Targeting Transcription Factor FOXM1 Interferes With The Discovery Of Peptide M1-21 And Preclinical Studies Of Tumor Suppression

As the second leading cause of death threatening human life,tumo r incidence and death are increasing year by year.With the development of science and technology and the establishment of a highly sound healt h and epidemic prevention system,human beings have established comprehensive prevention and control mechanisms and safeguarded treatment measures for viral and infectious pathogenic microbial diseases.Tumors,such as malignant diseases that may be driven by inherited genes,caused by the accumulation of ingestion in an unclean environment,or caused by the decline of the body’s immune function and the failure to remove malignant proliferating cells in a timely manner,have gradually become the most important cause of threats to human life.The process of tumor development inevitably involves overactivation and expression of multiple oncogenes.They can directly or indirectly promote malignant disorderly proliferation of normal cells or,when encountering t umor therapeutic drugs,contribute to drug resistance and immune tolerance of cancerous cells throug h tumor cell stemness and drug resistance mechanisms,which in turn promote cancerous cells to invade normal organs of the body,thus endangering life.Amon g them,FOXM1 transcription factor has been shown to be over-activated and expressed in a variety of tumor tissues and cells.It not only promotes tumor cell stemness directly(e.g.,by assisting β-catenin protein into the nucleus,thus activating the WNT p athway),but also promotes tumor cell proliferation(transcriptional activation of CDC25 B gene),invasion and migration(transcriptional activation of Vimentin,C-MYC PLK1 gene,etc.).Therefore,FOXM1 is considered an effective target for tumor therapeuti c drug development.Biologically targeted therapies are a promising therapeutic modality and research area,with low biotoxicity and the ability to target intracellular targets through a combination of appropriate delivery strategies.One such drug type th at should be explored is interfering peptides.And through the coupled cell penetration of peptides(e.g.,TAT,R9),also can achieve efficient delivery in and out of the cell.Based on the above research background,this paper further completes a number of important preclinical work based on the anti-tumor recombinant protein M1-138.The main research results are as follows:1.Key affinity peptide screening and lead drug validationIn this study,a computer-simulated peptide array(P1 to P24)was constructed based on the anti-tumor recombinant protein M1-138 and its FOXM1 N-terminal domain source(FOXM1(1-138aa)sequence).The peptide array was simulated by Rosetta Flex Pep Dock and calculated by Interface Analyzerm to screen for FOXM1C-terminal affinity peptides.After 50,000 simulated docking for each peptide,the binding affinity between the peptide and FOXM1 was analyzed,and the inhibitory activity of the peptide on cells was detected by experiments.Finally,candidate peptide segments 96-116,101-121,106-126 were selected.To verify the cytoinhibitory activity of the candidate peptides,we conjugated the cell penetrating peptide TAT,which was synthesized and purified by chemical solid phase synthesis.Subsequent Pulldown experiments showed that TAT-106-126 could target FOXM1 and inhibit the activity of 231 cells.2.Optimization of lead drug dosage form and precise mutat ion of target protein verified the target specificity of M1-21Subsequently,we designed and synthesized a D-type amino acid peptide by modifying the dosage form of TAT-106-126 peptide and named it M1-21.Compared to the parent peptide TAT-106-126,M1-21 has higher in vitro stability,longer intracellular metabolic cycle,and better tumor cell inhibitory activity.While M1-21 was targeted to inhibit a variety of tumor cells,the mutant peptide M1-21 mut targeting FOXM1’s C-terminal domain completely lost tum or cell consolidation function and activity.3.Unbiased transcriptomics-based analysis of regulatory pathways,and intracellular phenotypic validationTranscriptomic RNA-seq unbiased analysis of regulated signaling pathways showed that M1-21 inhibited the initiation of DNA replication and activation of WNT signaling pathways.It activates the cell adhesion signaling pathway,and the apoptosis signaling pathway.The results of cell and molecular biology experiments showed that M1-21 inhibited the proliferation of 231 breast tumor cells in vitro and in nude mice,inhibited 231 cell migration,and induced 231 cell apoptosis.4.The mechanism of FOXM1 inhibition by interfering peptide M1-21In molecular mechanism,M1-21 binds multiple FOXM1 domains(N-terminal domain,DNA binding domain,and C-terminal domain).It also interferes with FOXM1’s interaction with LIN9 and B-MYB in its N-terminal interaction complex,with PLK1,a kinase protein required for FOXM1 C-terminal activation,and with the interaction between FOXM1 and β-catenin,a key protein in the WNT signaling pathway,and β-catenin nuclear entry-associated function.Thus,in cancer cells,M1-21 inhibits FOXM1-associated direct transcriptional activity,FOXM1-associated indirect transcriptional activity,and FOXM1-mediated β-catenin nuclear entry-associated transcriptional activity of the WNT pathway.5.Validation of the efficacy of interfering peptide M1-21 in animal modelsAnimal experiments showed that M1-21 significantly inhibited tumor proliferation in the spontaneous mammary tumor FVB/N MMTV-Py VT model;inhibited organ invasion and colonization of 4T1-derived cells,and tumor cell proliferation in the BALB/c mouse model.6.Biosafety analysis of M1-21The drug metabolism test in mice showed that M1-21 was able to metabolize enriched organs and tissues such as heart,liver,bladder,mandible,spleen and lung,and the drug remained in the body after 24 h.Physiolo gical sections of organs after continuous administration of M1-21 showed that M1-21 did not cause significant organ damage.The acute toxicity dose of up to 200 mg/kg and the hemolytic toxicity test of 800 μg/m L showed high drug safety and an ideal safety threshold for administration.The final immunogenicity test assay showed that M1-21 did not cause excessive antigen-like immune activation and showed controlled immune tolerance.