Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha
Open accessAbstract
Target identification in natural products plays a critical role in the development of innovative drugs. Bufalin, a compound derived from traditional medicines, has shown promising anti-cancer activity; however, its precise molecular mechanism of action remains unclear. Here, we employ artificial intelligence, molecular docking, and molecular dynamics simulations to elucidate the molecular mechanism of Bufalin. Using an integrated multi-predictive strategy, we identify CYP17A1, ESR1, mTOR, AR, and PRKCD as the potential targets of Bufalin. Subsequent validation via surface plasmon resonance, biotin pulldown, and thermal shift assays confirms Bufalin's direct binding to ESR1, which encodes estrogen receptor alpha (ERα). Molecular docking analyses pinpoint Bufalin's selective interaction with Arg394 on ERα. Molecular dynamic simulations further show that Bufalin acts as a molecular glue, enhancing the interaction between ERα and the E3 ligase STUB1, thereby promoting proteasomal degradation of ERα. Given the therapeutic potential of ERα degradation in overcoming endocrine resistance, we investigate the inhibitory effect of Bufalin on endocrine-resistant models and prove Bufalin reverses Tamoxifen resistance in vitro, in vivo, and in patient-derived breast cancer organoids from tamoxifen-relapsed cases. Collectively, our findings indicate that Bufalin functions as a molecular glue to degrade ERα, offering a potential therapeutic strategy for reversing Tamoxifen resistance.
AI snapshot
Written from the paper’s full text.
TL;DR
Bufalin, a natural product, was identified via an integrated AI target-prediction strategy as a direct binder of estrogen receptor alpha (ERα/ESR1) that acts as a molecular glue to enhance ERα–STUB1 interaction and promote proteasomal ERα degradation. Bufalin reversed Tamoxifen resistance in ER+ breast cancer models in vitro, in vivo, and in patient-derived organoids.
Key findings
- An integrated AI/multi-predictive strategy nominated CYP17A1, ESR1, mTOR, AR, and PRKCD as Bufalin targets; SPR confirmed the strongest direct binding to ESR1/ERα with KD = 5.42E−06 M, versus PRKCD (1.29E−02 M) and CYP17A1 (2.94E−02 M).
- Bufalin selectively bound Arg394 of ERα: the ERα R394A mutant lost Biotin-Bufalin binding, showed reduced Bufalin-induced degradation, and had decreased ubiquitination compared with wild-type ERα.
- Bufalin acted as a molecular glue for ERα degradation by enhancing ERα–STUB1 interaction; MD simulations reported the ERα–STUB1 binding free energy changing from −4.9455 kcal/mol to −58.1918 kcal/mol with Bufalin, and STUB1 knockdown attenuated Bufalin-induced ERα degradation.
- Bufalin induced proteasomal ERα degradation, suppressed ERα transcriptional activity and target genes, and overcame Tamoxifen resistance in LCC2 cells, LCC2 xenografts (n = 7 mice per group), and Tamoxifen-relapsed patient-derived organoids (n = 3 independent experiments), with better anti-cancer effects than Fulvestrant in several assays.
Methods
- Target prediction combined FMBS, SwissTargetPrediction, SEA, PPB2, and ChEMBL, followed by KEGG enrichment and a Chemprop graph neural network trained on 11 targets (average 1187 compounds per target, 80% training/20% test, ROC-AUC 0.94, probability cutoff 0.8).
- Experimental validation used SPR, biotin pulldown, CETSA, western blot, cycloheximide chase, MG-132/MLN4924/HCQ treatments, Co-IP, ERE-luciferase, qPCR, CCK-8, colony formation, EdU, Annexin V apoptosis assays, IHC, and xenograft studies.
- Mechanistic modeling used Glide SP docking, Amber22 molecular dynamics simulations (100 ns and 200 ns), MM-GBSA/MM-PBSA energy calculations, alanine scanning, and protein–protein docking; patient-derived organoids were established from Tamoxifen-relapsed breast cancer specimens.
Limitations
- The paper does not state a dedicated limitations section.
- Evident from the text: only ERα among the five predicted targets was experimentally validated as the primary target, while mTOR and AR were noted as previously reported and PRKCD/CYP17A1 showed much weaker binding; the molecular glue mechanism relies heavily on docking and MD simulations rather than a solved ternary crystal structure.
- Evident from the text: efficacy is preclinical only, with in vivo work limited to LCC2 xenografts, organoid experiments reported as n = 3 independent experiments, and no clinical trial, pharmacokinetic, or long-term toxicity data beyond mouse serum markers.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
Statistics
Claims the paper makes
45 of 64 cited sources checked · 20 supported · 0 flagged · accuracy not yet measured
- Premisecitation
STUB1 downregulates ERα levels through the ubiquitin-proteasome system.
“On the contrary, the ubiquitin ligase STUB1 downregulates ERα levels through the ubiquitin–proteasome system33.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [33] Fan, M., Park, A. & Nephew, K. P. CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha. Mol. Endocrinol. 19, 2901–2914 (2005). source
- Findingcitation
Silencing STUB1 increased ERα levels.
“Consistent with previously published research, silencing STUB1 increased ERα levels (Fig. 5g).”
Quote in the paperSubstantial claim - Findingown data
Cells overexpressing ERα were more sensitive to Bufalin.
“whereas cells overexpressing ERα were more sensitive to Bufalin (Fig. 6h).”
Quote in the paperSubstantial claim - Findingown data
STUB1 knockdown attenuated Bufalin-induced ERα degradation.
“Furthermore, STUB1 knockdown attenuated Bufalin-induced ERα degradation (Fig. 5i), suggesting that STUB1 was required for ubiquitination of ERα induced by Bufalin.”
Quote in the paperSubstantial claim - Findingown data
Ligand binding reduces the flexibility of residues at the protein-protein interface, specifically residues 335-343, 417-425, and 515-538 of ESR1, and residues 25-49, 61-69, and 121-136 of STUB1.
“Moreover, RMSF profiles of ESR1 and STUB1 indicate that ligand binding reduces the flexibility of residues at the protein-protein interface. Specifically, decreased fluctuations were observed in residues 335–343, 417–425, and 515–538 of ESR1, and in residues 25–49, 61–69, and 121–136 of STUB1 (Fig. 5e).”
Quote in the paperSubstantial claim
- Findingown data
The binding of Biotin-Bufalin with ERα was dose-dependent.
“Moreover, the binding of Biotin-Bufalin with ERα was dose-dependent (Fig. 2f), and the addition of free Bufalin in cells significantly inhibited the binding of Biotin-Bufalin to ERα (Fig. 2g).”
Quote in the paperSubstantial claim - Findingown data
Bufalin treatment causes dose-dependent and time-dependent reduction of ERα protein in ER+ breast cancer cells.
“Western blot analysis showed dose-dependent and time-dependent ERα reduction upon Bufalin treatment (Fig. 3a, b).”
Quote in the paperSubstantial claim - Findingown data
Bufalin weakened the transcriptional activity of ERα.
“The ERE luciferase assay showed that Bufalin weakened the transcriptional activity of ERα (Fig. 3i), which was corroborated by downregulation of ERα target genes, including AGR2, CCND1, GREB1, NRIP1, PGR, and SIAH2 (Fig. 3j and Supplementary Fig. 1c).”
Quote in the paperSubstantial claim - Findingown data
Bufalin significantly inhibited MCF-7 and T47D cell growth.
“Bufalin significantly inhibited MCF-7 and T47D cells growth, as demonstrated by both colony formation and CCK-8 assays (Fig. 6a, b).”
Quote in the paperSubstantial claim - Findingown data
Bufalin selectively interacts with Arg394 on ERα.
“Molecular docking analyses pinpoint Bufalin’s selective interaction with Arg394 on ERα.”
- Also stated in: § Bufalin selectively binds to Arg394 (R394) of ERα · plain
“Our analysis revealed that residues with energy contributions better than −1 kcal/mol, specifically Leu354, Leu387, Arg394, Met528, and Ala350, are key residues for Bufalin binding to ERα.”
Quote in the paperSubstantial claim - Also stated in: § Bufalin selectively binds to Arg394 (R394) of ERα · plain
- Findingown data
Cells with silenced ERα were less sensitive to Bufalin treatment compared to control cells in colony formation assays.
“Consistently, colony formation assays demonstrated that cells with silenced ERα were less sensitive to Bufalin treatment compared to control cells (Fig. 6i).”
Quote in the paperSubstantial claim - Findingown data
ERα-knockdown models revealed a diminished therapeutic response to Bufalin, as indicated by decreased Annexin V staining.
