Screening Of MMP-2 Inhibitory Peptides And Anti-cancer Effects Of Their Mimetic Compounds In Vitro And In Vivo

Objectives:1.To screen MMP-2 inhibitory peptides from phage display library and observe their effects on tumor cell invasion.2.To design and synthesis compounds using bioinformatics according to the conformation of the selected peptides and MMP-2 active domain.3.To observe the effects of the compounds on tumor cell proliferation in vitro,and tumor growth,invasion and metastasis in vivo.Methods:1.Phages with high affinity to MMP-2 enzyme were selected from phage display library.2.MMP-2 fluorescent assay kit for drug discovery was used to screen the selected phage clones and determine their effect on MMP-2 activity.3.Extracting single-stranded phage DNA and sequencing.4.Synthesizing the peptides in vitro.5.Determining the synthetic peptide effect on MMP-2 activity using MMP-2 fluorescent assay kit for drug discovery.6.Matrigel-coated transwell inserts were used to observe the effects of synthetic peptides on cancer cell invasion.7.According to the conformation of selected peptides and MMP-2 active domain,a series of compounds were designed using bioinformatics and synthesized.All compounds were assayed with MMP-2 fluorescent assay kit for drug discovery and MTT method to determine their effects on MMP-2 activity and cancer cell proliferation.8.Breast cancer nude mice model was made to observe their treatment effects in vivo.Results:1.22 phage clones with high affinity to MMP-2 enzyme were obtained after screening of phage display library,and all of these clones could inhibit the activity of MMP-2 enzyme.19 peptides were obtained after sequencing.2.Two peptides were obtained after screening of phage display library and MMP-2 enzyme activity inhibitory effect assay.Their sequences were as follows:M204C4:HWWQWPSSLQLRGGGSM205C4:HNWTRWLLHPDRGGGS3.Those two peptides M204C4 and M205C4 inhibited the activity of MMP-2 in the different effects and the inhibitory effects were dose dependent. The median effective inhibiting concentration(IC₅₀)for M204C4 and M205C4 were 78.0 and 38.8 nmoml/L,respectively.4.M204C4 inhibited MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1 with a dose dependent manner.High concentration(200 nmol/L)of M204C4 significantly inhibited the invasion of these two cell lines than the control(P＜0.001).The other two concentrations 60 nmol/L and 20 nmol/L of M204C4 had similar inhibitory effects,but milder than high concentration.5.M205C4 inhibited MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1 with a dose dependent manner.The high and middle concentration(100 nmol/L and 30 nmol/L)of M205C4 could inhibit the pancreatic cancer cell invasion significantly.The low concentration (10nmol/L)could inhibit the pancreatic cancer cell invasion,but no difference was observed compared with the control group(P＞0.05).6.48 compounds were synthesized.According to the results of MTT assay and MMP-2 enzyme activity inhibitory assay,WB-1,WB-45 and WB-46 were selected to further investigate in vivo.7.No toxic reaction was observed between the control and the drug administration groups.In the control group,breast cancer metastases were observed in the lung pathologic section,but no metastasis in the WB-1 group. The average weight of the animals in WB-1 administration group was higher than the control group,but no difference was observed.The average tumor weight in the 200mg/kg group decreased to 24.7%of the control group,and the same phenomenon was observed between 100mg/kg and 10mg/kg groups. In the WB-45 and WB-46 group,tumor metastases were observed in almost all lung pathologic sections.In other organs,no metastasis was observed.8.The serum level of VEGF in the WB-1 group(100mg/kg and 10mg/kg) were lower than that in the control group,and a statistical difference was observed between 100mg/kg and the control group(P＜0.05).9.The serum levels of pro- and active MMP-2 were lower in the WB-1 100mg/kg administration group than that in the control group(P＜0.05),but no difference for pro-MMP-9 expression was observed between the drug administration group and the control group.Conclusions:1.Two peptides M204C4 and M205C4 were obtained after screening the phage display library.2.In vitro,M204C4 and M205C4 inhibited the activity of MMP-2 enzyme significantly,meanwhile inhibited MMP-2 mediated pancreatic cancer cell invasion.3.The mimetic compound WB-1 not only inhibited MMP-2 activity and cancer cell proliferation in vitro,but also inhibited tumor growth and metastasis and decreased the serum levels of VEGF,pro- and active MMP-2 in vivo.