The α4β2 nicotinic acetylcholine receptor (nAChR) is an important target for currently approved smoking cessation therapeutics. However, the development of highly selective α4β2 nAChR antagonists remains a significant challenge. α-Conotoxin GID is an antagonist of α4β2 nAChRs, though it is significantly more potent toward the α3β2 and α7 subtypes. With the goal of obtaining further insights into α-conotoxin GID/nAChR interactions that could lead to the design of GID analogues with improved affinity for α4β2 nAChRs, we built a homology model of the GID/α4β2 complex using an X-ray co-crystal structure of an α-conotoxin/acetylcholine binding protein (AChBP) complex. Several additional interactions that could potentially enhance the affinity of GID for α4β2 nAChRs were observed in our model, which led to the design and synthesis of 22 GID analogues. Seven analogues displayed inhibitory activity toward α4β2 nAChRs that was comparable to GID. Significantly, both GID[A10S] and GID[V13I] demonstrated moderately improved selectivity toward α4β2 over α3β2 when compared with GID, while GID[V18N] exhibited no measurable inhibitory activity for the α3β2 subtype, yet retained inhibitory activity for α4β2. In this regard, GID[V18N] is the most α4β2 nAChR selective α-conotoxin analogue identified to date. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 78-87, 2014.

Banerjee J., et al. (2014)  Design and synthesis of α-conotoxin GID analogues as selective α4β2 nicotinic acetylcholine receptor antagonists. Biopolymers. PMID: 24122487

Alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Millard E., et al. (2009)  Inhibition of Neuronal Nicotinic  Acetylcholine Receptor Subtypes by  α-Conotoxin GID and Analogues. JBC.  PMID: 19098004

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3 beta 2 nAChRs with IC(50) values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha 1 beta 1 gamma delta, alpha 3 beta 4, and alpha 4 beta 4 combinations. GID also potently inhibits the alpha 4 beta 2 subtype (IC(50) of 150 nm). Deletion of the N-terminal sequence (GID Delta 1-4) significantly decreased activity at the alpha 4 beta 2 nAChR but hardly affected potency at alpha 3 beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha 4 beta 2 and alpha 7 activity but not to alpha 3 beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha 7 versus alpha 3 beta 2 selectivity.

Nicke A., et al. (2003)  Isolation, Structure, and Activity of GID, a Novel 4/7-Conotoxin with an Extended N-terminal Sequence. JBC.  PMID: 12419800