Screening of a novel CRM1 targeting inhibitor and its effects on the proliferation and growth of extranodal NK/T cell lymphoma / 中国药科大学学报

ABSTRACT

@#The purpose of this study was to screen out the novel chromosome maintenance protein 1(CRM1)covalent targeting inhibitors by computer-assisted drug design(CADD), and to study their effects on the proliferation of extranodal nature killer/T cell lymphoma(ENKTL). A novel CRM1 inhibitor LFS-829 was designed based on the molecular structure of LFS-01 by means of ADME/T and covalent docking. The target binding of LFS-829 with CRM1 was analyzed by MALDI-TOF mass spectrometry. The effects of LFS-829 on the proliferation of extranodal NK/T cell lymphoma SNK6 and HANK-1 cells were detected by CCK-8. The cell morphology was observed by live cell workstation. Immunofluorescence experiments were used to analyze the effect of LFS-829 on nuclear export function of CRM1. The changes of NF-κB signaling pathway under different concentrations of LFS-829 were analyzed by Western blot, dual luciferase reporter gene assay and enzyme-linked immunosorbent assay. Apoptosis was detected by flow cytometry, and the expression of proteins related to apoptosis pathway was detected by Western blot. Tests of peripheral blood mononuclear lymphocyte(PBMC)toxicity, platelet toxicity and mouse acute toxicity were done to make sure that it is not harmful to human. LFS-829 could bind covalently to the cysteine residue of the hydrophobic active pocket of CRM1. LFS-829 could selectively kill SNK6 and HANK-1 cells, with IC50 of 366 nmol/L and 158 nmol/L in 72 h, respectively, and cell morphology was significantly changed. LFS-829 at 800 nmol/L significantly inhibited the nuclear export function of CRM1, promoted nuclear assembly of IκB-α, down-regulated the transcriptional activity of NF-κB signaling pathway, significantly up-regulated the expression of apoptotic pathway protein p53, cleaved Caspase 3 and cleaved Caspase 9 and induced apoptosis, with no obvious killing effect on PBMC or platelets. It did not cause substantial tissue damage to mice at the high dose of 300 mg/kg, which shows its great prospect of future application.