Targeting the NOT9 subunit of the CCR4-NOT complex inhibits mRNA deadenylation

The removal of poly(A) tail from mRNA sequences reduces their stability and translational activity. The deadenylation process is mainly regulated by the CCR4-NOT complex for which potent chemical inhibitors are rare. The subunit NOT9 binds various RNA binding proteins (RBPs) which recruit mRNA in a sequence-specific manner to the CCR4-NOT complex to promote their deadenylation. Several of these RBPs interact with NOT9 through an evolutionary conserved (C)NOT9 binding motif (CBM) which we used as a starting point for inhibitor design. A potent hydrocarbon stapled peptide (NIP-2) with a 60 nM binding affinity for NOT9 was identified and able to inhibit the deadenylation activity of the CCR4-NOT complex on poly(A) RNA in vitro. A co-crystal structure of NIP-2 bound to NOT9 was obtained and allowed further optimization of the peptide through point mutation which led to a variant with high cell permeability. The optimized NIP-2-H27A-N3 was able to increase the poly(A) tail length of target mRNAs by inhibiting the deadenylation activity of the CCR4-NOT complex in cells demonstrating the feasibility of increasing mRNA stability using deadenylation inhibitors.