Ilja Gordijenko

Therapeutic intervention targeting mRNA typically aims at reducing the levels of disease‐causing sequences. Achieving the opposite effect of blocking the destruction of beneficial mRNA remains underexplored. The degradation of mRNA starts with the removal of poly(A) tails, reducing their stability and translational activity, which is mainly regulated by the CCR4‐NOT complex. The subunit NOT9 binds various RNA binding proteins, that recruit mRNA in a sequence‐specific manner to the CCR4‐NOT complex to promote their deadenylation. These RNA binding proteins interact with NOT9 through a helical NOT9 binding motif, which we used as a starting point for development of the hydrocarbon stapled peptide NIP‐2. The peptide (K_D=60.4 nM) was able to inhibit RNA‐binding (IC₅₀=333 nM) as well as the deadenylation activity of the CCR4‐NOT complex in vitro while being cell‐permeable (cell‐permeability EC₅₀=2.44 μM). A co‐crystal structure of NIP‐2 bound to NOT9 allowed further optimization of the peptide through point mutation leading to NIP‐2‐H27A‐N₃ (K_D=122 nM) with high cell permeability (cell‐permeability EC₅₀=0.34 μM). The optimized peptide was able to inhibit deadenylation of target mRNAs when used in HeLa cells at a concentration of 100 μM, demonstrating the feasibility of increasing mRNA stability.

Therapeutic intervention targeting mRNA typically aims at reducing the levels of disease‐causing sequences. Achieving the opposite effect of blocking the destruction of beneficial mRNA remains underexplored. The degradation of mRNA starts with the removal of poly(A) tails, reducing their stability and translational activity, which is mainly regulated by the CCR4‐NOT complex. The subunit NOT9 binds various RNA binding proteins, that recruit mRNA in a sequence‐specific manner to the CCR4‐NOT complex to promote their deadenylation. These RNA binding proteins interact with NOT9 through a helical NOT9 binding motif, which we used as a starting point for development of the hydrocarbon stapled peptide NIP‐2. The peptide (K_D=60.4 nM) was able to inhibit RNA‐binding (IC₅₀=333 nM) as well as the deadenylation activity of the CCR4‐NOT complex in vitro while being cell‐permeable (cell‐permeability EC₅₀=2.44 μM). A co‐crystal structure of NIP‐2 bound to NOT9 allowed further optimization of the peptide through point mutation leading to NIP‐2‐H27A‐N₃ (K_D=122 nM) with high cell permeability (cell‐permeability EC₅₀=0.34 μM). The optimized peptide was able to inhibit deadenylation of target mRNAs when used in HeLa cells at a concentration of 100 μM, demonstrating the feasibility of increasing mRNA stability.

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.