
Molecular Determinants Conferring the Stoichiometric-Dependent Activity of α-Conotoxins at the Human α9α10 Nicotinic Acetylcholine Receptor Subtype.
2018,
Pubmed
,
Xenbase Zhangsun,
αO-Conotoxin GeXIVA disulfide bond isomers exhibit differential sensitivity for various nicotinic acetylcholine receptors but retain potency and selectivity for the human α9α10 subtype.
2017,
Pubmed
,
Xenbase Zouridakis,
Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain.
2014,
Pubmed
,
Xenbase
