Development Of Peptide-based Urokinase Inhibitors And The Studies On The Anti-tumor Effects

Urokinase-type plasminogen activator(uPA),also termed urokinase,is an important cancer-related serine protease that is overexpressed in most tumor tissues.The active uPA degrades the extracellular matrices and promote the metastasis of tumor cells by activating plasminogen,matrix metalloproteinase in direct or indirect ways.Meanwhile,uPA on the surface of the tumor also activates growth factors promoting the proliferation of tumor cells.uPA has been recommended as a diagnostic and prognostic indicator for breast and prostate cancer by the American Society of Clinical Oncology and the German Breast Cancer Society.Moreover,an uPA inhibitor,Mesupron,was FDA-approved for the clinical treatment of the pancreatic cancers in 2017.Based on our previous work,we developed a series of highly potent and specific uPA cyclic peptide inhibitors.Among them,the best peptide inhibited uPA with a K_i value of 6.8 nM,showing non-measurable inhibitory effects on most homologous serine hydrolase.In the following work,we elucidated the inhibitory mechanism of these peptide inhibitors by various biochemical and molecular biological methods.The kinetics of the interaction between cyclic peptides and uPA or various homologous proteases were studied by surface plasmon resonance analysis,isothermal titration calorimetry and enzyme kinetics.At the same time,we solved the crystal structure of these peptides in complex with uPA to study the detailed peptide-uPA interactions.However,the crystal structures did not completely elucidate the inhibitory mechanism of these peptide inhibitors.Therefore,based on the crystal structure,we performed the molecular dynamic simulations and studied the dynamic interactions between peptides and uPA.Combining the experimental results with the computational simulation,we revealed the inhibition mechanism of polypeptide inhibitors on uPA at the molecular level.In addition,we further demonstrated the effects of the series of polypeptide inhibitors on the proliferation,migration and invasion properties of tumor cells in the cellular level by cell scratch assay and Transwell invasion assay.Our inhibitors significantly inhibited the invasion of mouse colon cancer cells CT-26G in vitro,but did not affect the growth of tumor cells,indicating that the inhibitory effects of peptide-based uPA inhibitors on tumor metastasis was shown through the inhibition of the degradation of extracellular matrix or biofilm.Basing on the above experimental results in the molecular and cellular levels,we established animal tumor models to evaluate the inhibitory effects of peptide-based uPA inhibitors on tumor growth and metastases.Our peptide inhibitors significantly suppressed tumor growth in a subcutaneously inoculated CT-26G-grafted mice model.Moreover,in another lung-metastatic mice model,we demonstrated the remarkable antimetastatic effects of our peptides via the pathological section,real-time quantitative PCR,survival rates etc.In conclusion,we developed a series of peptide-based uPA inhibitors with high potency and specificity comparable to monoclonal antibodies,which effectively inhibited the proliferation and metastases of tumor cells by modulating the uPA-related signaling pathways.Our results not only demonstrate that peptide inhibitors can be used as highly potent antitumor lead drugs,but also clarify their inhibitory mechanism on uPA,which is of great significance for the development of other serine protease inhibitors.