Characterization and SAR studies of Novel and Selected α- and μ- Conotoxins

Kalyana Bharati Akondi (2011). Characterization and SAR studies of Novel and Selected α- and μ- Conotoxins PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Kalyana Bharati Akondi
Thesis Title Characterization and SAR studies of Novel and Selected α- and μ- Conotoxins
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-09
Thesis type PhD Thesis
Supervisor Prof. Paul F. Alewood
Total pages 282
Total colour pages 99
Total black and white pages 183
Language eng
Subjects 06 Biological Sciences
Abstract/Summary Conotoxins are small disulfide rich peptides present in cone snail venom. They target a wide array of receptors and ion-channels and exhibit extensive pharmacological diversity. The huge biodiversity displayed by these venom peptides holds considerable promise for novel ligand discovery. Conotoxins targeting nicotinic acetylcholine receptors (nAChRs) and voltage gated sodium (Nav) channels have received particular interest in view of their research and therapeutic potential. This thesis specifically focuses on the synthesis and structural and functional characterization of novel peptides from the α- and μ-conotoxin families. The α-conotoxins target a wide array of nAChR subtypes whereas the μ-conotoxins are Nav channel blockers. Chapter 1 includes a general introduction of conotoxins and the current status of conotoxin research. All of the peptides in this thesis were synthesized using Boc or Fmoc solid phase peptide chemistry. A brief overview of solid phase peptide synthesis along with the materials and general methods used for conotoxin structural and functional characterization throughout this thesis are described in Chapter 2. The process of identifying novel conotoxin sequences has accelerated significantly with the advent of DNA-based technology and MS/MS techniques. Though many conotoxin primary sequences are available, their pharmacological targets and structures still remain to be characterized. This issue is partially addressed in Chapter 3, which focuses on the characterization of novel α-conotoxins. The α4/3 subfamily of conotoxins were specifically selected as only three peptides have been characterized in this interesting subfamily to date. The characterization of α4/3 conotoxins isolated from C. regius not only aided in expanding the repertoire of this subfamily but also identified α4/3 RegIA to be the most potent α9α10 targeting α-conotoxin known. In addition another conotoxin RegIIA was characterized, which exhibited the highest potency towards the α3β4 nAChR subtype in the α4/7 subfamily. Chapter 4 focuses on a novel α4/7 conotoxin LtIA, which lacks the conserved Ser-X-Pro motif in loop 1. Despite the lack of the conserved motif, LtIA was found to potently and selectively block the α3β2 nAChR. Structural characterization showed that the peptide was flexible with multiple conformations in solution. Engineering the conserved motif back into LtIA caused the nascent helix to stabilize indicating its importance in maintaining conotoxin structural integrity. Receptor mutagenesis studies revealed that LtIA establishes a unique set of interactions at the receptor binding site suggesting it targets a novel microsite in the nAChR. Structure activity relationship (SAR) studies on the analgesic α4/6 conotoxin AuIB using alanine scan mutagenesis were carried out in Chapter 5. The interest in this peptide heightened with the recent finding that α-conotoxin AuIB that targets the α3β4 nAChR also inhibits GABAB receptor induced N-type Ca2+ channel currents similar to the therapeutically important α-conotoxins Vc1.1 and RgIA. The affect of each mutation on conotoxin structure and function was examined in order to identify residues of structural and functional importance. Electrophysiological analysis identified Pro 6 and Phe 9 residues to be crucial for α3β4 subtype activity. NMR data indicated that replacement of the conserved Pro 6 residue destabilized the structural integrity of AuIB thus leading to a loss of activity. The Phe 9 to Ala mutation however, did not impact the conotoxin structure, but significantly lowered α3β4 subtype potency. This indicates that Phe 9 residue is involved in crucial toxin-α3β4 receptor binding site interactions. Interestingly, the selectivity profile of this mutant also shifted towards recognition of the α9α10 nAChR subtype. Finally, in Chapter 6, a truncation mutagenesis strategy was used to identify the minimum functional scaffold of the μ-conotoxin SIIIA. Previous studies have shown that loop two and loop three residues in SIIIA are important for maintaining potency at Nav channels. We demonstrated that loop 1 truncation yielded a structurally minimized SIIIA scaffold that retains potency equivalent to that of the native peptide. The study also showed that SIIIA 3-20 N5K D15A which has the neutral Asn residue in loop 1 replaced with a positively charged residue had a significant 13.7-fold increase in potency towards the rat Nav 1.2 subtype when compared to native SIIIA. Also, the truncation mutants with only a single residue in loop 1 (SIIIA 3-20 desN5,G6;D15A) were found to retain potency equal to that of the native peptide. Thus, this study generated a synthetic SIIIA analog with enhanced potency, in addition to identifying the minimum SIIIA scaffold required to maintain near native binding potency at rat Nav1.2 subtypes. The work in this thesis has contributed towards expanding the characterized conotoxin repertoire and bridging the gap between identified conotoxin sequences and those that have actually been structurally and pharmacologically characterized. It has advanced our understanding of structure activity relationships of selected novel and therapeutically significant α- and μ-conotoxins. In the process, several α-conotoxins showing the highest known nAChR subtype potencies have been characterized. The thesis also provides advanced SAR information on μ-conotoxin SIIIA and identified analogs exhibiting enhanced Nav channel potencies.
Keyword conotoxin, nicotinic acetylcholine receptor, voltage gated sodium channels, structure activity relationships, CD spectroscopy, NMR spectroscopy, electrophysiology, peptides
Additional Notes Colour page numbers: 20,21,23,25,26,28,30,31-33,35,39,40,43,46,65,67,69,72,73,74,84,89,95,99-103,107-109,111-117,119,121-126,128,129,132,133,137,147,149,152-155,161,162,165,167,169,179,182,183,185-187,189-197,200,206,209,211-213,215-222,225,236,250,251,253,268-270 Landscape Pg 28

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