Design,Synthesis,Biological Property And Bioactivity Of Stapled Peptide Inhibitors Targeting Ras-SOS1 Interaction

The incidence of cancer is increasing year by year,which has become one of the major diseases that seriously affect human life and living quality.Ras gene is one of the earliest and most frequently mutated proto-oncogenes in the occurrence and development of various cancers.Ras protein is a class of intrinsic GTP binding proteins that constantly switched between the GDP-bound inactive state and the GTP-bound active state to achieve a dynamic balance.The guanine nucleotide exchange factor SOS1 catalyzes Ras into a continuously activated state by loading GTP onto Ras,and disturbs downstream signal pathways and ultimately leads to cancer.Inhibiting the Ras-SOS1 interaction attracts considerable attention as a potential strategy of anticancer treatment.Based on the crystal structure of Ras-SOS1 complex,we analyzed key sites in the interaction interface,and designed and synthesized seven linear peptides,nine all-hydrocarbon stapled peptides and nine fluorescent-labeled peptides derived from two importantα-helix fragments of SOS1 using the Fmoc solid phase peptides synthesis technique.All peptides were purified by high performance liquid chromatography（HPLC）and characterized by electrospray ionization high resolution mass spectrometry（ESI-HR-MS）,and the final purity was greater than 95%.All compounds were confirmed to be reported for the first time by a literature search.Firstly,we measured the binding affinity between the polypeptides and the target protein Ras by surface plasmon resonance（SPR）experiment.The SPR results showed that the binding affinity of stapled peptide SSOSH-5 with Ras was about 8 times higher than that of linear peptide SOSH,and the K_d value of binding constant was 3.92μM.Subsequently,we tested the cytotoxicity of all peptides against the KRas-mutant tumor cells A549 through the CCK-8 cell activity assay.The results showed that the most A549 cells were inhibited after treating with 10μM SSOSH-5,and the half inhibitory concentration(IC₅₀)was 6.264μM.Further,CCK-8 cell activity assay was performed to test the cytotoxicity of SSOSH-5against variable wild-type,NRas-mutant and HRas-mutant tumor cells.We found that SSOSH-5 also had extremely strong cytotoxicity against these cells.At a concentration of10μM,SSOSH-5 could inhibit wild-type and pan-Ras-mutant tumor cells.Then,we evaluatedα-helicity,stability and membrane permeability of stapled peptides.The circular dichroism experiment showed that stapled peptide SSOSH-5 had a helicity of32.4%,which is better than that of linear peptide SOSH.In order to test the stability of SOSH and SSOSH-5,we use chymotrypsin as a hydrolase to simulate the degradation of linear peptide SOSH and stapled peptide SSOSH-5 in vivo.The result showed approximately 80%of SSOSH-5 remained structurally intact after 24 hours,which is better than that of linear peptide SOSH.Further,the membrane permeability of the fluorescent-labeled peptides in Ras-mutant tumor cells was detected by flow cytometry and live cell fluorescence staining experiment.The results showed that SSOSH-5 had strong membrane permeability in both wild-type and Ras-mutant tumor cells,and its membrane permeability was much higher than that of linear peptide SOSH.Above results indicated that all-hydrocarbon stapled strategy could improveα-helicity,stability and membrane permeability.Western blot assay and Annexin-V/PI double staining assay were performed to explore the mechanism of the stapled peptide SSOSH-5 inhibiting the proliferation of tumor cells.Western blot assay showed that SSOSH-5 effectively downregulated the phosphorylation of AKT and ERK,thus inhibited the over-proliferation of Ras mutant tumor cells.Annexin-V/PI double staining assay showed that SSOSH-5 induced apoptosis of A549 cells in a time-dependent and concentration-dependent manner.In conclusion,based on the crystal structure of Ras-SOS1 complex,a series of stapled peptides were designed and synthesized.The stapled peptide SSOSH-5 exhibited improvedα-helicity,stability and membrane permeability and could effectively bind with multiple Ras-mutant proteins.SSOSH-5 inhibit proliferation of multiple tumor cells and down-regulate level of ERK phosphorylation in tumor cells such as A549（KRas G12S）,T24（HRas G12V）and H1299（NRas Q61K）.As a novel pan-Ras stapled peptide inhibitor,SSOSH-5 exhibits good application prospect and further research value.