“ERα-knockdown models revealed a diminished therapeutic response to Bufalin, as indicated by decreased Annexin V staining (Fig. 6d, e).”
Quote in the paperSubstantial claim - Findingown data
Residues Leu354, Leu387, Arg394, Met528, and Ala350 are key residues for Bufalin binding to ERα.
“Our analysis revealed that residues with energy contributions better than −1 kcal/mol, specifically Leu354, Leu387, Arg394, Met528, and Ala350, are key residues for Bufalin binding to ERα.”
Quote in the paperSubstantial claim - Findingown data
Bufalin directly binds to ESR1, which encodes estrogen receptor alpha (ERα).
“Subsequent validation via surface plasmon resonance, biotin pulldown, and thermal shift assays confirms Bufalin’s direct binding to ESR1, which encodes estrogen receptor alpha (ERα).”
- Also stated in: § Bufalin directly interacts with estrogen receptor α (ERα) · boosted
“These results collectively demonstrate a specific and direct binding between Bufalin and the ERα protein, supporting the potential of Bufalin as a targeted agent against ERα in ER+ breast cancer.”
Quote in the paperSubstantial claim - Also stated in: § Bufalin directly interacts with estrogen receptor α (ERα) · boosted
- Findingown data
The presence of Bufalin resulted in an increased number of hydrogen bonds between STUB1 and ESR1, further stabilizing their interaction.
“As the simulation progressed, the presence of Bufalin also resulted in an increased number of hydrogen bonds between STUB1 and ESR1, further stabilizing their interaction (Fig. 5f).”
Quote in the paperSubstantial claim - Findingown data
Biotin-Bufalin and ERα were colocalized in MCF-7 cells.
“and the Biotin-Bufalin and ERα were colocalized in the MCF-7 cells (Fig. 2i).”
Quote in the paperSubstantial claim - Findingown data
Addition of free Bufalin in cells significantly inhibited the binding of Biotin-Bufalin to ERα.
“and the addition of free Bufalin in cells significantly inhibited the binding of Biotin-Bufalin to ERα (Fig. 2g).”
Quote in the paperSubstantial claim - Findingown data
Silencing ERα expression diminished Bufalin's anti-cancer effects on breast cancer cells.
“We also observed that silencing ERα expression diminished Bufalin’s anti-cancer effects on breast cancer cells (Fig. 6g)”
Quote in the paperSubstantial claim - Findingown data
The ERα-STUB1 complex remains conformationally stable throughout the 200 ns simulation in the presence of Bufalin.
“RMSD analysis demonstrates that the ERα-STUB1 complex remains conformationally stable throughout the 200 ns simulation in the presence of Bufalin (Fig. 5c).”
Quote in the paperSubstantial claim - Findingown data
Bufalin had a superior ERα degradation efficacy compared to Fulvestrant.
“The results showed that Bufalin had a superior ERα degradation efficacy compared to that of Fulvestrant (Supplementary Fig. 1d).”
Quote in the paperSubstantial claim - Findingown data
The mRNA of ERα had no obvious change in cells exposed to Bufalin.
“Notably, the mRNA of ERα had no obvious change in cells exposed to Bufalin, suggesting that the downregulation of the ERα protein was not due to genetic transcription inhibition (Supplementary Fig. 1a, b).”
Quote in the paperSubstantial claim - Findingown data
MLN4924 reversed the Bufalin-mediated downregulation of ERα.
“As illustrated in Fig. 3e, MLN4924 reversed the Bufalin-mediated downregulation of ERα.”
Quote in the paperSubstantial claim - Findingown data
The binding free energy between ERα and STUB1 changed from −4.9455 kcal/mol to −58.1918 kcal/mol owing to the involvement of Bufalin.
“Our results showed the binding free energy between ERα and STUB1 was changed from −4.9455 kcal/mol to −58.1918 kcal/mol owing to the involvement of Bufalin (Supplementary Table 1), consistent with the experimental results.”
Quote in the paperSubstantial claim - Findingown data
ERα silencing attenuated Bufalin-induced apoptosis, confirmed by reduced expression of cleaved PARP.
“Moreover, ERα silencing attenuated Bufalin-induced apoptosis, which was confirmed by the reduced expression of cleaved PARP (Fig. 6f).”
Quote in the paperSubstantial claim - Findingown data
The degradation efficacy of Bufalin on the ERα Arg394A protein was significantly reduced.
“Furthermore, the degradation efficacy of Bufalin on the ERα Arg394A protein was significantly reduced (Fig. 4e).”
Quote in the paperSubstantial claim - Findingown data
Using a default cutoff of 0.8, five target proteins were identified as potential targets of Bufalin: CYP17A1, ESR1, mTOR, AR, and PRKCD.
“Using a default cutoff of 0.8, five target proteins were identified and marked as potential targets of Bufalin: CYP17A1, ESR1, mTOR, AR, and PRKCD.”
Quote in the paperSubstantial claim - Findingown data
Co-IP assays revealed enhanced ubiquitination of ERα in response to Bufalin treatment.
“Additionally, co-immunoprecipitation (Co-IP) assays revealed enhanced ubiquitination of ERα in response to Bufalin treatment (Fig. 3g, h), corroborating that Bufalin induces ERα degradation predominantly through the proteasomal pathway.”
Quote in the paperSubstantial claim - Findingown data
MG-132 effectively prevented the Bufalin-induced decrease in ERα protein levels.
“Furthermore, treatment with the proteasome inhibitor MG-132 effectively prevented the Bufalin-induced decrease in ERα protein levels (Fig. 3d).”
Quote in the paperSubstantial claim - Findingown data
Binding surface area analysis shows enhanced interaction between ERα and STUB1 when Bufalin is present.
“This finding is further supported by the binding surface area analysis (Fig. 5d), which shows enhanced interaction between ERα and STUB1 when Bufalin is present.”
Quote in the paperSubstantial claim - Findingown data
Bufalin combined with CHX significantly accelerated the degradation of ERα.
“The results demonstrated that Bufalin combined with CHX significantly accelerated the degradation of ERα (Fig. 3c).”
Quote in the paperSubstantial claim - Findingown data
Bufalin downregulates ERα target genes, including AGR2, CCND1, GREB1, NRIP1, PGR, and SIAH2.
“which was corroborated by downregulation of ERα target genes, including AGR2, CCND1, GREB1, NRIP1, PGR, and SIAH2 (Fig. 3j and Supplementary Fig. 1c).”
Quote in the paperSubstantial claim - Findingown data
KEGG enrichment analysis on 53 candidate targets led to the identification of 11 key targets.
“To further refine this target list and enhance the precision of our selection, KEGG enrichment analysis was conducted on 53 candidate targets (Fig. 1c). We focused on the pathways that were statistically significant and specifically related to cancer and visualized these pathways using a chord diagram to highlight their associated targets, ultimately identifying 11 key targets (Fig. 1d).”
Quote in the paperSubstantial claim - Findingown data
Bufalin exhibited stronger anti-cancer effects than Fulvestrant.
“In addition, Bufalin exhibited stronger anti-cancer effects than Fulvestrant (Supplementary Fig. 1e–g).”
Quote in the paperSubstantial claim - Findingown data
Intersection criteria narrowed the initial target predictions to 53 candidate targets.
“To ensure the reliability of these initial predictions, we implemented intersection criteria that narrowed down the results to 53 candidate targets (Fig. 1b).”
Quote in the paperSubstantial claim - Findingown data
Bufalin increased the thermal stability of ERα.
“Additionally, Bufalin increased the thermal stability of ERα (Fig. 2h), and the Biotin-Bufalin and ERα were colocalized in the MCF-7 cells (Fig. 2i).”
Quote in the paperSubstantial claim - Findingown data
Bufalin enhanced the binding of ERα and STUB1.
“The results showed that Bufalin enhanced the binding of ERα and STUB1 (Fig. 5h).”
Quote in the paperSubstantial claim - Findingown data
Using an integrated multi-predictive strategy, CYP17A1, ESR1, mTOR, AR, and PRKCD are identified as potential targets of Bufalin.
“Using an integrated multi-predictive strategy, we identify CYP17A1, ESR1, mTOR, AR, and PRKCD as the potential targets of Bufalin.”
- Also stated in: § Exploring pharmacological mechanisms of Bufalin based on a combinatorial target screening strategy · hedged
“Using a default cutoff of 0.8, five target proteins were identified and marked as potential targets of Bufalin: CYP17A1, ESR1, mTOR, AR, and PRKCD.”
Quote in the paperSubstantial claim - Also stated in: § Exploring pharmacological mechanisms of Bufalin based on a combinatorial target screening strategy · hedged
- Findingown data
Hydroxychloroquine (HCQ) failed to rescue ERα levels.
