Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: Anti-SARS-CoV-2 efficacy of ansatrienin analogs. (A) Anti-SARS-CoV-2 efficacy of ansatrienin analogs at 2.5 μmol/L in Vero E6 cells. (B) Cytotoxicity of ansatrienin analogs at 10 μmol/L in Vero E6 cells. (C) Dose-response relationship of ansatrienin analogs in Vero E6 cells. Vero E6 cells were treated with eight 2-fold serial dilutions of the indicated compounds and then infected with SARS-CoV-2. (D) Selective index (SI, CC 50 /IC 50 ) of ansatrienin analogs in vitro . (E) Anti-SARS-CoV-2 activity of ansatrienin analogs in Vero E6 cells by RT-qPCR. Data are represented as mean ± SD for three independent experiments. ∗∗∗ P < 0.001 compared to vehicle control. Vehicle control contained the same volume of DMSO as the corresponding concentrations of labeled compounds.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Infection, In Vitro, Activity Assay, Quantitative RT-PCR, Control, Labeling

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: Broad-spectrum antiviral activities of ansatrienin B. (A) Dose-response relationship of ansatrienin B in other cell lines. Hela-ACE2/Calu-3/Caco-2 cells were treated with eight 2-fold serial dilutions of ansatrienin B and then infected with SARS-CoV-2. (B) Dose-response relationship of ansatrienin B against SARS-CoV-2 Omicron variants. Vero-E6 cells were treated with eleven 2-fold serial dilutions of ansatrienin B and then infected with SARS-CoV-2 Omicron variants. (C) Effect of ansatrienin B on flaviviruses (YFV, WNV, DENV) and alphaviruses (CHIKV). Huh7 cells were treated with eight 2-fold serial dilutions (2.5‒0.0195 μmol/L) of ansatrienin B and then infected with YFV, WNV, DENV, or CHIKV, respectively. The non-structural (NS) protein and cell nuclei were visualized with an IF assay. (D) Chemical structure of ansatrienin B. Data was represented as mean ± SD for three independent experiments.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Infection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: Mechanistic study of ansatrienin B against SARS-CoV-2. (A) Effect of ansatrienin B on SARS-CoV-2 life cycles by a Time-of-addition assay. Vero E6 cells were incubated with SARS-CoV-2 for 2 h to synchronize the assay, then the culture medium was removed. Cells were treated with ansatrienin B or remdesivir at a final concentration of 2.5 μmol/L at 2 h before (−2) or 0, 2, 4, 6, 8, and 10 h after the supernatants were removed. The infection was qualified at 24 h post-inoculation after fixation and staining for SARS-CoV-2 N protein and cell nuclei were visualized with IF assay. Remdesivir served as a positive control. (B, C) Effect of ansatrienin B on SARS-CoV-2 entry in Vero E6 cells. Vero E6 cells were treated with vehicle control or ansatrienin B at indicated concentrations, and incubated with SARS-CoV-2 (MOI = 0.1) at 4 °C for 2 h. For binding assay (no incubation) or internalization assay (incubated at 37 °C for another 2 h), cells were washed with cold PBS 3 times to remove unbound viruses. Then, cells were supplemented with growth medium and incubated at 37 °C for 24 h to test the infection by an IF assay. TRIzol reagent was directly added to the 24-well plate to lyse the cells. The total RNA was extracted, and the level of SARS-CoV-2 RNA was detected via RT-qPCR. The vehicle control contained the same volume of DMSO as was used to dissolve the corresponding concentrations of ansatrienin B. (D, E) Ansatrienin B suppressed SARS-Cov-2 replication. Vero-E6 cells were transfected with 1 μg of SARS-CoV-2 replicon RNA for 6 h. The culture medium was replaced with fresh medium (vehicle control) or containing ansatrienin B or Remdesivir at indicated concentrations for 24 hpi. Cells were detected via luciferase assay, IF and RT-qPCR. (F) A parallel experiment was performed and the expression of replicon was detected using an IF assay. Data are represented as mean ± SD for three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 compared to vehicle control. Scale bar = 1000 μm.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Incubation, Concentration Assay, Infection, Staining, Positive Control, Control, Binding Assay, Quantitative RT-PCR, Transfection, Luciferase, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: SPR and molecular docking studies of the interaction between ansatrienin B and viral RdRp. (A, B) Evaluation of the binding affinity of ansatrienin B to SARS-CoV-2 RdRp and WNV RdRp by a SPR assay. (C, D) Predicted intermolecular interaction of ansatrienin B and SARS-CoV-2 RdRp (PDB 7BV2 ) and WNV RdRp (PDB 2HCN ). Left are the overall view and binding pocket of RdRp with ansatrienin B; right shows the individual residues lining the pocket. Hydrogen bonds are indicated with pink arrows.