The identification and characterisation of the target proteins of the anti-epileptic drug R-lacosamide

UoM administered thesis: Phd

  • Authors:
  • Onrapak Reamtong

Abstract

(2R)-N-Benzyl-2-acetamido-3-methoxypropionamide (lacosamide) is ananticonvulsant (Choi et al., 1996); under the brand name "Vimpat" this small molecule hasrecently been approved by the European Medicines Agency and the U.S. Food and DrugAdministration for the treatment of epilepsy. The purpose of the research reported here is todevelop and apply mass spectrometry approaches to the determination of protein targets ofthis novel therapeutic.The general strategy involves selecting potential target proteins using lacosamideanalogues incorporating an 'affinity bait' to enable covalent modification binding to targetproteins, and a 'chemical reporter' for the selective recovery of modified proteins.Lacosamide analogues are incubated with biological samples (primarily mouse brainextracts) and the modified proteins are recovered by introduction of a biotin tag (via thechemical reporter group). Streptavidin affinity chromatography is then used to enrich forbound molecules. The enriched proteins are subjected to tryptic digestion and the resultantpeptides analysed by reversed phase liquid chromatography coupled with tandem MS,enabling recognition of proteins via database searchingFirstly, mass spectrometric characterisation of the biotinyl (R)-lacosamide analoguebound to model compounds was performed. Adducts with protected lysine, neurotensin andenolase were analysed. The data showed that ESI was more suitable for ionisation ofmodified peptides and proteins than MALDI. The biotin enrichment strategy was applied tomouse brain lysate to identify putative candidate target proteins. Twenty-eight candidatetarget proteins were identified. Moreover, the 14-3-3 protein family, CRMP2 and thesodium/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 wereused to enrich candidate target proteins of lacosamide. Most of the identified target proteinssupported the findings of the analogue A. To indicate the binding sites, a method wasdeveloped for enriching peptides modified by the biotinyl (R)-lacosamide analogue, usingstreptavidin beads and subsequently analysed these biotinylated peptides using CID andETD fragmentation methods. Neither fragmentation technique was optimal for elucidation ofthe sequence or site of modification of unknown target peptides. Purified recombinantproteins were therefore adducted with an AB-(R)-lacosamide analogue lacking the biotinprobe. This smaller (R)-lacosamide analogue underwent less fragmentation than the biotinanalogue during CID and could be used for sequence and site identification of the modifiedpeptides.In summary, the studies illustrated the power of MS to study drug mechanisms viathe discovery of candidate protein targets.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
  • Simon Gaskell (Supervisor)
  • Claire Eyers (Supervisor)
Award date31 Dec 2009