Characterisation and structural biology of protein arginine methyltransferases

Abstract

Post-translational and epigenetic modifications of proteins and nucleic acids are known to play major roles in influencing cell fate. Enzymes that catalyse modifications such as phosphorylation, acetylation and methylation have been identified as promising drug targets. Protein methyltransferase 2 (PRMT2) and Coactivator-associated arginine methyltransferase 1 (CARM1) belong to the class of Type 1 PRMTs which catalyse the asymmetric dimethylation of substrate arginine residues. CARM1 has been shown to be overexpressed in different cancer types including breast and prostate cancer. PRMT2 has been identified as a potential target for oncology with reported links to androgen receptor signalling, NF-κB signalling and induction of apoptosis. However, selective chemical probes that could be used as tools for target validation and which could potentially be a starting point for drug discovery are still missing. The work presented here aims to identify selective CARM1 and PRMT2 inhibitors that target the cofactor- and substrate-binding sites. Crystal structures of mouse PRMT2 in the apo-state and in complex with Sinefungin are presented. Crystal structures of the catalytic domain of CARM1 in complex with the cofactor S-adenosyl Lhomocysteine (SAH) and different small molecule inhibitors were also determined. Surface plasmon resonance was used to characterise inhibitor binding to CARM1 and identify structure-activity relationships. To further map the CARM1 active site, ligand soaks of CARM1 with a library of small fragments called FragLites were performed. These small fragments can more readily find potential binding pockets than larger more drug-like inhibitors. A direct and label-free mass spectrometry-based assay was developed to measure CARM1 activity and its inhibition. Together these findings can be used to further develop inhibitors that target the PRMT family. These inhibitors will be useful tools to investigate the biology of PRMT2 and CARM1 and to understand their biological role in cancer.