Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors

Huynh, Peter N.; Harvey, P.J.; Gajewiak, Joanna; Craik, David J.; McIntosh, J. Michael

doi:10.1016/j.bcp.2020.114124

Cited by 12 publications

(16 citation statements)

References 36 publications

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“…A previous RgIA structure–activity relationship (SAR) study indicated that residues in the first loop ( 5 Asp, 6 Pro, and 7 Arg) were responsible for orienting the peptide for binding to nAChRs and 9 Arg, which was important for selectivity toward the α9α10 subtype of nAChR . Previous SAR data also revealed that an Arg13Tyr mutation can enhance RgIA potency. , We sought to potentially capitalize on these findings. β-amino acids are homologs of natural α-amino acids, with an extra methylene group immediately before the carboxylic group in the backbone of the amino acids.…”

Section: Resultsmentioning

confidence: 99%

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism

et al. 2021

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Venom-derived compounds are of broad interest in neuropharmacology and drug development. α-Conotoxins are small disulfide-containing peptides from Conus snails that target nicotinic acetylcholine receptors (nAChRs) and are in clinical development for non-opioid-based treatment of intractable pain. Although refined by evolution for interaction with target prey receptors, enhancements of pharmacological properties are needed for use in mammalian systems. Therefore, we synthesized analogues of α-conotoxin RgIA using a combination of selective penicillamine substitutions together with natural and non-natural amino acid replacements. This approach resulted in a peptide with 9000-fold increased potency on the human α9α10 nAChR and improved resistance to disulfide shuffling compared to the native peptide. The lead analogue, RgIA-5474, potently blocked α9α10 nAChRs, but not opioid- or other pain-related targets. In addition, RgIA-5474 effectively reversed chemotherapy-induced neuropathic pain.

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Section: Resultsmentioning

confidence: 99%

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism

et al. 2021

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“…By comparison, native RgIA inhibits ACh-evoked currents mediated by human α9α10 nAChRs with an IC 50 value of 510 nM due to the low affinity for the human receptor. 33,63 Differential effects were produced when methylene thioacetal was introduced to the sequence of a modified RgIA analogue, RgIA4. Specifically, RgIA-5533, which had the loop II [Cys II −Cys IV ] disulfide exchanged for methylene thioacetal, had low nanomolar potency (IC 50 = 6.1 nM).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…36 Similarly, the importance of the [Cys I − Cys III ] disulfide on the structure and activity was also demonstrated in another α-4/3-CTx ImI by analyzing disulfide-deficient analogues. 63,64 We then modified RgIA-5533 with mutants based on RgIA5 and a noncanonical amino acid, β-homotyrosine (bhTyr), to afford the most potent analogue RgIA-5524 with an IC 50 value of 0.9 nM. 30,46 Single residue mutation of bhTyr with β-Alanine (bAla) yielded an analogue, RgIA-5573, which was less potent (IC 50 = 2.9 nM), indicating the importance of a phenolic moiety at residue position 13, consistent with our recent research of critical residues in RgIA.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…30,46 Single residue mutation of bhTyr with β-Alanine (bAla) yielded an analogue, RgIA-5573, which was less potent (IC 50 = 2.9 nM), indicating the importance of a phenolic moiety at residue position 13, consistent with our recent research of critical residues in RgIA. 63 We next investigated the subtype selectivity of RgIA-5533 and RgIA-5524. TEVC electrophysiology showed that both analogues failed to inhibit a wide range of nAChR subtypes at 10 μM (IC 50 > 10 μM), including α1β1δε, α2β2, α2β4, α3β2, α3β4 α4β2, α4β4, α6/α3β2β3, and α6/α3β4 (Figures 2C, S2).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Discovery of Methylene Thioacetal-Incorporated α-RgIA Analogues as Potent and Stable Antagonists of the Human α9α10 Nicotinic Acetylcholine Receptor for the Treatment of Neuropathic Pain

et al. 2021

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α9-Containing nicotinic acetylcholine receptors (nAChRs) are key targets for the treatment of neuropathic pain. α-Conotoxin RgIA4 is a peptide antagonist of human α9α10 nAChRs with high selectivity. However, structural rearrangement reveals a potential liability for clinical applications. We herein report our designer RgIA analogues stabilized by methylene thioacetal as nonopioid analgesic agents. We demonstrate that replacing disulfide loop I [CysI–CysIII] with methylene thioacetal in the RgIA skeleton results in activity loss, whereas substitution of loop II [CysII–CysIV] can be accommodated. The lead molecule, RgIA-5524, exhibits highly selective inhibition of α9α10 nAChRs with an IC50 of 0.9 nM and much reduced degradation in human serum. In vivo studies showed that RgIA-5524 relieves chemotherapy-induced neuropathic pain in wild type but not α9 knockout mouse models, demonstrating that α9-containing nAChRs are necessary for the therapeutic effects. This work highlights the application of methylene thioacetal as a disulfide surrogate in conotoxin-based, disulfide-rich peptide drugs.

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“…In particular, in continuation of the work [ 151 ], which revealed key residues in α-conotoxin RgIA responsible for its affinity to α9α10 and α7 nAChR subtypes (Asp5, Pro6, Arg7, Arg9), a large series of analogues with substitutions for various residues at positions 7, 9–11, 13 was obtained [ 152 ]. As a result, the fundamental role of Arg7 was confirmed.…”

Section: Marine Origin Peptides Targeting Nachrsmentioning

confidence: 99%

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

et al. 2022

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The purpose of our review is to briefly show what different compounds of marine origin, from low molecular weight ones to peptides and proteins, offer for understanding the structure and mechanism of action of nicotinic acetylcholine receptors (nAChRs) and for finding novel drugs to combat the diseases where nAChRs may be involved. The importance of the mentioned classes of ligands has changed with time; a protein from the marine snake venom was the first excellent tool to characterize the muscle-type nAChRs from the electric ray, while at present, muscle and α7 receptors are labeled with the radioactive or fluorescent derivatives prepared from α-bungarotoxin isolated from the many-banded krait. The most sophisticated instruments to distinguish muscle from neuronal nAChRs, and especially distinct subtypes within the latter, are α-conotoxins. Such information is crucial for fundamental studies on the nAChR revealing the properties of their orthosteric and allosteric binding sites and mechanisms of the channel opening and closure. Similar data are provided by low-molecular weight compounds of marine origin, but here the main purpose is drug design. In our review we tried to show what has been obtained in the last decade when the listed classes of compounds were used in the nAChR research, applying computer modeling, synthetic analogues and receptor mutants, X-ray and electron-microscopy analyses of complexes with the nAChRs, and their models which are acetylcholine-binding proteins and heterologously-expressed ligand-binding domains.

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Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors

Cited by 12 publications

References 36 publications

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism

Discovery of Methylene Thioacetal-Incorporated α-RgIA Analogues as Potent and Stable Antagonists of the Human α9α10 Nicotinic Acetylcholine Receptor for the Treatment of Neuropathic Pain

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

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