“In contrast, the lysosomal inhibitor hydroxychloroquine (HCQ) failed to rescue ERα levels (Fig. 3f).”
Quote in the paperSubstantial claim - Findingown data
Bufalin exhibited the strongest binding affinity for ESR1 (KD = 5.42E−06 M) compared to PRKCD (1.29E−02 M) and CYP17A1 (2.94E−02 M).
“As shown in Fig. 2a–c, SPR assays revealed that Bufalin exhibited the strongest binding affinity for ESR1 (encodes Estrogen Receptor α, ERα) (KD = 5.42E−06 M) compared to the PRKCD (PKC delta) (1.29E−02 M) and CYP17A1 (2.94E−02 M) (Table 1).”
Quote in the paperSubstantial claim - Findingown data
Biotin-Bufalin specifically pulled down ERα in biotin-pull down assays.
“To further confirm these findings, we synthesized Biotin-Bufalin conjugates, and found that Biotin-Bufalin specifically pulled down ERα in biotin-pull down assays (Fig. 2d, e).”
Quote in the paperSubstantial claim - Findingown data
Treatment with Bufalin induced apoptosis in MCF-7 and T47D cells.
“Additionally, treatment with Bufalin induced apoptosis, as evidenced by the elevated Annexin V-positive populations (Fig. 6c).”
Quote in the paperSubstantial claim - Findingown data
Co-IP experiments indicated a decrease in ubiquitination of the ERα Arg394A mutant compared to the WT protein in the presence of Bufalin.
“Co-IP experiments indicated a decrease in ubiquitination of the ERα Arg394A mutant compared to the WT protein in the presence of Bufalin (Fig. 4f).”
Quote in the paperSubstantial claim - Findingown data
In this study, we applied an integrated multi-predictive strategy and identified CYP17A1, ESR1, mTOR, AR, and PRKCD as potential targets of Bufalin.
“In this study, we applied an integrated multi-predictive strategy and identified CYP17A1, ESR1, mTOR, AR, and PRKCD as potential targets of Bufalin”
Quote in the paperSubstantial claim - Findingown data
Bufalin promotes ERα degradation in Tamoxifen-resistant cell LCC2.
“As shown in Fig. 7b, the Bufalin could promote the ERα degradation in Tamoxifen-resistant cell LCC2”
Quote in the paperSubstantial claim - Findingown data
Tamoxifen-resistant PDOs showed a marked decrease in cell viability after Bufalin treatment.
“the Tamoxifen-resistant PDOs showed a marked decrease in cell viability after Bufalin treatment (Fig. 9c).”
Quote in the paperSubstantial claim - Findingown data
Bufalin selectively binds to R394 of ERα.
“Molecular docking experiments revealed that Bufalin selectively binds to R394 of ERα”
- Also stated in: § Discussion · hedged
“Structural analysis revealed that Bufalin could selectively bind to the R394 of the ERα protein”
Quote in the paperSubstantial claim - Also stated in: § Discussion · hedged
- Findingown data
Bufalin treatment reduced viability of LCC2 cells in a dose-dependent manner.
“As shown in Fig. 7c, Bufalin treatment reduced the viability of LCC2 cells in a dose-dependent manner”
Quote in the paperSubstantial claim - Findingown data
Protein expression of ERα was decreased in tumors following Bufalin treatment.
“Moreover, the protein expression of ERα was decreased in tumors following Bufalin treatment (Fig. 8f, g).”
Quote in the paperSubstantial claim - Findingown data
PDOs were successfully established from Tamoxifen-resistant patient specimens.
“PDOs were successfully established from Tamoxifen-resistant patient specimens”
Quote in the paperSubstantial claim - Findingown data
Calcein-AM/PI fluorescence staining revealed extensive cell death in Bufalin-treated organoids.
“Calcein-AM/PI fluorescence staining further revealed extensive cell death in the Bufalin-treated organoids (Fig. 9d).”
Quote in the paperSubstantial claim - Findingown data
Experimental validation confirmed ERα (ESR1) as the primary target of Bufalin in ER+ breast cancer.
“with experimental validation confirming ERα (ESR1) as the primary target in ER+ breast cancer”
Quote in the paperSubstantial claim - Findingown data
Immunohistochemical analysis revealed marked Ki67 suppression in Bufalin-treated tumors.
“Immunohistochemical analysis revealed marked Ki67 suppression in Bufalin-treated tumors (Fig. 8d).”
Quote in the paperSubstantial claim - Findingown data
Bufalin exhibited better anti-cancer effects than Fulvestrant against LCC2 cells.
“Moreover, Bufalin exhibited better anti-cancer effects than Fulvestrant against LCC2 cells (Fig. 7h, i).”
Quote in the paperSubstantial claim - Findingown data
Following intraperitoneal injection of Bufalin, tumor volumes and weights significantly decreased in Tamoxifen-resistant LCC2 xenografts.
“following intraperitoneal injection of Bufalin, the tumor volumes and weights significantly decreased”
Quote in the paperSubstantial claim - Findingown data
Bufalin induces apoptosis in LCC2 cells, evidenced by increased Annexin V staining, PARP cleavage, and Bcl-2 downregulation.
“Additionally, the activation of apoptosis induced by Bufalin was evidenced by increased Annexin V staining, PARP cleavage, and Bcl-2 downregulation in LCC2 cells (Fig. 7f, g).”
Quote in the paperSubstantial claim - Findingown data
Bufalin effectively inhibited estrogen response elements transcriptional activity.
“and effectively inhibited the estrogen response elements transcriptional activity.”
Quote in the paperSubstantial claim - Findingown data
No significant signs of drug-induced toxicity at therapeutic doses of Bufalin were found in mice serum biochemical markers related to liver and kidney function.
“we analyzed various serum biochemical markers related to liver and kidney function in mice, and found no significant signs of drug-induced toxicity at the therapeutic doses of Bufalin (Fig. 8h, i)”
Quote in the paperSubstantial claim - Findingown data
Bufalin demonstrated superior anti-tumor activity to Fulvestrant in Tamoxifen-resistant LCC2 xenografts.
“demonstrating superior anti-tumor activity to Fulvestrant.”
Quote in the paperSubstantial claim - Findingown data
Treatment with Bufalin resulted in a significant reduction in the size of Tamoxifen-resistant PDOs.
“treatment with Bufalin resulted in a significant reduction in the size of these organoids”
Quote in the paperSubstantial claim - Findingown data
Clinical samples from endocrine therapy-relapsed patients (n = 8) maintained ERα expression.
“Clinical samples from endocrine therapy-relapsed patients (n = 8) maintained ERα expression (Fig. 7a).”
Quote in the paperSubstantial claim - Findingown data
The deep learning model achieved a ROC-AUC of 0.94 on the test set.
“achieving a receiver operating characteristic area under the curve (ROC-AUC) of 0.94 on the test set (Supplementary Table 3).”
Quote in the paperSubstantial claim - Findingown data
In our study, we observed high ERα expression in samples from patients with endocrine treatment relapse.
“In our study, we observed high ERα expression in samples from patients with endocrine treatment relapse, aligning with previous findings.”
Quote in the paperNot a substantial claim - Interpretationown data
The anti-tumor activity of Bufalin mechanistically depends on ERα degradation.
“These findings suggest that the anti-tumor activity of Bufalin mechanistically depends on ERα degradation, identifying ERα as the critical therapeutic target of Bufalin.”
- Also stated in: § Discussion · boosted
“Our results demonstrate Bufalin-induced ERα degradation drives its anticancer activity”
Quote in the paperSubstantial claim - Also stated in: § Discussion · boosted
- Interpretationown data
ERα is the critical therapeutic target of Bufalin.
“identifying ERα as the critical therapeutic target of Bufalin.”
Quote in the paperSubstantial claim - Interpretationown data
This repositioning appears to induce subtle conformational changes in ERα that enhance its binding interface with STUB1.
“This repositioning appears to induce subtle conformational changes in ERα that enhance its binding interface with STUB1.”
Quote in the paperSubstantial claim - Interpretationown data
STUB1 was required for ubiquitination of ERα induced by Bufalin.
“suggesting that STUB1 was required for ubiquitination of ERα induced by Bufalin.”
Quote in the paperSubstantial claim - Interpretationown data
Bufalin acts as a molecular glue, enhancing the interaction between ERα and the E3 ligase STUB1.
“Molecular dynamic simulations further show that Bufalin acts as a molecular glue, enhancing the interaction between ERα and the E3 ligase STUB1, thereby promoting proteasomal degradation of ERα.”
