CCR4-NOT is a multi-subdomain protein complex consisting of eight subunits (CCR4, CAF1, NOT1, NOT2, NOT3, NOT9, NOT10, and NOT11, Fig. ). NOT1 acts as the main scaffold subunit where other subunits and modules dock onto. CCR4 and CAF1 are the exonucleases responsible for the catalytic activity of the CCR4-NOT complex and this heterodimeric complex and work in the 3' to 5' direction. The NOT2/3 and 10/11 subunits are non-catalytic and provide a protein-protein interaction (PPI) platform for sequence-specific RNAbinding proteins (RBPs) as well as non-specific RNA-binding capacity. The NOT9 (also known as CNOT9 or Caf40) subunit binds to the (C)NOT9-binding domain (CN9BD) of NOT1 in close proximity to the deadenylases and similar to the NOT2/3 and NOT10/11 subunits it is both a RBP binding site and a site for non-specific RNA binding. Since poly(A) tails have a high sequence similarity across all transcripts, deadenylation selectivity is regulated by RBPs that interact with mRNA in a sequence-specific manner while the non-specific RNA binding of the various subunits provides basal non-transcript-specific deadenylation (Fig. ). Although various studies have investigated which CCR4-NOT subunits are involved in deadenylation, a clear picture of which mRNAs they regulate is still missing. Selective inhibitors for each individual site involved in mRNA recruitment could help to provide further insights into which mRNA sequences are recruited by which site using a chemical biology-based approach. Currently, the only inhibitors for the CCR4-NOT complex available are for the deadenylases which do not help to answer this question as they would shut down deadenylation in general. These include several inhibitors for the exonuclease CAF1 and the most potent molecule has an IC50 value of 0.59 μM. The only inhibitors described for CCR4 are nucleotide monophosphates with the most potent being adenosine monophosphate with an IC50 value of 453 μM. Although these compounds were demonstrated to be able to inhibit deadenylation in vitro, no cellular activity has been reported. Since two exonucleases are present, it is possible that inhibition of only one of them is not sufficient which is highlighted by the fact that inactivating CAF1 or CCR4 through mutation still allows deadenylation. To develop a first site selective inhibitor that does not target the deadenylases directly, we aimed to design inhibitory peptides using a structure-based design approach. As the NOT9 subunit was shown to promote deadenylation and its protein-protein interactions have been well studied from a structural perspective we selected it as a starting point for development of a such a compound. In Drosophila melanogaster (Dm) three RBPs (Bam, Roquin and NOT4) have been described to recruit the CCR4-NOT complex by interacting with the NOT9 subunit. In humans, RNF219 has recently been identified as a regulator of the human CCR4-NOT complex via NOT9 but rather inhibits deadenylation as it has no RNA-binding capacity. High-quality structural data showed that the majority of these RBPs have single α-helical domains to interact with NOT9 referred to as (C)NOT9 binding motifs (CBMs). In contrast to RBP regulated transcript specific deadenylation, mechanistic studies demonstrated the existence of a bulk deadenylation mechanism, where the RNA gets directly recruited to the CCR4-NOT complex via interaction with NOT9 (Fig. ). Additionally, the protein-protein and protein-RNA interactions involved in both mechanisms share the same surface on NOT9 and are mutually exclusive to each other. We hypothesized that inhibiting NOT9 and thereby simultaneously inhibiting its protein-protein and protein-RNA interactions could protect and stabilize poly(A) tail containing RNA from being digested by the CCR4-NOT complex (Fig. ). Such an approach would open a new way to modulate RNA degradation and overall gene regulation. Additionally, NOT9 was found to be overexpressed in breast cancer cells and acted as a part of the oncogenic EGFR pathway making new chemical entities to target NOT9 interesting future drug candidates. Here we report the structure-based design of hydrocarbon stapled α-helical peptides as first-in-class inhibitors of NOT9 to modulate the activity of the CCR4-NOT complex. A hydrocarbon stapled α-helical peptide (NIP-2) has been identified with low nanomolar affinity for NOT9 which competes with RNA-binding and efficiently inhibits the activity of the CCR4-NOT complex in vitro. A crystal structure of the CBM-derived stapled peptide bound to NOT9 was obtained in support of the design strategy and confirmed the helical fold adopted by the stapled peptide. Furthermore, NIP-2 exhibited excellent proteolytic stability but limited cell permeability. To overcome this last issue, specific residues were selected and mutated to alanine which led to NIP-2-H27A with high cell permeability. Treatment of HeLa cells with this compound increased the maximum length of poly(A) tail containing mRNAs by inhibiting the activity of CCR4-NOT in cellulo.