The results of the in vitro deadenylation experiments demonstrated that we likely needed concentrations exceeding 25 μM to assess the effect of CCR4-NOT inhibition in cells. To improve the uptake of NIP-2-N3 we prepared the octaarginine modified NIP-2-R8-N3 and evaluated it in the NanoClick assay. The conjugate had a 3.4-fold improve cell uptake but polycationic cell-penetrating peptides are known to be toxic in the micromolar range and can displace RNA-binding proteins from RNA. Indeed, when evaluated in a cell viability assay, we found that NIP-2-R8-N3 had an IC50 value of 29.2 ± 0.57 μM limiting its use (supplemental Fig. ). To improve on both cell permeability while avoiding toxicity issues we designed three variants of NIP-2 where a polar residue that was not determined to be a hot-spot in the virtual alanine scan was changed to alanine following previously reported guidelines. All three peptides were modified with an N-terminal azidolysine and evaluated using the NanoClick assay as well as fluorescently labelled to measure their affinity for the NOT9-NOT1 complex. Mutation of aspartic acid in position 18 or glutamic acid in position 26 did result in a moderate 2.1 or 1.6-fold increase in uptake respectively (Fig. ) but the affinity of these compounds was decreased (NIP-2-D18A-F: KD = 393 ± 180 nM, NIP-2-E26A-F: KD = 195 ± 147 nM). Mutation of histidine in position 27 (NIP-2-H27A-N3/F) led to a much stronger increase in uptake of nearly 7-fold (Fig. , supplemental Fig. ). In combination with a moderate 2-fold loss of affinity in comparison to the parent peptide NIP-2-F with a KD of 122 ± 8 nM (supplemental Fig. ) we deemed this sufficient for cellular experiments. Furthermore, we found that the peptide was non-toxic to HeLa cells (IC50 value > 100 μM) (supplemental Fig. ) and was able to inhibit deadenylation by the CCR4-NOTCORE complex at 10 μM (supplemental Fig. ).