- Also stated in: § Bufalin facilitates ERα degradation by enhancing interaction between ERα and the E3 ligase STUB1 · hedged
“Collectively, these findings suggest that Bufalin functions as a molecular glue, facilitating the interaction between ERα and STUB1 and thereby inducing proteasomal degradation of ERα.”
Quote in the paperSubstantial claim - Also stated in: § Bufalin facilitates ERα degradation by enhancing interaction between ERα and the E3 ligase STUB1 · hedged
- Interpretationown data
The downregulation of ERα protein by Bufalin was not due to genetic transcription inhibition.
“Notably, the mRNA of ERα had no obvious change in cells exposed to Bufalin, suggesting that the downregulation of the ERα protein was not due to genetic transcription inhibition (Supplementary Fig. 1a, b).”
Quote in the paperSubstantial claim - Interpretationown data
Bufalin promotes proteasomal degradation of ERα.
“thereby promoting proteasomal degradation of ERα.”
- Also stated in: § Bufalin induces ERα degradation and suppresses its transcriptional activity · boosted
“corroborating that Bufalin induces ERα degradation predominantly through the proteasomal pathway.”
Quote in the paperSubstantial claim - Also stated in: § Bufalin induces ERα degradation and suppresses its transcriptional activity · boosted
- Interpretationown data
Bufalin induces ERα degradation predominantly through the proteasomal pathway.
“corroborating that Bufalin induces ERα degradation predominantly through the proteasomal pathway.”
Quote in the paperSubstantial claim - Interpretationown data
Molecular dynamics simulations suggest Bufalin's molecular glue function stabilizes the interaction between ERα and STUB1.
“while molecular dynamics simulations suggest its molecular glue function stabilizes the interaction between ERα and STUB1”
Quote in the paperSubstantial claim- Rests on an assumption: Molecular docking and molecular dynamics simulations accurately model Bufalin-ERα binding and molecular glue function.
- Interpretationown data
Bufalin has a potent inhibitory effect on the growth of Tamoxifen-resistant tumors.
“indicating its potent inhibitory effect on the growth of Tamoxifen-resistant tumors (Fig. 9b).”
Quote in the paperSubstantial claim - Interpretationown data
Bufalin has a favorable safety profile.
“suggesting a favorable safety profile.”
Quote in the paperNot a substantial claim- Rests on an assumption: Toxicity results in mice are predictive of safety in humans.
- Interpretationreasoning
Bufalin suggests selective cytotoxicity in ERα-expressing malignancies, including cervical and colorectal cancers.
“suggesting selective cytotoxicity in ERα-expressing malignancies, including cervical and colorectal cancers.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 42
- May combine two claims. 35
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBufalin is a potent anticancer agent used clinically in China to treat various cancers.
“Bufalin, an active monomer extracted from the Chansu, is a potent anticancer agent used clinically in China to treat various cancers1,2.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [1] Zhang, H. et al. Bufalin targeting CAMKK2 inhibits the occurrence and development of intrahepatic cholangiocarcinoma through Wnt/β-catenin signal pathway. J. Transl. Med. 21, 900 (2023). source
The Introduction (page unavailable) calls bufalin a potent anticancer drug, and the Discussion describes antitumor effects across several cancers. But the paper does not say bufalin is used clinically in China to treat cancer; it describes bufalin as a promising drug candidate and reports cell and mouse experiments. The clinical-use portion of the claim is unsupported. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [2] Yu, Z. et al. Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma. J. Immunother. Cancer 10, e004297 (2022). source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTamoxifen acts by competitively blocking the binding of estrogen to ERα and has significantly improved prognosis in patients with ER+ breast cancer.
“Among these agents, Tamoxifen, a selective estrogen receptor modulator, acts by competitively blocking the binding of estrogen to ERα and has significantly improved prognosis in patients with ER+ breast cancer15,21,22.”
Quote in the paperNot a substantial claim2 of 3 sources checked- cites [15] Zhao, W. et al. Ginsenoside Rg3 overcomes tamoxifen resistance through inhibiting glycolysis in breast cancer cells. Cell Biol. Int. 48, 496–509 (2024). source
- cites [21] Chen, X. S. et al. UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer. Theranostics 10, 1833–1848 (2020). source
The Introduction says ERα-positive breast cancers respond to tamoxifen and usually have a better prognosis; the Discussion identifies tamoxifen as a selective estrogen receptor modulator. Neither passage states that tamoxifen competitively blocks estrogen binding to ERα or establishes that tamoxifen significantly improved patients’ prognosis. Page numbers were not available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [22] Liu, S. et al. MCM2 is involved in subtyping and tamoxifen resistance of ERα-positive breast cancer by acting as the downstream factor of ERα,. Biotechnol. J. 19, e2300560 (2024). source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationBufalin exerts effective anti-tumor action against lung cancer, liver cancer, colorectal cancer, and glioma.
“Accumulating evidence indicates that Bufalin exerts effective anti-tumor action against lung cancer, liver cancer, colorectal cancer, and glioma3,4.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [3] Farooqi, A. A. et al. Bufalin-mediated regulation of cell signaling pathways in different cancers: spotlight on JAK/STAT, Wnt/β-Catenin, mTOR, TRAIL/TRAIL-R, and non-coding RNAs. Molecules 28, 2231 (2023). source
- cites [4] Chen, J. et al. Bufalin targets the SRC-3/MIF pathway in chemoresistant cells to regulate M2 macrophage polarization in colorectal cancer. Cancer Lett. 513, 63–74 (2021). source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationBufalin inhibits tumor growth, regulates metastasis, and enhances antitumor immune responses by polarizing tumor-infiltrating macrophages toward the M1 phenotype.
“Recent studies have demonstrated that Bufalin inhibits tumor growth, regulates metastasis, and enhances antitumor immune responses by polarizing tumor-infiltrating macrophages toward the M1 phenotype2.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [2] Yu, Z. et al. Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma. J. Immunother. Cancer 10, e004297 (2022). source
The Results section reports that bufalin shifts tumor-infiltrating macrophages toward M1, and the Conclusions link that shift to antitumor T-cell responses and an antitumor effect. The Background mentions earlier studies associating suppressed HCC metastasis with immune-cell modulation, but this paper does not establish that M1 polarization regulates metastasis. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationBufalin could suppress multiple signaling pathways, including Hippo-YAP, Wnt/β-Catenin, JNK, SRC3/MIF, mTOR, and MAPK, leading to apoptosis, necrosis, and autophagy in different cancer cells.
“Current pharmacological research suggests that Bufalin could suppress multiple signaling pathways, including Hippo-YAP, Wnt/β-Catenin, JNK, SRC3/MIF, mTOR, and MAPK, leading to apoptosis, necrosis, and autophagy in different cancer cells3,5–8.”
Quote in the paperNot a substantial claim3 of 5 sources checked- cites [3] Farooqi, A. A. et al. Bufalin-mediated regulation of cell signaling pathways in different cancers: spotlight on JAK/STAT, Wnt/β-Catenin, mTOR, TRAIL/TRAIL-R, and non-coding RNAs. Molecules 28, 2231 (2023). source
Section 3 reports that bufalin inhibits AKT/mTOR signaling, with evidence of apoptosis and autophagy in cancer cells; section 4 reports reduced β-catenin signaling. The copy does not substantiate suppression of Hippo-YAP or SRC3/MIF, or bufalin-induced necrosis. Section 5 instead describes enhanced JNK and p38 MAPK activation with bufalin and TRAIL, not their suppression. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [5] Soumoy, L., Ghanem, G. E., Saussez, S. & Journe, F. Bufalin for an innovative therapeutic approach against cancer. Pharm. Res. 184, 106442 (2022). source
Section 7 reports inhibition of mTOR and SRC-3, including SRC-3’s target MIF as discussed in section 16.1, but says bufalin activates JNK and can either decrease or increase MAPK activity depending on the cancer type. Sections 2–4 describe apoptosis, necroptosis and autophagy; the review calls the second process necroptosis rather than necrosis. Searches for Hippo-YAP and Wnt/β-catenin did not establish suppression of those pathways in our copy, so the full list and its stated link to cell death are not supported. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [6] Qian, Z. et al. Bufalin inhibits the proliferation of lung cancer cells by suppressing Hippo-YAP pathway. Cell Signal 109, 110746 (2023). source
- cites [7] Chen, G. et al. Bufalin targeting BFAR inhibits the occurrence and metastasis of gastric cancer through PI3K/AKT/mTOR signal pathway. Apoptosis 28, 1390–1405 (2023). source
- cites [8] Wang, Y. et al. Bufalin is a potent small-molecule inhibitor of the steroid receptor coactivators SRC-3 and SRC-1. Cancer Res. 74, 1506–1517 (2014). source
The Results sections on SRC-3 degradation and cancer-cell proliferation report that bufalin reduces SRC-3 protein levels and inhibits growth of the cancer cells tested. The section on targeted therapeutics links SRC-3 to PI3K/AKT/mTOR signaling, but does not establish that bufalin suppresses mTOR. These passages do not support the other named pathways, the SRC3/MIF pairing, or the specified outcomes of apoptosis, necrosis, and autophagy. (LLM, conf=0.89)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationApproximately 30% of ER+ breast cancer cases recur with resistance to Tamoxifen, and this recurrence can lead to more aggressive neoplasms.
