Design of Bam-derived stapled peptides. The D. melanogaster protein Bam has a conserved 22 amino acid N-terminal CBM sequence ( 13 DDQQLDHNFKQMEEHLALMVEG ) that was previously found to serve as a binding motif for the NOT9 subunit of the CCR4-NOT complex. Interestingly, it was found that it also binds human NOT9 with high affinity and co-crystal structure is available (Fig. ). The CBM domain adopts an amphipathic α-helical structure that is bound across the conserved hydrophobic surface of the crescent-shaped NOT9 ARM domain (Fig. ) that is also thought to be responsible for RNA binding. The affinity of the DmBam CBM peptide for human NOT9 was not reported but found to be in the nanomolar range for DmBam (KD = 183 nM for DmNOT9). We assumed that because of the high similarity between DmNOT9 and human NOT9 (79% sequence similarity) the affinity would be similar. Furthermore, the existence of a high resolution crystal structure of its bound state provided a good starting point for the design of a specific inhibitor via hydrocarbon stapling. An initial virtual alanine scan of the wild-type DmBam CBM (named here: NOT9 Inhibitory Peptide -wild type (NIP-WT)) peptide was conducted to identify the hotspot residues that are responsible for NOT9 binding (supplemental Table ). The analysis found that the majority of the identified hotspot residues was hydrophobic in nature (L17, N20, F21, M24, L28, M31, V32, and E33). Next, we sought to truncate the NIP-WT peptide to improve synthetic accessibility and potentially reduce the strong negative charge to promote future cell-uptake. To this end, we performed a short MD simulation (100 ns) of the CBM-NOT9 complex to identify any flexible regions of the NIP-WT peptide that are not making any productive contacts with NOT9 when bound. Indeed, we observed that the N-terminal DDQQ region of the NIP-WT peptide is unstructured during the entire duration of the simulation (Fig. and supplemental Fig. ). This result prompted us to remove this region from the NIP-WT sequence for further peptide design. Additionally, the C-terminal G34 was removed as glycine is known to disturb helix formation resulting in the linear peptide NIP-1 (Fig. ). To evaluate the binding affinity, we fluorescently labelled the full-length and truncated peptides to produce NIP-WT-F and NIP-1-F and measured their affinity for the NOT9-NOT1 complex (consisting of NOT1 1351-1588 and NOT9 19-285) using fluorescence polarization (FP). The complex was co-expressed in E. coli, purified and its formation confirmed using mass photometry (supplemental Fig. ). Interestingly, the truncation significantly affected the binding affinity when comparing NIP-WT-F (KD = 40.7 ± 4.6 nM) to the truncated NIP-1-F sequence (KD = 876 ± 43 nM) (Fig. and supplemental Fig. ). We assumed that although the DDQQ sequence doesn't make any contacts with NOT9, it still plays a key role in helix stabilization as the carbonyl groups of D14, Q15 and Q16 act as a hydrogen bond donors for intramolecular backbone hydrogen bonds and provide a nucleation site for helix propagation. Therefore, we anticipated that introducing a helical constraint in the form of a hydrocarbon staple into the truncated peptide would regain the helical fold and thus recover the binding affinity for NOT9. Furthermore, a hydrocarbon staple is known to have additional benefits such as providing stability against proteolytic cleavage and improved cell-permeation compared to the native peptide sequence. To fulfill this goal, several hydrocarbon stapling sites were explored to induce the native α-helical conformation into the NIP-1 peptide sequence. To install the hydrocarbon staple, two C α -disubstituted amino acids containing an alkene side chain were introduced at i and i+n positions of the linear peptide, followed by cyclizing them by treatment with Grubbs catalyst while the peptide was still on solid support. We initially employed the i,i+4 strategy where two (S)-2-(4-pentenyl)alanines (S5) are incorporated at the i and i+4 positions. To avoid any steric clash with NOT9, the hydrocarbon linkers were installed orthogonal to the CBM-NOT9 binding interface and three different amino acid partners (H19/Q23, K22/E26 and E26/L30) were chosen. We replaced the two methionine residues (M24 and M31) with the chemically stable analogue norleucine as we observed oxidation of the sulfur atoms during the metathesis reaction. All three peptides were fluorescently labelled at the N-terminus to determine their binding affinity for NOT9. NIP-2-F was found to have the highest affinity among all three with a KD of 60.4 ± 15.5 nM compared to NIP-3-F and NIP-4-F (2299 ± 1223 nM and 588 ± 21 nM respectively) (Fig. and supplemental Fig. ). Seeking further optimization of the peptides, NIP-2-F was selected as a starting point where we kept the first stapling position constant, but used a longer linker to connect the i and i+7 residues (H19 = (R)-2-(7-octenyl)alanine and E26 = (S)-2-(4-pentenyl)alanine) to provide peptide NIP-5-F. Furthermore, two double-stapled peptides were designed again using NIP-2-F as a starting point. In the first peptide (NIP-6-F) residue Q23 was replaced with the double alkene amino acid bis-pentenylglycine, while H27 was replaced with (R)-2-(4-pentenyl)alanine to obtain a so-called stitched peptide (Fig. and supplemental Fig. ). For NIP-7-F, the residues H19, Q23, E26 and L30 were replaced with (S)-2-(4-pentenyl)alanine to obtain a double stapled peptide. For these three analogues an accurate affinity could only be measured for NIP-5-F (KD = 601 ± 147 nM), but the determination of the direct binding affinities of NIP-6-F and NIP-7-F was not possible due to their poor solubility in aqueous buffer. Since NIP-2-F still had the highest binding affinity and good solubility properties, it was used for further studies. NIP-MUT-F, a mutant version of the NIP-WT peptide, was generated as a negative control where both L17 and M24 were substituted by glutamic acid which were previously described to disrupt the interaction. FP analysis of FITC-labelled NIP-MUT-F indeed demonstrated it had negligible binding (KD > 10000 nM) to NOT9 (Fig. and supplemental Fig. ).