Journal: Acta Pharmaceutica Sinica. B

Article Title: Dimeric natural product panepocyclinol A inhibits STAT3 via di-covalent modification

doi: 10.1016/j.apsb.2024.10.001

Figure Lengend Snippet: PecA inhibits the transcription activity of STAT3. (A) Cell viability screen of an in-house dimer compound collection. After treatment with 10 μmol/L compounds for 48 h, cell viabilities were triplicate tested by MTS and then normalized to the value of the DMSO-treated group. (B) Structure of PecA. (C) Transcription factors activation profiling was used to analyze the activities of indicated transcription factors in Karpas299 cells upon DMSO or 10 μmol/L PecA treatment. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (D) Fluorescence polarization assay measured the affinity of diverse compounds for purified full-length STAT3. (E) GSEA analysis for the RNA-seq results of Karpas299 and SU-DHL-1 cells upon DMSO or 10 μmol/L PecA treatment. (F) The genome-wide profiles of STAT3 ChIP-seq peaks in Karpas299 cells upon DMSO or 10 μmol/L PecA treatment. Gene ontology analysis for the significantly reduced genes upon PecA treatment was shown (fold change <0.5). (G) ChIP-seq tracks in DMSO or PecA-treated Karpas299 cells along the indicated gene loci.

Article Snippet: STAT3 inhibitors Stattic (Cat.# HY13818), C188-9 (Cat.# HY112288), BP-1-102 (Cat.# HY-100493), S3I-201 (Cat.# HY-15146) were purchased from MedChemExpress, Shanghai, China.

Techniques: Activity Assay, Activation Assay, Fluorescence, Purification, RNA Sequencing, Genome Wide, ChIP-sequencing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dimeric natural product panepocyclinol A inhibits STAT3 via di-covalent modification

doi: 10.1016/j.apsb.2024.10.001

Figure Lengend Snippet: PecA selectively induces covalent homodimer of STAT3. (A) Karpas299 (upper) and SU-DHL-1 (lower) cells were treated for different concentrations of PecA for 2 h. Cell lysates were subjected to SDS-PAGE gel for Western blot. (B) Karpas299 cells were treated with various STAT3 inhibitors for 4 h. STAT3 and p-STAT3 were analyzed by Western blotting. (C) Purified full-length STAT3 protein (20 nmol/sample) was incubated with a series concentration of PecA at room temperature for 1 h. The mixture was then applied to SDS-PAGE gel followed by commassie blue staining. (D) Karpas299 cells were treated with 30 μmol/L of PecA or its analogs for 2 h. Cell lysates were then subjected to SDS-PAGE for Western blot. (E) Karpas299 cells upon PecA treatment were lysed for Western blot. Appropriate antibodies were used as indicated. (F) Karpas 299 cells were treated with 20 μmol/L of PecA for a time course. (G) Full-length STAT3 or truncated STAT3 (127–688 aa) proteins were incubated with series concentration of PecA for 1 h. A biotin-labeled DNA probe was used to detect the DNA binding affinity for STAT3 by EMSA. Mutant probe to STAT3 (mut) was used as a negative control.

Article Snippet: STAT3 inhibitors Stattic (Cat.# HY13818), C188-9 (Cat.# HY112288), BP-1-102 (Cat.# HY-100493), S3I-201 (Cat.# HY-15146) were purchased from MedChemExpress, Shanghai, China.

Techniques: SDS Page, Western Blot, Purification, Incubation, Concentration Assay, Staining, Labeling, Binding Assay, Mutagenesis, Negative Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dimeric natural product panepocyclinol A inhibits STAT3 via di-covalent modification

doi: 10.1016/j.apsb.2024.10.001

Figure Lengend Snippet: Cysteins 712 and 718 of STAT3 are responsible for the covalent modifications of PecA. (A) The dimer bands and untreated STAT3 bands were analyzed by LC‒MS/MS. The raw data were analyzed by pLink2.0. Potential cross-linking spectra presented in dimer samples but not in untreated control samples were listed. (B) Karpas299 cells harboring different STAT3 mutations were treated with 20 μmol/L PecA for 2 h. Cell lysates were then subjected to SDS-PAGE. (C) STAT3 wildtype or a C712A/C718A mutant (CA) was stably introduced into Karpas299 or SU-DHL-1 cells by Crispr-Cas9. IC 50 values of PecA were measured by MTS assay. (D, E) Karpas299 (D) or SU-DHL-1 cells (E) harboring wildtype or C712A/C718A mutant STAT3 were treated as indicated concentrations of PecA for 4 h. The mRNA levels of STAT3 target genes were quantified by real-time PCR. Data are presented as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS, not significant.

