Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki = 44 nM) though selectivity reverted to α3β2 (IC50 = 0.7 nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants

AChBP-targeted α-conotoxin correlates distinct binding orientations with nAChR subtype selectivity

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases

Synthesis, Structure and Activity of Disulfide-Rich Conus Peptides

There is great interest in investigating structural and activity relationships between conotoxins and their biological targets. The information obtained using these techniques has added to our knowledge of how these molecules interact with receptors, and allowed us to pursue the synthesis of more selective and/or potent analogues. Chapters 1 and 2 serve as an introduction to the field of conotoxin research, and to the diversity of biological targets for which they are selective. They also give a general introduction to solid-phase peptide synthesis, the powerful tool used for the synthesis of peptides and analogues used in this study. Chapter 3 contains the materials and methods used throughout this thesis. Common methods used throughout the thesis have been included in one chapter to help minimise repetition. A brief introduction to many of the specialised techniques encountered is also given. Chapter 4 involves the SAR study of á-Ctx [A10L] PnIA through alaninescanning mutagenesis. Peptides were synthesised and their affinity for the á7 nAChR evaluated in a rat brain homogenate, and potency was determined with electrophysiological studies. This provides information as to which residues are important for interacting with the á7 subtype of the neuronal nicotinic acetylcholine receptor. Preliminary structural studies were performed to determine whether structural changes accompanied any loss in affinity of the peptide to the receptor, to distinguish important interactions from structural perturbations. Chapter 5 probes the importance that the side-chain of position 10 has on affinity to the á7 nAChR. A series of seventeen point mutations were synthesised, with side chains containing aromatic, aliphatic, polar and charged residues. Previous work demonstrated that the side-chain at position 10 could discriminate between two different subtypes of the nAChR, therefore a functional screen was performed to estimate the activity and selectivity of these mutated peptides at different subtypes of the receptor. Chapter 6 describes the synthesis and structural studies of á-conotoxin ImII, a conotoxin that is functionally active at the á7 nAChR, but does not bind to the classical conotoxin binding site. It is the only á-conotoxin described to date that does not contain a proline residue at position 6, and makes an excellent candidate for structural studies. Chapter 7 investigates a novel method for the synthesis of conotoxins from the O-superfamily. These conotoxins all contain adjacent cysteine residues located between the second and third loops, making them excellent targets for synthesis by native chemical ligation of two peptide segments. This novel strategy was applied to synthesise chimeras of N-type VGCC selective CVID and P/Q-type VGCC selective MVIIC, two peptides that are pharmacologically well characterised. It is hoped that this methodology will enable synthesis of peptides that have either been difficult to synthesise or oxidise in the past. The overall aim of this thesis is to investigate the structure and activity of some disulfide-rich conus peptides, and determine the residues responsible for selectivity and affinity toward their biological target. The information obtained from this study will further our knowledge of how these peptides interact with these receptors and receptor subtypes. Subtype selective ligands can be used as pharmacological tools to dissect and characterise the roles individual subtypes play in the complex mixtures of receptors found in native tissues

Designed Trpzip‑3 β‑Hairpin Inhibits Amyloid Formation in Two Different Amyloid Systems

The trpzip peptides are small, monomeric, and extremely stable β-hairpins that have become valuable tools for studying protein folding. Here, we show that trpzip-3 inhibits aggregation in two very different amyloid systems: transthyretin and Aβ(1–42). Interestingly, Trp → Leu mutations renders the peptide ineffective against transthyretin, but Aβ inhibition remains. Computational docking was used to predict the interactions between trpzip-3 and transthyretin, suggesting that inhibition occurs via binding to the outer region of the thyroxine-binding site, which is supported by dye displacement experiments

Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer\u27s disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki = 44 nM) though selectivity reverted to α3β2 (IC50 = 0.7 nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants

Rapid access to omega conotoxin chimeras using native chemical ligation

Grafting different regions of related peptides together to form a single protein chimera is a valuable tool in rapidly elucidating regions of activity or selectivity in peptides and proteins. To conveniently evaluate the contributions of the N- and C-terminal segments of ω-conotoxins CVID and MVIIC to activity, we employed native chemical ligation in CVID-MVIIC chimera design. Assembly of these peptide segments via the ligation method improved overall yield and coupling efficiency, with no difficult sequences encountered in contrast to the traditional full-length chain assembly of CVID. Radio-ligand binding assays revealed regions of importance for receptor recognition

Nature versus design: the conformational propensities of D-amino acids and the importance of side chain chirality

NoD-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of L- and D-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host-guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the D-amino acids, including D-allo-isoleucine and D-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate L-amino acids verifies that the intrinsic backbone conformational propensities of the D-amino acids are the inverse of their cognate L-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties.NI