Inhibitory Function And Mechanism Of OXPHOS Inhibitor SMIP004-7 On Malignant Melanoma In Vivo

Background:SMIP004-7 is an OXPHOS inhibitor,which blocks mitochomdrial complex I(NADH:ubiquinone oxidoreductase)and induces reactive oxygen species(ROS),thereby causing tumor cell death.The autonomic effects of cells include:targeting the respiratory phenotype of bulk of tumor mass,targeting the tumor stem cell compartment and targeting metabolic inflexibility.Beyond cancer cell autonomous effects,OXPHOS inhibitors may have tumor microenviromental effects.Hypoxia is an important factor in the tumor microenvironment,and is considered to be one of the most relevant factors for tumor response of tumor cells.At the same time,hypoxia is related to immunosuppression.It is currently important to combine cell autonomic and microenvironmental effects,for example to design rationale combination therapies(for example with immune checkpoint modulators:anti-PD-1).In this project,we propose to explore potential microenvironmental and immune modulatory functions of SMIP004-7.Methods and results:Flow cytometry was used to measure intracellular ROS levels of B16 melanoma cells in vitro,this showed that SMIP004-7 led to a marked increase in ROS levels of B16 cells.The effect of SMIP004-7 on the growrh of B16 melanoma xenografts by measuring tumor sizes.H&E staining and IHC staining were performed on the melanoma tissue to detect differences in tumor morphology as well as and HIF1 alpha protein expression in response to SMIP004-7.Changes in the tumor mocroenvironment were assessed in frozen sections of tumor tissues.Flow cytometry was used to detect SMIP004-7-induces changes in the populations of CD4+and CD8+tumor-infiltrating lymphocytes.These showed that SMIP004-7 effectively inhibited the growth of B16 melanomas in mice.Compound-induced reduction of tumor hypoxia correlated with increased recruitment of CD4+T lymphocytes thus indicating activation of the innate anti-tumor immune response.In agreement with this findings,SMIP0047 combined with anti-PD-1 had a synergistic effect on the inhibition of B16 melanoma xenografts compared to the single agents.In vivo nuclide imaging showed that SMIP004-7 could enter the hypoxic environment inside the mouse tumor and significantly inhibit mitochondrial complex I.Conclusion:Our data showed that SMIP004-7 potently inhibits the growth of melanoma in vivo by changing the tumor microenvironment and activating the antitumor immune response.The result underscore the potential of SMIP004-7 as an anti-cancer drug candidate,which may serve as the theoretical basis for further clinical development of this novel anti-cancer agent.