Synthesis Of Stapled Derivatives Of Wasp Venom Peptide Anoplin And The Mechanism Of Its Oncolysis Induced Anti-tumor Immunotherapeutic Effects

ObjectiveIt was previously found that Anoplin,a natural wasp venom peptide derived from traditional Chinese medicine,exists intrinsic drawbacks in anti-tumor efficacy and medicinal properties.A series of Anoplin derivatives(Anos)were synthesized via peptide stapling technology,and the structure-activity relationships and preliminary druggability of Anos were examined.Further,we comprehensively evaluated oncolytic effect of Anos.Tumor-bearing mice were used to investigate their dual mechanism of efficient oncolysis and activated systematic anti-tumor immunity.This study provided important preliminary data to elucidate new oncolytic immune mechanism of natural antitumor wasp venom peptides,and provided experimental evidences to realize novel tumor treatment patterns with efficient oncolysis and enhanced immunity.Methods1.We prepared a series of Anoplin derivatives(Anos)via peptide stapling technology.Their physicochemical properties and α-helix structures were characterised by high performance liquid chromatography,high resolution mass spectroscopy and circular dichroism.The antiproliferative activities of Anos toward triple-negative breast cancer(TNBC)4T1 and melanoma B16F10 cell lines were detected by Cell Counting Kit-8(CCK-8).With in silico prediction,structure-activity relationships and hemolytic index of Anos were further analysed.Red blood cell hemolysis assays were performed to verify the actual hemolytic side effects of Anos and the preferred compound(Ano-3)with best therapeutic index was obtained.2.To verify that Ano-3 could mediate membrane lysis of cancer cells,we investigated the release characteristics of extracellular lactate dehydrogenase(LDH)and adenosine triphosphate(ATP)from tumor cells after treatment with Ano-3 at different concentrations and time.The oncolytic process of Ano-3 was continuously observed with high resolution fluorescence microscopes.The candidate oncolytic peptide LTX-315 in clinical phase II trial served as a standard positive control.3.In order to further improve the solubility of Ano-3,we designed and synthesized the Ano-3s,the derivate of Ano-3,inspired by double side-chain-retention stapling strategy.Previously established methods were applied to test physicochemical property,druggability(water solubility,hemolytic side effects,and protease resistance),and oncolytic potency of Ano-3 s.4.The antitumor effects of Ano-3 and Ano-3s were investigated in vivo using the 4T1/B16F10 cell-based mouse cancer models.Their antitumor mechanisms in vivo were explored to explain significantly different therapeutic effects in the two models.We performed immunohistochemical stainings to determine cytotoxic lymphocyte(CTL)infiltration in tumors after treatment of Ano-3 and Ano-3 s.CD8+T cell depletion was used to study the significance for dual oncolytic-immunotherapy in Ano-3 s treated mice.Results1.We successfully designed and prepared Anos.Compared to the parent Anoplin,the Anos demonstrated α-helicity with a 1.3-to 15.8-fold increase.More importantly,the half-maximal inhibitory concentration(IC50)of Anos ranged from 5.8-to 47.8-fold enhanced potency for tumor cells.The structure-activity relationship study of Anos indicated that the increased α-helical structure and strong hydrophobic network were the critical reasons for improved anti-proliferative activities of Anos in vitro.Moreover,several Anos with enhanced anti-tumor effects exhibited lower hemolytic toxicity,and Ano-3 had the best therapeutic window.2.In vitro experiments showed that Ano-3 could rapidly kill tumor cells,and significantly inhibited proliferation in a time-and concentration-dependent manner.We also detected the instant release of LDH and ATP in the supernatant after the cells were exposed to Ano-3.With high resolution fluorescence microscopes,we observed that Ano3 induced rapidly contraction and lysis of tumor cell membranes,and Ano-3 exerted superior oncolytic activity relative to positive control LTX-315.3.To further optimize the water solubility and other druggabilities of Ano-3,Ano3 s,a stapled derivative with the retention of double side chains of lysine and leucine,was successfully synthesized by using novel stapling technology.The oncolytic effect and druggability studies of Ano-3 s in vitro showed that Ano-3 s still maintained significant oncolytic effect and antiproliferative activity,and no significant difference from Ano-3.In addition to improved water solubility,the hemolytic side effect of Ano-3 s was significantly reduced.Subsequently,both Ano-3 and Ano-3s were distinctly more stable toward protease-mediated degradation when compared to parent Anoplin and positive control LTX-315.All these results suggested that Ano-3s could serve as a lead compound for subsequent studies.4.Ano-3 and Ano-3s achieved high efficiency to inhibit tumor growth in melanoma B16F10 tumor-bearing mice,but failed inhibiting tumor growth in TNBC 4T1 cell-based mouse models effectively.We further found that Ano-3 and Ano-3s could mediate immunogenic cell death of tumor cells through efficient oncolysis,and the infiltration of CD8+T cells in TNBC tumors was significantly lower than that in melanoma.We speculated that the tumor immunesuppressive microenvironment was a limit for the efficacy of oncolytic peptides in vivo.In addition,CD8+T cell depletion in Ano-3 s treated mice did not prevent initial B16F10 tumor progression.The above results suggested that Ano-3 and Ano-3 s could induce tumor immunogenic cell death through oncolytic effect,thereby achieving dual oncolytic-immunotherapy through CD8+T cell infiltration mediated by systemic antitumor immunity.ConclusionTo solve the problems of the insufficient stability and anti-tumor activity of Anoplin,a natural anti-tumor wasp venom peptide derived from traditional Chinese medicine,a series of structurally constrained Anos were designed and synthesized by stapling technology.Systematic experiments proved that these Anos all had high helicity and exerted superior tumor cytotoxicity than parent Anoplin.With the structure information of Anos,their structure-activity relationships were further elucidated.Ano-3,with the best therapeutic window,was selected as the lead molecule,and its oncolytic mechanism was evaluated in vitro.To further optimize the druggability of Ano-3,Ano-3 s was synthesized via novel stapling strategy with the retention of double side chains.As expected,Ano-3 s maintained prominent oncolytic activity while exhibiting significantly improved water solubility and lower hemolytic side effects.The in vivo antitumor assay showed that Ano3s could activate the systemic anti-tumor immunity through efficient oncolysis,and significantly inhibit tumor growth in melanoma tumor-bearing mice.This study provided important preliminary data and innovative ideas for the application of stapling technology in the development of peptide drugs derived from traditional Chinese medicine,and provided important basis for the innovative application of the peptide-based oncolytic immunotherapy in melanoma.