α-Conotoxin Dendrimers Have Enhanced Potency and Selectivity for Homomeric Nicotinic Acetylcholine Receptors

Abstract

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

Inhibition of the norepinephrine transporter by χ‐conotoxin dendrimers

“…Moreover, a truncated analogue χ-MrIA with residue asparagine-1 deleted maintained its 3D structure and binding affinity comparable with amidated χ-MrIA [22,33]. To avoid the formation of aspartimide [32], the truncated χ-MrIA analogue 1 ([Pro] 11 MrIA [2][3][4][5][6][7][8][9][10][11][12][13], GVCCGYKLCHPC-NH 2 , 1-4, 2-3 connectivity) was used to build the azido-modified χ-MrIA moieties 4 and 5. The azido group was introduced to the N-terminus of 1 by coupling of azido-PEG(9)-COOH or azido-PEG(24)-COOH with chain lengths of approximately 35 and 90 Å, respectively.…”

“…To ensure correct folding of 4 and 5, pairs of Fmoccysteine(Acm)-OH and Fmoc-cysteine(Trt)-OH were utilized in the synthesis to direct regioselective disulfide bond formation (Scheme 2) [46]. The crude peptides were oxidized stepwise, purified after each oxidation step, and then subjected to the RP-HPLC for purification to give >90% pure peptides 4 and 5 (denoted as azido-PEG(9)-[Pro] 11 MrIA [2][3][4][5][6][7][8][9][10][11][12][13] and azido-PEG(24)-[Pro] 11 MrIA [2][3][4][5][6][7][8][9][10][11][12][13], respectively) with native (1-4, 2-3) disulfide connectivity ( Figure S2, see Supporting Information).…”

“…Hence, the click reaction was selected as the preferred conjugation chemistry for the synthesis of χ-MrIA dendrimers. This was achieved by coupling under a nitrogen atmosphere of a twofold excess of azido-PEG(9)-[Pro] 11 MrIA [2][3][4][5][6][7][8][9][10][11][12][13] peptide 4 per dendrimer arm of the alkyne dendron 2 in the presence of CuSO 4 with sodium ascorbate as the reducing reagent for the formation of the catalytic Cu(I). TBTA ligand, which stabilizes Cu(I) toward disproportionation and oxidation [48], was also added to the mixture to enhance the Cu (I) catalytic effect.…”

“…NMR is very sensitive to minor local variations in the chemical environment and is widely used to detect structural changes in specific residues of bioactive well-structured peptides [49]. Hence, structural changes may be routinely determined by comparing the H α chemical shifts with chemical shifts derived from random coil values [10]. Here, one-dimensional and 2D NMR spectroscopy at 900 MHz was employed to characterize the structures of three multivalent χ-MrIA dendrimers (6)(7)(8) and the truncated analogue [Pro] 11 MrIA [2][3][4][5][6][7][8][9][10][11][12][13] (1).…”

“…In this study, we undertook the synthesis of χ-MrIA dendrimers of differing-size PEG spacers with molecular weights ranging from 8 to 22 kDa and further investigated their structural and functional features. Initially, χ-MrIA dendrimers carrying four and eight copies of the χ-MrIA analogues ([Pro] 11 MrIA [2][3][4][5][6][7][8][9][10][11][12][13], 1) were successfully synthesized through the CuAAc reaction, based on the alkynemodified polylysine dendrons (2 and 3, Scheme 1) and azido-PEG chain-modified χ-MrIA analogues [N 3 -PEG(9 or 24)-[Pro] 11 MrIA[2-13]-NH 2 ] (4 and 5, Scheme 2). The CuAAc reaction is a widely used strategy in peptide conjugation promising high efficiency and high selectivity [35,36].…”