Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma
- School of Bioengineering, Dalian University of Technology, China
- School of Software, Dalian University of Technology, China
- Department of Oncology, Shengjing Hospital of China Medical University, China
Figures
Figure 1 with 4 supplements
The discovery of sulforaphene synthetic analogue LFS-1107.
(A) The identification of ten commercial-accessible aromatic fragments aided by deep reinforcement learning model; (B) Synthesis of LFS-1107 via the installation of aromatic tetrazole moiety selected from the previous step to the sulforaphene parent structure; (C) Assessment of protein-ligand binding kinetics and binding affinity of tetrazole aromatic fragments via Bio-layer interferometry (BLI) assay; (D) Binding affinity of LFS-1107 and KPT-330 determined via BLI assay: LFS-1107, Kd~1.25E-11 M; KPT-330: Kd~5.29E-09 M.
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Figure 1—source data 1
The chemical structure of 10 commercial-accessible aromatic fragments.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-data1-v2.zip
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Figure 1—source data 2
The synthesis of compound LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-data2-v2.zip
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Figure 1—source data 3
The data of affinities and binding kinetics of CRM1 to S5 and S8.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-data3-v2.zip
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Figure 1—source data 4
The data of affinities and binding kinetics of CRM1 to LFS-1107 and KPT-330.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-data4-v2.zip
Figure 1—figure supplement 1
Binding affinities and binding kinetics of ten commercial-accessible fragments with CRM1 were determined using Bio-layer interferometry (BLI) assay.
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Figure 1—figure supplement 1—source data 1
The data of affinities and binding kinetics of CRM1 to ten commercial-accessible aromatic fragments.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
The BLI results of two control proteins Keap1 (A) and IκBα (B).
No binding affinities were detected for the two control proteins during BIL assay with compound LFS-1107.
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Figure 1—figure supplement 2—source data 1
The data of affinities and binding kinetics of Keap1 and IκBα to LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-figsupp2-data1-v2.zip
Figure 1—figure supplement 3
Organic synthesis scheme of compound LFS-1107.
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Figure 1—figure supplement 3—source data 1
Organic synthesis scheme of compound LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-figsupp3-data1-v2.zip
Figure 1—figure supplement 4
Expression of CRM1 mRNA in different tumor types.
It shows that CRM1 was significantly elevated in patients with Lymphoma.
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Figure 1—figure supplement 4—source data 1
The expression of CRM1 mRNA in different tumor types.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig1-figsupp4-data1-v2.zip
Figure 2 with 3 supplements
LFS-1107 strongly suppresses the growth of ENKTL cells acting through the nuclear retention of IkBα and subsequent attenuation of NF-κB signaling.
(A) Suppression of different human NK/T cell lymphoma cells; (B) GSH/GSSG ratio detection upon the treatment of LFS-1107; (C) The effect of LFS-1107 on normal PBMC cell lines; (D) The viability of platelets treated with LFS-1107; (E) Western blot result of the CRM1 with β-actin as loading control; (F) The cellular activities of LFS-1107 on siCRM1-293T and the wild-type 293T cell line with different concentrations of treatment for 48 h; (G) Nuclear retention of IκBα by confocal microscopy (Scale bar, 20 μM); (H) Quantification of the nuclear IΚB-α ratio by fluorescence intensity per cell (Data presented as mean ± SEM); (I) Representative western blots of p65, Cox-2, c-Myc, and Survivin; (J) Western blots showing the protein level of IκBα in nucleus and cytoplasm; (K) ELISA detection of cytokine production after treated with LFS-1107. Data were either presented as representative images or expressed as the mean ± SD of each group. Statistical analysis were performed via Student t-test, ***p<0.001, ****p<0.0001.
