Periodic Reporting for period 1 - StapPep (Stapled peptide analogues as inhibitors of the CCR4-NOT complex to regulate the mRNA degradation)
Periodo di rendicontazione: 2022-06-01 al 2024-05-31
Human body functions are controlled by several biomolecules (eg. DNA, RNA, proteins and lipids). Malfunction of any of these biomolecules is responsible for numerous diseases including cancers. Among these biomolecules, RNA has gained a significant attention recently as a therapeutic target due to its evolutionary role in gene regulations. In the gene regulation process, RNA seldom works as an individual entity but rather associated with several protein-protein complexes (PPCs) and these complexes play a key role in mRNA maturation. Almost all mRNA contains a stretch of adenosine (called poly(A) tail) at the 3’ end which plays an important role in mRNA stability and translation process. Typically, mRNA undergoes different post-transcriptional modifications and deadenylation is the key post-transcriptional event in which the shortening of the poly(A) tail at the 3’ end of mRNA occurs and therefore influences the overall mRNA stability. Deadenylation is often regulated by different PPCs and dysregulation of any of these complexes severely impaired this process. In fact, deadenylation process is considered to be the surveillance mechanism for the cell and uncontrolled deadenylation could drives the cell to the oncogenic state. In many cancer cells shortening of poly(A) tail has been found due to the non-specific deadenylation event. Therefore, chemical modulators that can regulate the deadenylation process have immense therapeutic importance. The major focus of the ‘StapPep’ project is to stabilize mRNA containing poly(A) tail by identifying suitable inhibitors against CCR4-NOT complex which is a crucial player of the mRNA degradation. Therefore, the key object of this project is to develop peptidic modulators to inhibit the mRNA deadenylation process in target specific manner by directly inhibiting the activity of CCR4-NOT complex. This approach is unique in the sense that only weak inhibitors without any cellular activities were reported. Moreover, the better stability, larger surface area, versatile side-chain functionalities and low cytotoxicity are advantageous for peptide drugs over the traditional small molecules. We anticipate that the current strategy would open a new way of tackling cancer by modulating the mRNA stability and thus revolutionize the therapeutics invention for the future cancer treatments.
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.
Post-transcriptional regulations of gene expressions is immensely important for the proper function of cells and organisms. Many oncogenes or growth factors are regulated post-transcriptionally and mRNA turnover seems to have impact on these processes. Deadenylation is considered as the rate limiting step of mRNA degradation and thus significantly influences the overall mRNA turnover. In fact, these processes are overreactive in various types of cancers. Therefore, understanding the underlying mechanisms of these post-transcriptional processes and perturbing them by any chemical modulators has great importance in precise gene expression modulation and eventually it will lead us to tackling deadly diseases like cancers. Nonetheless, gene expressions can be regulated at different levels either DNA, RNA level or protein level. Although gene expression at DNA and protein level by traditional drug molecules have been quite successful, targeting RNA is relatively underexplored. ‘StapPep’ project undertook the challenge to target the protein-protein and protein-RNA interactions associated with the CCR4-NOT complex functions. CCR4-NOT complex is known for its regulatory activities in the deadenylation process. ‘StapPep’ project identified the first stapled peptide inhibitor against the CCR4-NOT complex. The peptide inhibitor has the ability to modulate the gene expressions by regulating the overall stability of certain mRNAs. ‘StapPep’ project overcome several challenges to find the potent peptide with excellent stability, improved cell-permeability and outstanding inhibitory activity at both in vitro and cellular level and paved an alternative way of targeting gene expressions.