“Approximately 30% of ER+ breast cancer cases recur with resistance to Tamoxifen, and this recurrence can lead to more aggressive neoplasms23,24.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [23] Lai, X. et al., HGF/c-Met Promotes Breast Cancer Tamoxifen Resistance Through the EZH2/HOTAIR-miR-141/200a Feedback Signaling Pathway. Mol. Carcinog. 64, 769–783 (2025). source
The Introduction says approximately 30% of ER+ breast cancer patients have primary tamoxifen resistance, not that 30% recur with resistance. Section 3.1 reports that tamoxifen-resistant cells showed more aggressive biological functions than parental cells, but does not establish that recurrence leads to more aggressive tumors in patients. Page numbers are unavailable in our copy. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [24] Melone, V. et al. LncRNA PVT1 links estrogen receptor alpha and the polycomb repressive complex 2 in suppression of pro-apoptotic genes in hormone-responsive breast cancer. Cell Death Dis. 16, 80 (2025). source
The Introduction (page unavailable) says that approximately 30% of patients with ERα-positive breast cancer acquire resistance to hormone therapies. It does not give that figure specifically for tamoxifen-resistant recurrence or say that recurrence leads to more aggressive neoplasms. (LLM, conf=0.87)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTRIM56, RNF181, and RNF2 stabilize ERα expression.
“It has been reported that the ubiquitin ligases TRIM56, RNF181, and RNF2 stabilize ERα expression30–32.”
Quote in the paperNot a substantial claim3 of 3 sources checked- cites [30] Yuan, L., Li, X., Yang, H. & Li, H. The ubiquitin ligase RNF2 stabilizes ERα and modulates breast cancer progression. Hum. Cell 36, 353–365 (2023). source
- cites [31] Zhu, J. et al. The ubiquitin ligase RNF181 stabilizes ERα and modulates breast cancer progression. Oncogene 39, 6776–6788 (2020). source
- cites [32] Xue, M. et al. Regulation of estrogen signaling and breast cancer proliferation by an ubiquitin ligase TRIM56. Oncogenesis 8, 30 (2019). source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationBufalin induced apoptosis, necroptosis, ferroptosis, autophagy, and senescence through AMPK, RIP1/ROS, DECR1, p62, and p53 pathways.
“Bufalin induced apoptosis, necroptosis, ferroptosis, autophagy, and senescence through AMPK, RIP1/ROS, DECR1, p62, and p53 pathways5,44–48.”
Quote in the paperNot a substantial claim4 of 6 sources checked- cites [5] Soumoy, L., Ghanem, G. E., Saussez, S. & Journe, F. Bufalin for an innovative therapeutic approach against cancer. Pharm. Res. 184, 106442 (2022). source
Sections 2–5 report bufalin-induced apoptosis, RIP1/ROS-dependent necroptosis, autophagy associated with p62 and AMPK, and p53-related senescence; page numbers are unavailable in our copy. The copy does not substantiate the claim that bufalin induces ferroptosis through DECR1 (searched: “ferroptosis” and “DECR1”). If the pathways are intended to correspond to the effects in the order listed, the copy links AMPK to autophagy, not apoptosis. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [44] Wu, S. et al. Bufalin induces ferroptosis by modulating the 2,4-dienoyl-CoA reductase (DECR1)-SLC7A11 axis in breast cancer. Phytomedicine 135, 156130 (2024). source
- cites [45] Li, C. et al. Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway. J. Cardiovasc. Transl. Res. 18, 221–236 (2024). source
- cites [46] Zheng, Y. D. et al. Bufalin induces apoptosis and autophagy via the Ca2+/CaMKKβ/AMPK/Beclin1 signaling pathway in osteosarcoma cells. Cell Biol. Int. 47, 1344–1353 (2023). source
The Abstract and section 3.4 report bufalin-induced autophagy through the Ca²⁺/CaMKKβ/AMPK/Beclin1 pathway, and section 3.3 reports bufalin-induced apoptosis in osteosarcoma cells. The passages do not establish bufalin-induced necroptosis, ferroptosis, or senescence through RIP1/ROS, DECR1, p62, or p53; the sentence’s multiple citations do not specify which source supports those parts. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [47] Liu, X. D., Song, C. Y., Kong, C. C. & Tian, X. Bufalin Induces Programmed Necroptosis in Triple-Negative Breast Cancer Drug-Resistant Cell Lines through RIP1/ROS-Mediated Pathway. Chin. J. Integr. Med. 28, 900–908 (2022). source
- cites [48] Zhang, Y. et al. Role of P53-Senescence induction in suppression of LNCaP prostate cancer growth by cardiotonic compound bufalin. Mol. Cancer Ther. 17, 2341–2352 (2018). source
The Results report bufalin-induced apoptosis and increased senescence in LNCaP cells, with p53/p21 signaling promoting senescence. They do not establish necroptosis, ferroptosis, or the AMPK, RIP1/ROS, DECR1, and p62 pathways; the Results also report no increase in the autophagy marker LC-3-II after bufalin exposure. The citation therefore supports only part of the multi-paper sentence. (LLM, conf=0.90)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationTargeted protein degradation (TPD), including molecular glues and PROTACs, has emerged as a promising strategy to target proteins previously considered undruggable.
“Targeted protein degradation (TPD), including molecular glues and PROTACs, has emerged as a promising strategy to target proteins previously considered undruggable53.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [53] Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat. Rev. Drug Discov. 21, 181–200 (2022). source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationTargeting ERα signaling is widely regarded as the most effective therapeutic strategy for ERα-positive breast cancer, such as Tamoxifen, Fulvestrant, and Letrozole, which have brought significant prognostic improvements for ER+ breast cancer.
“Targeting ERα signaling is widely regarded as the most effective therapeutic strategy for ERα-positive breast cancer, such as Tamoxifen, Fulvestrant, and Letrozole, which have brought significant prognostic improvements for ER+ breast cancer52.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [52] Wang, Y. et al. Discovery of novel covalent selective estrogen receptor degraders against endocrine-resistant breast cancer. Acta Pharm. Sin. B. 13, 4963–4982 (2023). source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationBufalin has been shown to downregulate expression of transcriptional coactivators SRC-1 and SRC-3, as well as nuclear transcription factor E2F1.
“Furthermore, Bufalin has been shown to downregulate the expression of transcriptional coactivators SRC-1 and SRC-3, as well as the nuclear transcription factor E2F18,43.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [8] Wang, Y. et al. Bufalin is a potent small-molecule inhibitor of the steroid receptor coactivators SRC-3 and SRC-1. Cancer Res. 74, 1506–1517 (2014). source
The Results section ('Effects of digoxin and bufalin on cellular protein concentrations') reports that bufalin significantly reduced SRC-1 and SRC-3 protein levels in MCF-7 and A549 cells. The Introduction mentions E2F1 only as a transcription factor coactivated by SRC proteins; our copy does not report that bufalin downregulates E2F1. Page numbers were not available in the indexed passages. (LLM, conf=0.90)
Experimental — accuracy not yet measured - cites [43] Wang, T. et al. The effects of bufadienolides on HER2 overexpressing breast cancer cells. Tumour Biol. 37, 7155–7163 (2016). source
May combine two claims.
PremisecitationCompared to SRC, ERα constitutes the central oncogenic driver in ER+ breast cancer, as ERα overexpression promotes tumorigenesis.
“However, compared to SRC, the ERα constitutes the central oncogenic driver in ER+ breast cancer, as ERα overexpression promotes tumorigenesis50,51.”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [50] Chen, P., Li, B. & Ou-Yang, L. Role of estrogen receptors in health and disease. Front Endocrinol.13, 839005 (2022). source
The “ERs in breast cancer” section reports that ERα is overexpressed in breast cancer tissue and promotes tumor growth through its interaction with estrogen. It does not compare ERα with SRC or establish that ERα is the central oncogenic driver relative to SRC. No page number is available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [51] Bak, M. J., Das Gupta, S., Wahler, J. & Suh, N. Role of dietary bioactive natural products in estrogen receptor-positive breast cancer. Semin Cancer Biol. 40-41, 170–191 (2016). source
The Introduction and section 3 describe ER-dependent estrogen signaling as a contributor to breast carcinogenesis, including increased proliferation and DNA damage. They do not establish that ERα overexpression itself promotes tumorigenesis or that ERα is a more central oncogenic driver than SRC. Page numbers were not available in our copy. (LLM, conf=0.87)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMechanistic studies reveal Bufalin's pleiotropic anti-tumor effects through cancer-type-specific targets.