Article Snippet: STAT3 inhibitors Stattic (Cat.# HY13818), C188-9 (Cat.# HY112288), BP-1-102 (Cat.# HY-100493), S3I-201 (Cat.# HY-15146) were purchased from MedChemExpress, Shanghai, China.

Techniques: Control, SDS Page, Mutagenesis, Stable Transfection, CRISPR, MTS Assay, Real-time Polymerase Chain Reaction

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dimeric natural product panepocyclinol A inhibits STAT3 via di-covalent modification

doi: 10.1016/j.apsb.2024.10.001

Figure Lengend Snippet: MD simulation of the STAT3-PecA complex. (A) A scheme illustrates the covalent connection of the two carbon atoms of the PecA to the two sulfur atoms of the C712 residues on the chains A and B of the STAT3_ligand complex. The covalently connected PecA is shown in the ball-and-stick representation. (B) An illustration of the STAT model immersed in a cubic water box. (C) Calculated RMSD curves of the STAT3 and STAT3_ligand complexes from 500 MD simulation trajectories. (D) A comparison of top 3 representative structures of the STAT3 and STAT3_ligand model derived from clustering analysis of simulated trajectories. (E) Comparison of the major conformations of the complexes shows that the loops at the binding sites of the STAT3 and STAT3_ligand complexes maintain closed and open conformations, respectively. The PecA bound at the binding site of the STAT3_ligand complex was colored in purple. (F) A comparison of the conformations of the DNAs in the STAT3 and STAT3_ligand complexes. The binding free energies of the two complexes were calculated.

Article Snippet: STAT3 inhibitors Stattic (Cat.# HY13818), C188-9 (Cat.# HY112288), BP-1-102 (Cat.# HY-100493), S3I-201 (Cat.# HY-15146) were purchased from MedChemExpress, Shanghai, China.

Techniques: Comparison, Derivative Assay, Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dimeric natural product panepocyclinol A inhibits STAT3 via di-covalent modification

doi: 10.1016/j.apsb.2024.10.001

Figure Lengend Snippet: PecA is efficacious against STAT3 wt and STAT3 Y640F ALCL. (A) Karpas299 cells with STAT3 mutations were treated with series concentrations of PecA for 48 h. Cell viability was tested by MTS. (B) Karpas299 cells harboring different STAT3 mutations were treated with PecA. STAT3 dimerization was analyzed by Western blot. (C) Karpas299 cells harboring wildtype STAT3 or STAT3 Y640F mutations were treated with 20 μmol/L PecA for 2 h. Cell lysates were then subjected to SDS-PAGE. (D, E) Wildtype STAT3 or STAT3 Y640F mutations engineered Karpas299 (D) or SU-DHL-1 cells (E) were treated with 20 μmol/L PecA for 4 h. The mRNA levels of STAT3 target genes were quantified by real-time PCR. Data are presented as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: STAT3 inhibitors Stattic (Cat.# HY13818), C188-9 (Cat.# HY112288), BP-1-102 (Cat.# HY-100493), S3I-201 (Cat.# HY-15146) were purchased from MedChemExpress, Shanghai, China.

Techniques: Western Blot, SDS Page, Real-time Polymerase Chain Reaction

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dimeric natural product panepocyclinol A inhibits STAT3 via di-covalent modification

doi: 10.1016/j.apsb.2024.10.001

Figure Lengend Snippet: PecA suppresses tumor growth in xenograft models of STAT3 wt and STAT3 Y640F lymphoma. (A‒C) Lymphoma Jeko-1 xenograft tumor models were administrated with the vehicle, 20 mg/kg PecA once daily (qd) or twice daily (bid) by tail vein injection. Data are presented as mean ± SEM ( n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS, not significant. The tumor volumes were monitored daily (A). Tumors were separated. Weights of tumors (B) and representative photos (C) for each experimental group are shown. (D‒H) xenograft mouse models harboring wildtype or Y640F STAT3 transfected Karpas299 cells were treated with vehicle or 20 mg/kg PecA twice a day by tail vein injection. The tumor volumes and body weights were measured every day (D). The tumors were harvested 7 days after drug treatment. Weights of tumors (E) and representative photos (F) for each experimental group are shown. The levels of STAT3 and p-STAT3 were analyzed by Western blot (G). H&E staining was shown (H), scale bar = 50 μm.

Article Snippet: STAT3 inhibitors Stattic (Cat.# HY13818), C188-9 (Cat.# HY112288), BP-1-102 (Cat.# HY-100493), S3I-201 (Cat.# HY-15146) were purchased from MedChemExpress, Shanghai, China.

Techniques: Injection, Transfection, Western Blot, Staining