(2R)-N-Benzyl-2-acetamido-3-methoxypropionamide (lacosamide) is an anticonvulsant (Choi et al., 1996); under the brand name "Vimpat" this small molecule has recently been approved by the European Medicines Agency and the U.S. Food and Drug Administration for the treatment of epilepsy. The purpose of the research reported here is to develop and apply mass spectrometry approaches to the determination of protein targets of this novel therapeutic. The general strategy involves selecting potential target proteins using lacosamide analogues incorporating an 'affinity bait' to enable covalent modification binding to target proteins, and a 'chemical reporter' for the selective recovery of modified proteins. Lacosamide analogues are incubated with biological samples (primarily mouse brain extracts) and the modified proteins are recovered by introduction of a biotin tag (via the chemical reporter group). Streptavidin affinity chromatography is then used to enrich for bound molecules. The enriched proteins are subjected to tryptic digestion and the resultant peptides analysed by reversed phase liquid chromatography coupled with tandem MS, enabling recognition of proteins via database searching. Firstly, mass spectrometric characterisation of the biotinyl (R)-lacosamide analogue bound to model compounds was performed. Adducts with protected lysine, neurotensin and enolase were analysed. The data showed that ESI was more suitable for ionisation of modified peptides and proteins than MALDI. The biotin enrichment strategy was applied to mouse brain lysate to identify putative candidate target proteins. Twenty-eight candidate target proteins were identified. Moreover, the 14-3-3 protein family, CRMP2 and the sodium/potassium-transporting ATPase family showed preference for the biologically active(R)- isomer over the (S)- lacosamide analogue using a fluorescence tag. Three more biotinyl lacosamide analogues containing different affinity baits were used to enrich candidate target proteins of lacosamide. Most of the identified target proteins supported the findings of the analogue A. To indicate the binding sites, a method was developed for enriching peptides modified by the biotinyl (R)-lacosamide analogue, using streptavidin beads and subsequently analysed these biotinylated peptides using CID and ETD fragmentation methods. Neither fragmentation technique was optimal for elucidation of the sequence or site of modification of unknown target peptides. Purified recombinant proteins were therefore adducted with an AB-(R)-lacosamide analogue lacking the biotinprobe. This smaller (R)-lacosamide analogue underwent less fragmentation than the biotin analogue during CID and could be used for sequence and site identification of the modified peptides. In summary, the studies illustrated the power of MS to study drug mechanisms via the discovery of candidate protein targets.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:518815 |
| Date | January 2010 |
| Creators | Reamtong, Onrapak |
| Contributors | Gaskell, Simon ; Eyers, Claire |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-identification-and-characterisation-of-the-target-proteins-of-the-antiepileptic-drug-rlacosamide(2e2e4b3b-4101-46e3-886f-ff56c62d2b59).html |
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