AlNasir, Jamal

Austen, Brian

Neurodegenerative diseases feature neurochemical and neuropathological changes which are intimately linked with excitotoxicity. PSD-95 a post-synaptic scaffold protein of the NMDA receptor complex, containing PDZ domains coupling to a PMCa2B calcium channel, NMDAR2, and i-nos, has been found to mediate Glutamate induced excitotoxicity. Neuronal damage may be mediated by calcium intrusion and oxidative stress and PSD-95 is a potential therapeutic target. PDZ binding ligands have been designed based on the C-terminal sequence of the PMCa2b calcium channel sub-unit in order to disrupt its interaction with PSD95 via PDZ domain 1. Current PDZ binding ligands currently have Kd constants in the micromolar range and tighter binding is required (Kd constants in nanomolar range) for therapeutic use. Structural alterations have been proposed by various researchers to this end including cyclisation of PDZ peptide ligands. Putative PDZ binding ligands were modeled in Silico based on existing structures but with important refinements to improve binding affinity. Ligand-Protein docking simulations were performed in order to select a group of ligands to synthesise. Solid Phase peptide synthesis was employed following the Fmoc protocol. Cyclisation was via amide formation between orthogonally protected side-chains. The PDZ binding peptides produced were tested in vitro on SHSY-5Y neuroblastoma cell lines in the presence of 2mmol Glutamate, and insult and cell viability quantified via MTT assay. The results indicate that the modification of a linker residue and side-chain on the PDZ ligand improves efficacy as seen by protection against excitotoxic cell death. Immunohistochemical studies of biotinylated PDZ peptides and their PSD-95 target demonstrated co-localisation and cell protection for an asparaginyl bridged peptide compared to a glutamate -bridged peptide. Our results also show that cyclisation alone does not confer improvement in PDZ binding affinity contrary to what has been postulated by other researchers.<br/

Royal Holloway - Pure

Characterisation of cyclic neuroprotective PDZ binding ligands

https://pure.royalholloway.ac.uk/ws/files/25080016/Booklet_Research_Day2011.pdf

Characterisation of cyclic neuroprotective PDZ binding ligands

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Royal Holloway - Pure