“Mechanistic studies reveal its pleiotropic anti-tumor effects through cancer-type-specific targets5,37.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [5] Soumoy, L., Ghanem, G. E., Saussez, S. & Journe, F. Bufalin for an innovative therapeutic approach against cancer. Pharm. Res. 184, 106442 (2022). source
- cites [37] Ye, Q. et al. An overview of the past decade of bufalin in the treatment of refractory and drug-resistant cancers: current status, challenges, and future perspectives. Front. Pharm. 14, 1274336 (2023). source
The Discussion’s summary describes bufalin as a multi-targeting or multi-functional agent with several anticancer mechanisms. But the Future perspectives section says it remains unknown how its targets work together and whether its cytotoxicity is cancer-type-dependent; the review does not establish that the targets are cancer-type-specific. (LLM, conf=0.84)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBufalin has been reported to inhibit key signaling pathways such as mTOR, Wnt/β-catenin, and Akt, which are necessary for cancer cell survival, proliferation, and migration.
“Bufalin has been reported to inhibit key signaling pathways such as mTOR, Wnt/β-catenin, and Akt, which are necessary for cancer cell survival, proliferation, and migration3.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [3] Farooqi, A. A. et al. Bufalin-mediated regulation of cell signaling pathways in different cancers: spotlight on JAK/STAT, Wnt/β-Catenin, mTOR, TRAIL/TRAIL-R, and non-coding RNAs. Molecules 28, 2231 (2023). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRecent studies indicated that Bufalin played an important role in the tumor microenvironment by inducing activation of the NF-κB pathway.
“Recent studies indicated that Bufalin played an important role in the tumor microenvironment by inducing the activation of the NF-κB pathway2.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [2] Yu, Z. et al. Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma. J. Immunother. Cancer 10, e004297 (2022). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationVarious computational models for target prediction have been developed, leading to notable improvements in predictive accuracy and efficiency.
“To this end, various computational models for target prediction have been developed, leading to notable improvements in predictive accuracy and efficiency38–40.”
Quote in the paperNot a substantial claim3 of 3 sources checked- cites [38] Zhang, Z., He, X., Long, D., Luo, G. & Chen, S. Enhancing generalizability and performance in drug-target interaction identification by integrating pharmacophore and pre-trained models. Bioinformatics 40, i539–i547 (2024). source
- cites [39] Song, W., Xu, L., Han, C., Tian, Z. & Zou, Q. Drug-target interaction predictions with multi-view similarity network fusion strategy and deep interactive attention mechanism. Bioinformatics 40, btae346 (2024). source
The Introduction describes several computational approaches to drug–target prediction, and Results §3.2 reports improved predictive accuracy for the paper’s MIDTI model against baselines. The Introduction says computational approaches aim to accelerate screening, but the passages do not establish a notable improvement in predictive efficiency; that part of the claim is not supported. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [40] Lin, X., Li, X. & Lin, X. A Review on Applications of Computational Methods in Drug Screening and Design. Molecules 25, 1375 (2020). source
Section 5.1 identifies random forests as a method used for drug target prediction, supporting the development of computational approaches for that task. The Conclusions say machine learning may improve efficiency and precision in drug screening and design generally, but do not establish that target-prediction models have already produced notable improvements in both accuracy and efficiency. Page numbers were not available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim. May combine two claims.
PremisecitationPersistent ERα expression post-Tamoxifen therapy motivates SERD development, such as Fulvestrant, which improved clinical benefits after failure of other endocrine therapies.
“It has been reported that persistent ERα expression post-Tamoxifen therapy motivates SERD development, such as Fulvestrant, which improved clinical benefits after failure of other endocrine therapies36.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [36] Shomali, M. et al. SAR439859, a Novel Selective Estrogen Receptor Degrader (SERD), demonstrates effective and broad antitumor activity in wild-type and mutant ER-Positive breast cancer models. Mol. Cancer Ther. 20, 250–262 (2021). source
The Introduction says tamoxifen stabilizes ERα and gives ERα degradation as a rationale for SERDs. It also identifies fulvestrant as a SERD and reports clinical benefits after failure of other hormonal therapies. It does not establish that ERα expression persists specifically after tamoxifen therapy; the Discussion describes benefit for post-tamoxifen patients as a potential outcome for SAR439859, not a demonstrated finding about persistent expression. Page numbers were not available in our copy. (LLM, conf=0.83)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOur prior work has successfully developed a variety of strategies to predict drug targets by integrating multiple biological signatures.
“Our prior work has successfully developed a variety of strategies to predict drug targets by integrating multiple biological signatures13”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [13] Wei, X. et al. Predicting novel targets with Bayesian machine learning by integrating multiple biological signatures. Chem. Sci. 15, 14471–14484 (2024). source
The Introduction (page not available) describes one Bayesian approach that combines 25 biological characterizations to predict drug targets and reports its validation. This supports the integration and target-prediction parts of the claim, but the paper presents a single FMBS strategy, not a variety of strategies developed in prior work. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationBufalin has emerged as a clinically validated natural compound with therapeutic potential across multiple cancer types.
“Bufalin has emerged as a clinically validated natural compound with therapeutic potential across multiple cancer types.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [5] Soumoy, L., Ghanem, G. E., Saussez, S. & Journe, F. Bufalin for an innovative therapeutic approach against cancer. Pharm. Res. 184, 106442 (2022). source
The Conclusions discuss bufalin’s antitumor effects across many cancer types and its potential to improve treatment. But §1.1, “Clinical trials on HuaChansu,” states that purified bufalin has never been administered to humans; the trials discussed tested HuaChansu, an extract containing bufalin. The claim’s “clinically validated” qualifier is therefore not supported for bufalin itself. (LLM, conf=0.92)
Experimental — accuracy not yet measured - cites [37] Ye, Q. et al. An overview of the past decade of bufalin in the treatment of refractory and drug-resistant cancers: current status, challenges, and future perspectives. Front. Pharm. 14, 1274336 (2023). source
The Introduction describes bufalin as a natural compound with anticancer potential across multiple cancer types. It does not establish that bufalin is clinically validated: the Introduction says a phase II pancreatic-cancer trial posted no results, and the Discussion calls for further animal testing before clinical trials. (LLM, conf=0.91)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationOur prior work successfully discovered the target of Ixabepilone, Mitoxantrone, and Tubeimoside-1 are Bcl-2, eEF2K, and AKT, respectively.
“and successfully discovered the target of Ixabepilone, Mitoxantrone, and Tubeimoside-1 are Bcl-2, eEF2K, and AKT, respectively12,41,42.”
Quote in the paperNot a substantial claim2 of 3 sources checked- cites [12] Cao, D. S. et al. A multi-scale systems pharmacology approach uncovers the anti-cancer molecular mechanism of Ixabepilone. Eur. J. Med. Chem. 199, 112421 (2020). source
- cites [41] Jiang, S. L. et al. Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway. Acta Pharm. Sin. 40, 919–928 (2019). source
- cites [42] Guan, Y. et al. Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy. Cell Death Dis. 11, 948 (2020). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPrevious studies have reported that Bufalin exerts anti-tumor effects by inhibiting SRC in MCF-7 cells.
“Previous studies have reported that Bufalin exerts anti-tumor effects by inhibiting SRC in MCF-7 cells8.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [8] Wang, Y. et al. Bufalin is a potent small-molecule inhibitor of the steroid receptor coactivators SRC-3 and SRC-1. Cancer Res. 74, 1506–1517 (2014). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOur previous study has reported that the UCH-L1 inhibitor LDN significantly enhanced the efficacy of Tamoxifen both in vivo and in vitro.
“Our previous study has reported that the UCH-L1 inhibitor LDN significantly enhanced the efficacy of Tamoxifen both in vivo and in vitro21.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [21] Chen, X. S. et al. UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer. Theranostics 10, 1833–1848 (2020). source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTamoxifen remains the standard first-line treatment for ER-positive breast cancer and has significantly improved patient prognosis.
