Computational design and studies of neuro-active peptides and the effects of calcium on nicotinic acetylcholine receptors in relation to neurodegenerative disorders

摘要

Neuronal nicotinic acetylcholine receptors (nAChRs) are important ligand gated ion-channels in the brain that play important roles in inter-cellular communication and the release of neurotransmitters. However, these channels are also implicated in common neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. The α7 and α4β2 subtypes of nAChRs are amongst the most prevalent subtypes in the brain. Given that Alzheimer’s disease (AD) is associated with a significant loss in α7 and α4 subunits, these subtypes are likely to play especially important roles in the pathophysiology of this disorder. A deeper understanding of the specific roles of these two receptors in AD would be possible with the availability of ligands which can modulate the activity of α7 and α4β2 in a subtype-specific manner. α-Conotoxins are a family of cysteine-rich peptides found in marine snails from the genus Conus. These small peptides act as antagonists to nAChRs, and some are able to discriminate between distinct nAChR subtypes. The α-conotoxin [γ4E]-GID is highly potent towards the human α7 nAChR, and is amongst the few known conotoxins which also inhibit α4β2. In this thesis, we use computer modelling and simulation techniques to 1) understand the interaction of α-conotoxin [γ4E]-GID with both α7 and α4β2 nAChRs at the canonical ligand binding site; 2) elucidate subtype-dependent conotoxin unbinding pathways, leading to identification of possible alternative sites which may serve as targets for rational conotoxin design; and 3) examine the influence of elevated calcium ion concentration on α7 structure and its interactions with [γ4E]-GID. Based on simulation data, we propose mutations to [γ4E]-GID which may lead to the design of a novel [γ4E]-GID-derived peptide with enhanced selective inhibition for α4β2. An introduction to neuronal nicotinic receptors and conotoxins along with a literature review of the important studies on receptor-conotoxin binding are presented in Chapter 1. The simulation methods, parameters used for this study and other derivative techniques, including umbrella sampling, that were employed in this study are described in Chapter 2. Current knowledge of the structure of the extracellular domain of the human α7 and human/rat α4β2 nicotinic receptors are presented in Chapter 3 along with protein interaction data derived from the simulation of the modelled toxin-bound receptor structures. From our simulations, several [γ4E]-GID residues were identified as being potentially amenable to improve selectivity of α4β2 over α7 by mutation. Results from recent experimental evidence are discussed, in which some of the [γ4E]-GID sites and mutants identified from simulations appeared to hold promise as selectivity enhancing modifications. In Chapter 4 atomistic molecular dynamics (MD) simulations and umbrella sampling methods were used to elucidate differences in the binding free energies and possible energetically favourable unbinding pathways of [γ4E]-GID at α7 and α4β2 which may be responsible for the marked difference in potency between the two receptors. This also helped identify important alternative binding sites on the receptor that differed between the two receptor subtypes, which could be exploited as targets for design of novel [γ4E]-GID-derived mutants. High stress levels in the brain could lead to increases in calcium levels. This elevation in calcium concentration in specific regions of the brain has been reported in some Alzheimer’s and Parkinson’s disease affected human and rat brain models. Thus, in a physiological context, especially in diseased brains, the development of novel conotoxins should take into account the presence of calcium. Chapters 5 and 6 discuss our attempts to understand the influence of calcium on nAChR structure, as well as the resultant effects on binding between [γ4E]-GID and α7. In Chapter 5, molecular dynamics simulation methods have shown that an abnormal level of calcium ions around the neuronal nicotinic receptor causes changes in receptor structure which resemble those associated with activation, even in the absence of agonist binding, especially in the M2-M3 transmembrane regions, leading to partial opening of the channel pore. Free energy calculations revealed that a calcium-affected partially-open channel displayed less resistance to a calcium ion permeation compared to a receptor channel simulated in a calcium-free system. We propose that these changes in the presence of elevated calcium concentration might be related to chronic activation of the receptor, resulting in continuous signal firing due to prolonged opening of the ion channel. In addition, the influence of calcium to the receptor structure and allosteric changes suggests that the potency of [γ4E]-GID towards the receptor may also be altered. Preliminary results that described the interaction of [γ4E]-GID at the extracellular ligand binding site of the respective receptor in a calcium-ion affected system is presented in Chapter 6. The study showed that [γ4E]-GID interacted more strongly at the calcium-affected receptor in comparison to a calcium-free simulation system. This was related to the tighter closure of the ligand binding pocket due to the presence of calcium ions. Finally, in Chapter 7 an outlook on the research presented and ideas in context to continuation of the current research in improving [γ4E]-GID conotoxin-receptor binding potency is discussed in the Closing thoughts and future directions section of this thesis.