Screening And Mechanism Research Of Novel Small-Molecule Anticancer Inhibitors Targeting CD147

Cancer remains a major cause of mortality worldwide and also a common dieases which is a severe threat to human health. The biological capabilities of cancer, including sustaining proliferative signaling, resisting cell death and activating invasion and metastasis, bring a huge challenge for cancer therapy. Traditional treatments can hardly cure this disease fundamentally and always accompany with some side effects, which affect the quality life of patients. In resent years, as a novel therapeutic strategy, TBCT has been identified to be a potential effective treatment because of its high specificity and low side-effect. Many of them have been approved by FDA and have gone into service, presenting good clinical curative effect. Targets play key role in molecular targeted therapy strategy, therefore it is necessary to cure cancer with increasing knowledge of tumor molecular mechanism and finding accurate targets.Recestly, CD147, a member of immunoglobulin superfamily have been mostly identified as a tumor associated molecule. It is reported that CD147 is overexpressed in nearly 20 kinds of tumor types and involved in their biological events such as tumorigenesis, proliferation, differation, invasion, apoptosis and abnormal metabolism. While all these events above can be reversed when CD147 is interfered, blocked or even knocked out. Moreover, the identification of CD147 structure is of great help to screen drugs specifically. All above, CD147 has been qualified to be a potential drug target in cancer. However no small-molecule inhibitors of CD147 have been developed to date. Therefore, our present study focused on the screening for novel small-molecule anticancer inhibitors targeting CD147 and their molecular mechanism.This present work includes three parts:Part I virtual screening of small molecule compounds targeting CD147 and identification of lead compound AC-73.We used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays including SPR assay and zymography test, and the most potent one, dubbed AC-73(ID number: AN-465/42834501), was identified to bind CD147 and studied further. In addition, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147.Part II AC-73 inhibits the motility and invasion of hepatocellular carcinoma cells.In this part, we evaluated the function and molecular mechanism of AC-73 in hepatocellular carcinoma cells(HCC), whose progress is reported frequently to correlate with CD147. Native SDS-PAGE/Western blot assay indicated that AC-73 could disrupt CD147 dimerazation significantly. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, using RT-qPCR, zymography test and western blot asssy, we found AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase(MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3(STAT3) signaling pathway. Finally, AC-73 represented well tolerance in vivo and attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/ STAT3/MMP-2 pathways, which are crucial for cancer progression.Part III a novel modified compound targeting CD147 affects cancer cell viability and induces apoptosis.In this exploratory study, 8 candidate compounds were obtained based on the modification of AC-73. Only one compound, dubbed HA-08, was identifed to bind CD147 through molecular docking simulation and SPR assay, and studied further. After chemical synthesis and purification, we carried on the functional assay with HA-08. Using transwell assay, we found HA-08 could also inhibit cancer cell invasion. But this results might due to the inhibition of cell viability of HA-08. Meanwhile, through native SDS-PAGE/western blot assay, we demonstrated that HA-08 didn’t inhibit CD147 dimerization, suggesting HA-08 might have different mechanism to AC-73. HA-08 was observed to affect cancer cell viability by using WST-1 assay with 9 different cancer cell lines. Additionally, we also found HA-08 could promote cancer cell apoptosis through inducing apoptotic phenotype and change of apoptosis-related proteins expression. Further, these anti-cancer activity of HA-08 was also proved in in vivo assay. Finally, we confirmed all the characterizations of HA-08 in cancer were correlated with CD147.