“Although Tamoxifen remains the standard first-line treatment for ER-positive breast cancer and has significantly improved patient prognosis”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [57] Zhu, J. et al. Involvement of RFC3 in tamoxifen resistance in ER-positive breast cancer through the cell cycle. Aging 15, 13738–13752 (2023). source
The Introduction states that clinical use of tamoxifen has brought significant prognostic improvements in patients with estrogen-positive breast cancer. It describes tamoxifen as a treatment for hormone-dependent breast cancer, but does not say it remains the standard first-line treatment. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationAn increasing number of endocrine-resistance mechanisms have been characterized, and several small molecules have been reported to overcome Tamoxifen resistance.
“An increasing number of endocrine-resistance mechanisms have been characterized, and several small molecules have been reported to overcome Tamoxifen resistance58,59.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [58] Song, T. et al. Small molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance. Breast Cancer Res. 26, 33 (2024). source
The Introduction (page not provided) describes multiple pathways involved in tamoxifen resistance, and the Results (page not provided) report that the small molecule S1g-2 could overcome resistance in the tested breast cancer cells. The paper does not support the claim that several small molecules overcome resistance: its Results say MKT-077 did not, and VER-155008 showed no synergy with tamoxifen. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [59] Hanker, A. B., Sudhan, D. R. & Arteaga, C. L. Overcoming endocrine resistance in breast cancer. Cancer Cell 37, 496–513 (2020). source
The Introduction describes recent discoveries of endocrine-resistance mechanisms. The ‘Aberrant co-factor activity’ section says tamoxifen-resistant cells may remain sensitive to selective estrogen receptor degraders, but it does not report that several small molecules have overcome tamoxifen resistance specifically. (LLM, conf=0.84)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationDevelopment of acquired resistance to Tamoxifen continues to pose a major clinical challenge.
“the development of acquired resistance continues to pose a major clinical challenge57.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [57] Zhu, J. et al. Involvement of RFC3 in tamoxifen resistance in ER-positive breast cancer through the cell cycle. Aging 15, 13738–13752 (2023). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFulvestrant is a standard therapeutic option for endocrine-resistant or advanced metastatic ER+ breast cancer.
“Fulvestrant is a standard therapeutic option for endocrine-resistant or advanced metastatic ER+ breast cancer59,60.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [59] Hanker, A. B., Sudhan, D. R. & Arteaga, C. L. Overcoming endocrine resistance in breast cancer. Cancer Cell 37, 496–513 (2020). source
- cites [60] Lu, Y. & Liu, W. Selective Estrogen Receptor Degraders (SERDs): A promising strategy for estrogen receptor positive endocrine-resistant breast cancer. J. Med Chem. 63, 15094–15114 (2020). source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationPrevious research has revealed that Bufalin as a molecular glue that targets E2F2 and inhibits the growth of hepatoma.
“Previous research has revealed that Bufalin as a molecular glue that targets E2F2 and inhibits the growth of hepatoma56.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [56] Liu, T. T. et al. Atypical E3 ligase ZFP91 promotes small-molecule-induced E2F2 transcription factor degradation for cancer therapy. EBioMedicine 86, 104353 (2022). source
May combine two claims.
Findingown dataOnly the ERα Arg394A mutant lost the ability to bind Biotin-Bufalin, while the other mutants retained similar binding affinities to Biotin-Bufalin compared to WT ERα.
“The results showed that only the ERα Arg394A mutant lost the ability to bind Biotin-Bufalin, while the other mutants retained similar binding affinities to Biotin-Bufalin compared to WT ERα (Fig. 4d).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBufalin reverses Tamoxifen resistance in vitro, in vivo, and in patient-derived breast cancer organoids from tamoxifen-relapsed cases.
“we investigate the inhibitory effect of Bufalin on endocrine-resistant models and prove Bufalin reverses Tamoxifen resistance in vitro, in vivo, and in patient-derived breast cancer organoids from tamoxifen-relapsed cases.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBufalin repositions itself deeper within the ERα binding pocket, forming a hydrogen bond with Arg394 of ERα.
“Detailed structural analysis of the 200 ns simulation trajectory reveals that Bufalin repositions itself deeper within the ERα binding pocket, forming a hydrogen bond with Arg394 of ERα (Fig. 5b).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBufalin inhibits proliferation of LCC2 cells, confirmed by colony formation and EdU assays.
“with colony formation and EdU assays further confirming its inhibitory effect on cell proliferation (Fig. 7d, e).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataH&E staining revealed xenograft tumor cells treated with Bufalin appeared more loosely arranged and exhibited smaller nuclei compared to vehicle-treated group.
“H&E staining revealed that xenograft tumor cells treated with Bufalin appeared more loosely arranged and exhibited smaller nuclei compared to those in the vehicle-treated group (Fig. 8e).”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationActive ER signaling promotes the proliferation and survival of ER+ breast cancer cells, with oncogenesis primarily mediated through ERα, making it a key therapeutic target.
“Active ER signaling promotes the proliferation and survival of ER+ breast cancer cells, with oncogenesis primarily mediated through ERα, making it a key therapeutic target17.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [17] Kumar, N. et al. Most recent strategies targeting estrogen receptor alpha for the treatment of breast cancer. Mol. Divers 25, 603–624 (2021). source
Classified as probably not a substantial claim.
PremisecitationArtificial intelligence, incorporating machine learning, deep learning, and network-based algorithms, is a powerful tool for predicting the potency, toxicity, and mechanism of action of compounds, thereby accelerating drug target discovery.
“Artificial intelligence (AI), which incorporates machine learning, deep learning, and network-based algorithms, has emerged as a powerful tool for predicting the potency, toxicity, and mechanism of action of compounds, thereby accelerating drug target discovery9,10.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [9] Qiu, Y. & Cheng, F. Artificial intelligence for drug discovery and development in Alzheimer’s disease. Curr. Opin. Struct. Biol. 85, 102776 (2024). source
- cites [10] Pun, F. W., Ozerov, I. V. & Zhavoronkov, A. AI-powered therapeutic target discovery. Trends Pharm. Sci. 44, 561–572 (2023). source
May combine two claims.
Interpretationown dataBufalin functions as a molecular glue to degrade ERα, offering a potential therapeutic strategy for reversing Tamoxifen resistance.
“Collectively, our findings indicate that Bufalin functions as a molecular glue to degrade ERα, offering a potential therapeutic strategy for reversing Tamoxifen resistance.”
Quote in the paperSubstantial claim- Rests on an assumption: ERα degradation has therapeutic potential in overcoming endocrine resistance.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBufalin has been previously reported to associate with mTOR and AR.
“Given that the association of Bufalin with mTOR and AR has been previously reported26,27, we evaluated the binding of Bufalin to recombinant human ESR1, PRKCD, and CYP17A1 proteins using surface plasmon resonance (SPR) assays.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [26] Su, S., Dou, H., Wang, Z. & Zhang, Q. Bufalin inhibits ovarian carcinoma via targeting mTOR/HIF-α pathway. Basic Clin. Pharm. Toxicol. 128, 224–233 (2021). source
- cites [27] Yu, Z. et al. Bufalin inhibits hepatitis B virus-associated hepatocellular carcinoma development through androgen receptor dephosphorylation and cell cycle-related kinase degradation. Cell Oncol. 43, 1129–1145 (2020). source
May combine two claims.
Interpretationown dataThe Arg394 residue of ERα is critical for the binding interaction between ERα and Bufalin, and is important in Bufalin-mediated ERα degradation.
“These findings indicate that the Arg394 residue of ERα is critical for the binding interaction between ERα and Bufalin, underscoring its importance in Bufalin-mediated ERα degradation.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningBufalin leads to ERα degradation and overcomes endocrine resistance.
“leading to ERα degradation and overcoming endocrine resistance (Fig. 9e).”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataBufalin has potential as an effective therapeutic agent for Tamoxifen resistance in ER+ breast cancer and validates its translational potential in cancer therapy.
“These results underscore the potential of Bufalin as an effective therapeutic agent for Tamoxifen resistance in ER+ breast cancer and validate its translational potential in cancer therapy.”
- Also stated in: § Discussion · hedged
“By degrading ERα, Bufalin has the potential to reverse Tamoxifen resistance, offering a promising therapeutic strategy for patients with limited treatment options.”
Quote in the paperSubstantial claim- Rests on an assumption: Preclinical models (cell lines, xenografts, patient-derived organoids) predict clinical responses in patients.
- Also stated in: § Discussion · hedged
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBufalin appears to regulate particular non-coding RNAs, including microRNA-203 (miR-203), to suppress tumor cell proliferation.
“Bufalin also appears to regulate particular non-coding RNAs, including microRNA-203 (miR-203), to suppress tumor cell proliferation49.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [49] Liu, T. et al. Bufalin inhibits cellular proliferation and cancer stem cell-like phenotypes via upregulation of MiR-203 in Glioma. Cell Physiol. Biochem. 44, 671–681 (2017). source
May combine two claims.