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Binding Assay, SPR Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: Effect of ansatrienin B with viral RdRp. (A) Ansatrienin B inhibited the interaction between RdRp and SARS-CoV-2 genome. Psoralen-PEG3-Biotin labeled SARS-CoV-2 genomic RNA (10 μg) was mixed with SARS-CoV-2 RdRp (10 μg), ansatrienin B (10 μmol/L), incubated with 10 μL streptavidin magnetic beads at 4 °C overnight and washed, RdRp protein in the pulled-down fractions was analyzed by immuneblotting. The gray intensity of immunoblotting bands was analyzed using ImageJ software. (B) Gel image showed elongation of the RNA duplex by the purified SARS-CoV-2 RdRp complex and its inhibition by ansatrienin B across a concentration range (50 to 15 mmol/L). (C) Quantitative analysis of RdRp inhibition by ansatrienin B. Data are presented as the mean ± SD of four replicates within one experiment. The WNV RdRp inhibition by ansatrienin B was shown as (D) luciferase activity and (E) WNV replicon RNA levels. In vitro -transcribed WNV replicon RNA was transfected into Huh7 WNV−NS5 (WNV-NS5) or Huh7 Vector (Vector). Six hours post-transfection, the cells were treated with either ansatrienin B (20 μmol/L) or DMSO (vehicle control). At 24 h post-transfection, luciferase activity was measured using a luciferase substrate (5D), and the levels of WNV replicon RNA were quantified by RT-PCR (5E). ns: no significant; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Labeling, Incubation, Magnetic Beads, Western Blot, Software, Purification, Inhibition, Concentration Assay, Luciferase, Activity Assay, In Vitro, Transfection, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: Ansatrienin B protects hamster from SARS-CoV-2 infection-caused injury. (A) Scheme and drug administration of the animal experiment. The hamsters were challenged via i.n. injection with inoculation of 10 5 TCID50 SARS-CoV-2 Wuhan strain and followed by daily i.p. administration of solvent buffer or test compounds, respectively, for five days. Three hamsters were monitored daily for body weights. The other three hamsters in each group were sacrificed and sampled four days after infection. (B) Percentage of body weight changes (compared to starting weight) were monitored daily for 15 days. Four groups were included: Mock group (uninfected and i.p. administration of solvent buffer); Vehicle control group (infected with SARS-CoV-2 and i.p. administration of solvent buffer); Ansatrienin B group (infected with SARS-CoV-2 and i.p. administration of ansatrienin B); Remdesivir group (infected with SARS-CoV-2 and i.p. administration of remdesivir). (C, D) Lungs were harvested at 4 dpi and viral load was assessed by plaque assay (viral titre) or RT-qPCR (viral load). The expression of inflammatory cytokines was tested by RT-qPCR. (E) Representative images of H&E-stained lung tissue section from SARS-CoV-2 infected hamsters. Red arrows: sites of inflammatory infiltration and alveolar wall thicken or congestion. (F) Histological analysis of lung pathology. Histology scores were given to each lung tissue to distinguish comprehensive lung pathological changes ( n = 6). Data are represented as mean ± SD for three independent experiments. ∗∗∗ P < 0.001 compared to vehicle control.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Infection, Injection, Solvent, Control, Plaque Assay, Quantitative RT-PCR, Expressing, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mutasynthetic optimization of ansatrienin B: A new broad-spectrum inhibitor against RNA viruses

doi: 10.1016/j.apsb.2026.02.021

Figure Lengend Snippet: Structures and of anti-SARS-CoV-2 efficacy of new ansatrienin derivatives. (A) Structures of ansatrienin derivatives As–Ds. (B) Anti-SARS-CoV-2 efficacy of ansatrienin derivatives As–Ds at 2.5 μmol/L in Vero E6 cells. (C) IC 50 s of ansatrienin derivatives As–Ds against SARS-CoV-2 in Vero E6 cells. Data are represented as mean ± SD for three independent experiments. ∗∗∗ P < 0.001 compared to vehicle control.

Article Snippet: Reaction mixtures containing 10 μg of biotin-labeled RNA and 10 μg of SARS-CoV-2 RdRp (with or without 10 μmol/L ansatrienin B) were incubated with 10 μL of streptavidin magnetic beads (HY-K0208, MedChemExpress) at 4 °C overnight.

Techniques: Control