Design of allele-specific protein methyltransferase inhibitors

Protein arginine methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to arginine side chains in target pr oteins, regulate transcription. RNA processing, and receptor-mediated signa ling. To specifically address the functional role of the individual members of this family, we took a "bump-and-hole" approach and designed a series o f N-6-substituted S-adenosylhomocysteine (SAH) analogues that are targeted toward a yeast protein methyltransferase RMT1. A point mutation was identif ied (E117G) in Rmt1 that renders the enzyme susceptible to selective inhibi tion by the SAH analogues. A mass spectrometry based enzymatic assay reveal ed that two compounds, N-6-benzyl- and N-6-naphthylmethyl-SAH, can inhibit the mutant enzyme over the wild-type with the selectivity greater than 20. When the E117G mutation was introduced into the Saccharomyces cerevisiae ch romosome, the methylation of Np13p, a known in vivo Rmt1 substrate, could b e moderately reduced by N-6-naphthylmethyl-SAH in the resulting allele. In addition, an N-6-benzyl-SAM analogue was found to serve as an orthogonal SA M cofactor. This analogue is preferentially utilized by the mutant methyltr ansferase relative to the wild-type enzyme with a selectivity greater than 67. This specific enzyme/inhibitor and enzyme/substrate design should be ap plicable to other members of this protein family and facilitate the charact erization of protein methyltransferase function in vivo when combined with RNA expression analysis.