Engineering Bisubstrates to Target m⁶Am RNA Methyltransferases: Synthesis and Computational Studies

Phosphorylated CTD-interacting factor 1 (PCIF1) has been recently discovered to introduce a methyl group at the N6 position of the first transcribed Am-adenosine in nascent capped mRNA (m⁶Am modification), modulating mRNA stability, transcription, and translation. In addition, the activity of PCIF1 is involved in various diseases, making PCIF1 a potential therapeutic target. In the absence of complete crystal structures of PCIF1 with an RNA substrate and/or cofactor and with the aim of accessing potent inhibitors, the bisubstrate strategy was employed to synthesize a series of bisubstrate analogs of PCIF1 that contain an analog of the cofactor S-adenosyl-L-methionine (SAM) covalently linked to an Am-modified RNA substrate. The versatility of the synthesis was exploited to increase structural diversity at the amino acid chain of SAM, and a cap analog was introduced by click chemistry. Also, AlphaFold 3 was used to generate a complete structure of PCIF1 with an RNA substrate. Docking studies carried out with the bisubstrates indicate that the SAM analog occupies the cofactor binding site while the Am-RNA substrate interacts with the putative RNA pocket. We think that these results could be a valuable starting point for the design of potent inhibitors of PCIF1.