Carbon Catabolite Repression 4—Negative On TATA-less (CCR4-NOT) is the main deadenylase complex that actively involves in the mRNA deadenylation process. Heterodimeric CCR4:CAF1 protein-protein complex (PPC) is responsible for the catalytic activity of the CCR4-NOT complex and directly docks into the scaffold protein NOT1 through the MIF4G region. Additionally, CNOT9 is another subunit of the complex which is responsible for the recruitment of the RNA as a substrate to the catalytic site. Several adaptor proteins (Bam, Roquin and NOT4) assists into this RNA recruitment process by interacting with the CNOT9 through the evolutionary conserved site called CNOT9 binding motif (CBM) domain. In some cases, the RNA directly interacts with the same surface of the CNOT9 and gets recruited to the CCR4-NOT complex. Therefore, these protein-protein and protein-RNA interactions are attractive target to design any inhibitors for the CCR4-NOT complex. In the ‘StapPep’ project, a structure-based design approach was utilized to identify suitable peptidic inhibitor derived from the CBM domain of the Bam protein. At the beginning, a molecular dynamic simulation was carried out using the existing crystal structure of the CBM peptide bound to the surface of the CNOT9. It was found that wild type (WT) full length CBM peptide (CBM-WT) retains its helical structure throughout the simulation period (100 ns) except for the four residues at the N-terminus (called DDQQ domain). Furthermore, this DDQQ domain doesn’t directly interact with the CNOT9 surface. For that reason, a truncated version of the CBM-WT peptide (CBM-short) was synthesized and interestingly CBM-short lost ~20 times binding affinity to the CNOT9 protein compared to the CBM-WT. In order to recover the potency, a panel of hydrocarbon stapled peptides were synthesized derived from the CBM-short peptide. The hydrocarbon stapling strategy was chosen because this strategy has been very efficient to retain the α-helical conformation in various peptide sequences and hydrocarbon part of the linker often induces better cellular uptake of peptides in numerous cancer cells. Eventually, a potent peptide binder was identified (CBM-pot) based on the fluorescence polarization experiment against the purified CNOT9-CNOT1 complex. Next, a co-crystal structure was obtained of CBM-pot bound to CNOT9-CNOT1 complex and it was found that CBM-pot specifically binds to the CNOT9 surface only and CNOT1 subunit interacts to the orthogonal surface of the CBM-pot…CNOT9 binding surface. To further understand the specificity of the CBM-pot…CNOT9 interaction, a pull-down experiment was performed where the CBM-pot was covalently connected to the DBCO-beads through click chemistry and the beads were incubated with Hela cell lysate. A western blot analysis exhibited that the CBM-pot was able to recognize CNOT9 selectively, however, a negative control peptide (CBM-neg) with negligible binding affinity to the CNOT9 barely showed any recognition. CBM-pot peptide showed excellent thermal stability and cell lysate stability compared to the CBM-WT peptide which were very important factors for any further use of CBM-pot peptide as a commercial drug. Additionally, CBM-pot was able to block the interaction between CNOT9 and RNA containing poly(A) tail. Having these results in hand, an in vitro inhibitory experiment was carried out in which short poly(A) tail RNA was incubated with purified CCR4-NOTcore complex and various concentration of CBM-pot peptide, interestingly, 10 μM concentration of CBM-pot was able to block the activity of CCR4-NOTcore complex and stabilized the RNA. However, CBM-pot exhibited lower cellular uptake in Hela cells as evidenced by Nanoclick experiment. Therefore, a panel of second-generation peptides were synthesized by keeping CBM-pot as a starting point and CBM-pot1 peptide was identified with ~10 times better cellular uptake without compromising the binding affinity. Next cellular RNAs were extracted from the CBM-pot1 peptide treated Hela cells and a cellular poly(A) tail length determination assay showed the stabilization of poly(A) tail of certain genes by CBM-pot1 peptide. Overall, ‘StapPep’ project adopted a very systematic approach starting from designing and synthesizing peptides, identifying the best peptide binder to the target protein and proving the stabilization of RNA by inhibiting the activity of CCR4-NOT complex at both in vitro and cellular level.
