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Searching for inhibitors of the protein arginine methyl transferases : synthesis and characterisation of peptidomimetic ligands Knuhtsen, Astrid

Abstract

Within the last two decades research in the field of epigenetics has increased significantly as targeting the epigenetic enzymes has the potential to alter the transcription of genes. Aberrant regulation of transcription is seen in several disease states, and drugs targeting the epigenetic histone deacetylases and DNA methylases are already marketed for cancer treatment. The Protein Arginine Methyl Transferases (PRMTs) belong to an epigenetic enzyme family that is upregulated in several cancers. However, currently no inhibitors of the PRMTs have been marketed. In this thesis several peptidomimetic strategies were utilised to modify the tryptophan residues in two peptide leads in order to discover new inhibitors of the PRMTs. One of these strategies involved constraining the side chain indole of tryptophan to the peptide backbone, thus producing a seven membered azepinone mimetic, Aia. The peptidomimetic efforts resulted in a structure-activity relationship study from which a constrained peptidomimetic containing two Aias was discovered to be a low micromolar inhibitor of several PRMTs. To characterise the inhibitor the conformation of the inhibitor was examined using solution-phase NMR and was shown to display an interesting turn-structure. The original peptide lead was fluorescently tagged and investigated in a cellular setting, but did not reveal any PRMT-specific localisation. In an effort to study the binding of the discovered inhibitor with the PRMTs, protein expression in E. coli and purification was performed. This resulted in the optimisation of PRMT6 purification in order to obtain highly pure PRMT6 for isothermal titration calorimetry (ITC) studies. Unfortunately these ITC studies were unsuccessful. Furthermore, as the constrained tryptophan mimetic had proven very useful in the peptidomimetic inhibitors of the PRMTs, we attempted to synthesise a lysine/arginine dipeptide mimetic using aziridine chemistry.

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