α-Conotoxins targeting neuronal nAChRs: understanding molecular pharmacology and potential therapeutics.

Kompella, S 2013, α-Conotoxins targeting neuronal nAChRs: understanding molecular pharmacology and potential therapeutics., Doctor of Philosophy (PhD), Medical Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title α-Conotoxins targeting neuronal nAChRs: understanding molecular pharmacology and potential therapeutics.
Author(s) Kompella, S
Year 2013
Abstract α-Conotoxins, a new class of peptides that act as nicotinic acetylcholine receptor (nAChR) antagonists have been identified from the venom of predatory marine cone snails. α-Conotoxins specifically target various nAChRs subtypes and are excellent molecular probes for identifying the physiological role of nAChR subtypes in both normal and disease states. nAChRs are ligand-gated ion channels expressed in both central nervous system (CNS) and peripheral nervous system (PNS) and are shown to contribute to the physiological roles of neurotransmitter release and synaptic plasticity. Further, they are also implicated in various conditions including Alzheimer’s disease and schizophrenia.

The α3β4 subtype is shown to be involved in lung cancer and nicotine addiction. Despite this, the knowledge of the pathophysiological role of α3β4 subtypes is limited by the lack of adequate subtype specific probes. I report the discovery of new α4/7-conotoxin RegIIA which was isolated from Conus regius. Using alanine scanning mutagenesis, I report the synthesis of [N11A,N12A]RegIIA, a selective α3β4 nAChR antagonist (IC50 of 370 nM) that could potentially be used in the treatment of lung cancer and nicotine addiction. In this study, I also identified critical residues of α-conotoxin RegIIA that interact with the acetylcholine-binding site of α3β2, α3β4 and α7 nAChRs.

My research also describes the pharmacological properties of two novel conotoxins: LsIA and GeXXA. α-Conotoxin LsIA is the first peptide isolated from Conus limpusi, a species of worm-hunting cone snail. LsIA exhibited selective and potent α7 and α3β2 nAChR subtype antagonism. These subtypes play vital roles in various functions, such as neuronal plasticity and synaptic transmission. In this report, I examined the structure–function relationship of a unique N-terminal serine and C-terminal carboxylation of LsIA. Furthermore, I also investigated the effect of α5 subunit incorporation, towards the inhibition of α3β2 nAChR subtype by LsIA.

GeXXA is a novel αD-conotoxin isolated from the venom of Conus generalis. This toxin is a disulfide-linked homodimer of a 10-cysteine-containing peptide with each peptide chain made of 50 amino acid residues. αD-GeXXA is a non-selective inhibitor of muscle and neuronal nAChRs. Here I describe the functional characterization of monomeric αD–conotoxin which shows selective and reversible inhibition of α9α10 nAChR subtype. These results provide insight into the novel blocking mechanism of α-D conotoxins.

α-Conotoxins inhibiting nAChRs have potential therapeutic applications. However, peptidic nature of α-conotoxins affects their stability and bioavailability. Various strategies to improve α-conotoxin stability have been implemented. Here, I explore the functional implications of dicarba modified cysteine bridges on novel analgesic α-conotoxins Vc1.1 and RgIA, which inhibit HVA calcium channel currents via GABAB receptor activation and α9α10 nAChR subtypes. My results revealed disulphide stacking interaction between the Cys2–Cys8 bond and disulphide of the C-loop of the principal subunit of nAChRs.

My research describes the discovery, characterization and development of a novel α3β4 antagonist as neurophysiological and potential therapeutic tool for lung cancer. Further, dicarba modification of α-conotoxins and characterization of new class of α- and αD-conotoxins provide future insights towards drug development.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Medical Sciences
Keyword(s) Conus regius
Conus generalis
Conus limpusi
α-conotoxin
nicotinic acetylcholine receptor
alanine scanning mutagenesis
electrophysiology
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