Study On The Mechanism Of Exportin-1 Small Molecule Antagonist In The Treatment Of Extranodal NK/T Cell Lymphoma

Extranodal natural killer cell(NK)/T cell lymphoma(ENKTL)has been extensively studied for its aggressive nature and poor prognosis in search of more effective treatment regimens.Chromosome region maintenance protein 1(CRM1),was initially detected in Schizosaccharomyces pombe.XPO1 was first identified through a mutation in its gene.XPO1 was later found to be an important nuclear output receptor.This project explored and studied the novel targeted small molecule compound inhibitor for XPO1 protein,and explored its in vitro binding ability to XPO1 protein and its therapeutic potential on ENKTL cells.The specific results are as follows:(1)Small molecule inhibitors with XPO1 targeting was established by means of computer aided drug design.COVDOCK was used to conduct segment docking,scoring identification,false positive screening and other methods on the small molecule database,and finally the target was reduced to AN-988.(2)SPR method was used to verify the highly targeted binding of small molecule An-988 to XPO1 protein in vitro.The results showed that the Kd value of AN-988 binding to XPO1 in the Biacore experiment was 2.132 μM,which verified that AN-988 had a strong targeting and in vitro binding ability to XPO1.(3)CCK-8 cytotoxicity test,immunofluorescence test,Western-blot test and ELISA test were used to carry out verification.Through the analysis of experimental data,AN-988 can significantly inhibit the cell viability of SNK6 cells.By immunofluorescence assay,we verified that the nuclear aggregation of IκB-α protein associated with XPO1 protein in the cells was obvious under the effect of AN-988,which indicated that AN-988 could significantly inhibit the nuclear output of XPO1 protein.Meanwhile,Western-blot assay confirmed that AN-988 could inhibit the expression of XPO1 protein,and also inhibit the expression of Bcl-XL and Survivin related proteins in the downstream NF-κB signaling pathway.The above results fully indicate that AN-988 may provide a new and promising therapeutic strategy for XPO1-mediated ENKTL.