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Figure 2—source data 1
Inhibition of the cell growth of SNK6 and Hank-1 cells by LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data1-v2.zip
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Figure 2—source data 2
GSH/GSSG ratio detection upon the treatment of LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data2-v2.zip
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Figure 2—source data 3
Suppression of the cell growth of PBMC cells by LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data3-v2.zip
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Figure 2—source data 4
Suppression of the cell growth of platelets by LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data4-v2.zip
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Figure 2—source data 5
Immunoblot of CRM1 expression after LFS-1107 treatment.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data5-v2.zip
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Figure 2—source data 6
The cellular activities of LFS-1107 on siCRM1-293T and the wild-type 293T cell line.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data6-v2.zip
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Figure 2—source data 7
Nuclear accumulation of IκBα induced by treatment with LFS-1107 for 3 hr.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data7-v2.zip
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Figure 2—source data 8
Quantification of the nuclear IκBα ratio by fluorescence intensity per cell.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data8-v2.zip
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Figure 2—source data 9
Immunoblot of expression p65, Cox-2, c-Myc, and Survivin after LFS-1107 treatment.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data9-v2.zip
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Figure 2—source data 10
Immunoblot of IκBα in nucleus and cytoplasm expression after LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data10-v2.zip
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Figure 2—source data 11
ELISA detection of TNF-α, IFN-1γ, p65, IL-1α, IL-1β, IL-6, IL-8, and MCP-1 after treated with LFS-1107.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-data11-v2.zip
Figure 2—figure supplement 1
Proteomics analysis of SNK6 cells (control vs. LFS-1107 treatment) indicates that CRM1 was downregulated upon LFS-1107 treatment (FC~1.3).
Moreover, the Gene Ontology (GO) analysis suggests that Biological Process (regulation of cellular component organization) and Molecular Function (protein binding) related to CRM1 were modulated upon the treatment of LFS-1107.
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Figure 2—figure supplement 1—source data 1
Proteomics analysis of SNK6 cells.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Cellular activities of KPT-330 towards two ENKTL cell lines.
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Figure 2—figure supplement 2—source data 1
Cellular activities of KPT-330 towards two ENKTL cell lines.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-figsupp2-data1-v2.zip
Figure 2—figure supplement 3
Representative immunofluorescence images of IκBα (stained with Cy3) localization in Hela cells.
Nuclear accumulation of IκBα induced by treatment with LFS-1107 (500nM) for 3 hours. The medium (containing LFS-1107) was removed and replaced with new medium in the Wash group. Nuclei was stained with DAPI. Scale bars, 50μM.
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Figure 2—figure supplement 3—source data 1
Nuclear accumulation of IκBα induced by treatment with 500 nM LFS-1107 for 3 hr.
The medium (containing LFS-1107) was removed and replaced with new medium in the Wash group. Fixed cells were stained for IκBα (orange) and DAPI (blue).
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig2-figsupp3-data1-v2.zip
Figure 3
LFS-1107 can ameliorate the symptoms of ENKTL in xenograft mouse model.
(A) Scheme of the xenograft mouse model. The mice were randomly divided into three groups (n = 6-13 per group). SNK6 cells were injected i.v. in female NOD SCID mice, and mice were injected per week with LFS-1107; (B) Survival rate of different animal groups; (C–E) Flow cytometry of human ENKTL cell lines in mouse bone marrow with the use of FITC anti-human CD45 antibody (P1, red) or no primary antibody control (black); (F–G) The symptoms of splenomegaly in each group of euthanized mice. From left to right: normal group, control ENKTL xenograft model group(splenomegaly), LFS-1107 treatment group (10 mg/kg). Data were either presented as representative images or expressed as the mean ± SEM of each group. Statistical analysis were performed via Student t-test, *p<0.05, ***p<0.001.
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Figure 3—source data 1
The process for the in the xenograft mouse model study.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig3-data1-v2.zip
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Figure 3—source data 2
Survival rate of normal group, control group and LFS-1107 treatment group.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig3-data2-v2.zip
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Figure 3—source data 3
Flow cytometry of human ENKTL cell lines in mouse bone marrow.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig3-data3-v2.zip
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Figure 3—source data 4
The symptoms of splenomegaly in normal group, control group and LFS-1107 treatment group mice.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig3-data4-v2.zip
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Figure 3—source data 5
The spleen weight of normal group, control group and LFS-1107 treatment group mice.
- https://cdn.elifesciences.org/articles/80625/elife-80625-fig3-data5-v2.zip
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/80625/elife-80625-mdarchecklist1-v2.pdf
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Source code 1
Deep reinforcement learning model for molecular de-novo design.
- https://cdn.elifesciences.org/articles/80625/elife-80625-code1-v2.zip
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Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma
eLife 12:e80625.
https://doi.org/10.7554/eLife.80625