InterpretationreasoningOur research uncovers a promising ERα-targeting therapeutic strategy by demonstrating that Bufalin can selectively induce degradation of ERα.
“Our research uncovers a promising ERα-targeting therapeutic strategy by demonstrating that Bufalin can selectively induce the degradation of ERα.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataBufalin acts as a molecular glue-type ERα degrader that effectively overcomes Tamoxifen resistance in preclinical models.
“Taken together, these results indicate that Bufalin acts as a molecular glue-type ERα degrader that effectively overcomes Tamoxifen resistance in preclinical models.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataBufalin acts as a molecular glue that strengthens the ERα-STUB1 interaction, thereby driving degradation of ERα.
“Here, we found that Bufalin may serve as a molecular glue that strengthens the ERα-STUB1 interaction, thereby driving the degradation of ERα.”
- Also stated in: § Discussion · boosted
“Here, we demonstrate that Bufalin enhances ERα’s association with the ubiquitin E3 ligase STUB1 by acting as a molecular glue, which results in proteasome-mediated degradation of ERα.”
- Also stated in: § Discussion · plain
“Additionally, Bufalin functions as a molecular glue, facilitating the interaction between ERα and the ubiquitin E3 ligase STUB1, ultimately leading to proteasomal degradation of ERα.”
Quote in the paperSubstantial claim- Rests on an assumption: Molecular docking and molecular dynamics simulations accurately model Bufalin-ERα binding and molecular glue function.
- Also stated in: § Discussion · boosted
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe experiments were randomized.
“The experiments were randomized.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll samples in the study represent biological replicates.
“All samples represent biological replicates, and the statistical measurements are presented as mean ± SD as specified in each figure.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenNo statistical method was used to predetermine the sample size.
“No statistical method was used to predetermine sample size.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenNo data were excluded from the analyses.
“No data were excluded from the analyses.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataBufalin is a molecular glue for ERα degradation and has potential as a promising lead compound for the treatment of Tamoxifen resistance.
“Collectively, our findings not only establish Bufalin as a molecular glue for ERα degradation but also highlight its potential as a promising lead compound for the treatment of Tamoxifen resistance.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenTissue samples were collected from Xiangya Hospital of Central South University, and all individuals provided informed consent prior to participating in the study.
“Tissue samples were collected from the Xiangya Hospital of Central South University (Changsha, China), and all individuals provided informed consent prior to participating in the study.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll representative experiments were repeated at least three times independently with similar results.
“All representative experiments are repeated at least three times independently with similar results.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataBufalin exhibits potent anti-cancer activity against Tamoxifen-resistant breast cancer in both preclinical models and patient-derived organoids.
“Furthermore, Bufalin exhibits potent anti-cancer activity against Tamoxifen-resistant breast cancer in both preclinical models and patient-derived organoids.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataAs a molecular glue of ERα, Bufalin exhibited potent anti-cancer effects in vitro, in vivo, and in Tamoxifen-resistant patient-derived organoids.
“Moreover, as a molecular glue of ERα, Bufalin exhibited potent anti-cancer effects in vitro, in vivo, and in Tamoxifen-resistant patient-derived organoids.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe experiments were approved by the Medical Ethics Review Committee of Xiangya Hospital of Central South University (Ethics code: 2023121169).
“The experiments were approved by the Medical Ethics Review Committee of Xiangya Hospital of Central South University (Ethics code: 2023121169).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe investigators were blinded to allocation during experiments and outcome assessment.
“The Investigators were blinded to allocation during experiments and outcome assessment.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataBufalin selectively binds to the R394 residue of ERα and promotes its degradation based on AI-driven methods.
“In summary, our study demonstrates that Bufalin selectively binds to the R394 residue of ERα and promotes its degradation based on the AI-driven methods.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAnimal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University (CSU-2023-0462).
“Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University (CSU-2023-0462)”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll cell lines were tested negative for mycoplasma contamination.
“All cell lines were tested negative for mycoplasma contamination.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenWhen tumors were palpable, mice were randomly divided into designated groups and received the indicated treatments.
“When the tumors were palpable, the mice were randomly divided into designated groups and received the indicated treatments.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationUnlike conventional inhibitors, molecular glues facilitate protein degradation by inducing proximity between the target and E3 ligases and have gained attention as a potential strategy for targeting proteins.
“Unlike conventional inhibitors, molecular glues facilitate protein degradation by inducing proximity between the target and E3 ligases and have gained attention as a potential strategy for targeting proteins54,55.”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRecent studies have shown that ERα-targeting PROTACs can also effectively degrade ERα and combat endocrine-resistant breast cancer.
“In line with this, recent studies have shown that ERα-targeting PROTACs can also effectively degrade ERα and combat endocrine-resistant breast cancer61,62.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [61] Xie, B. et al. Discovery of a novel class of PROTACs as potent and selective estrogen receptor α degraders to overcome endocrine-resistant breast cancer in vitro and in vivo. J. Med Chem. 66, 6631–6651 (2023). source
- cites [62] Deng, X. et al. Discovery of novel bicyclic phenylselenyl-containing hybrids: an orally bioavailable, potential, and multiacting class of estrogen receptor modulators against endocrine-resistant breast cancer. J. Med Chem. 65, 7993–8010 (2022). source
The paper relies on this without support.
The precise molecular mechanism of Bufalin's anti-cancer activity remains unclear.
“Bufalin, a compound derived from traditional medicines, has shown promising anti-cancer activity; however, its precise molecular mechanism of action remains unclear.”
“However, the molecular mechanisms underlying Bufalin’s anti-tumor activity remain to be elucidated.”
The paper relies on this without support.
The accurate identification of drug targets remains challenging.
“However, the accurate identification of drug targets remains challenging.”
The paper relies on this without support.
ERα degradation has therapeutic potential in overcoming endocrine resistance.
“Given the therapeutic potential of ERα degradation in overcoming endocrine resistance, we investigate the inhibitory effect of Bufalin on endocrine-resistant models and prove Bufalin reverses Tamoxifen resistance in vitro, in vivo, and in patient-derived breast cancer organoids from tamoxifen-relapsed cases.”
The paper relies on this without support.
Molecular docking and molecular dynamics simulations accurately model Bufalin-ERα binding and molecular glue function.
“Molecular docking experiments revealed that Bufalin selectively binds to R394 of ERα, while molecular dynamics simulations suggest its molecular glue function stabilizes the interaction between ERα and STUB1”
The paper relies on this without support.
Toxicity results in mice are predictive of safety in humans.
“suggesting a favorable safety profile.”
The paper relies on this without support.
Preclinical models (cell lines, xenografts, patient-derived organoids) predict clinical responses in patients.
“These results underscore the potential of Bufalin as an effective therapeutic agent for Tamoxifen resistance in ER+ breast cancer and validate its translational potential in cancer therapy.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Binding-energy calculation is described as both MM-GBSA and MM-PBSA
MajorThe Results attribute the ESR1-Bufalin energy and residue-decomposition analysis to MM-GBSA, while the Methods describe MM-PBSA calculations and residue decomposition for the same complex. These are different calculation methods.
“The docked structure of the ESR1-Bufalin complex was then subjected to 100 ns molecular dynamics simulations with Amber22 software, followed by MM-GBSA energy calculations and residue energy decomposition.”
“MM-PBSA binding free energy calculations were performed using MMPBSA.py from AmberTools202368, using only the last 100 ns of the trajectory. We extracted 100 frames from the trajectory for subsequent energy calculations. In addition, residual energy decomposition was performed for the Bufalin-ESR1 complex.”
ESR1-Bufalin simulation duration differs between Results and Methods
MinorThe Results describe a 100 ns simulation of the ESR1-Bufalin complex, whereas the Methods list that complex among the simulated systems and specify a 200 ns production simulation.
“The docked structure of the ESR1-Bufalin complex was then subjected to 100 ns molecular dynamics simulations with Amber22 software, followed by MM-GBSA energy calculations and residue energy decomposition.”
“Finally, a 200 ns production classical MD simulation was conducted with a time step of 2 fs.”
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Self-citationThe effects of bufadienolides on HER2 overexpressing breast cancer cells
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“Additionally, they confirm that bufalin effectively overcomes resistance to Tamoxifen, suggesting its potential as a therapeutic agent in treating hormone-resistant breast cancer.”
Zheng, Y., He, Z., Su, Z., Wang, H., Jiang, X., Fang, X., Lu, S., & Li, Y. (2023) · Cell Biology International · doi:10.1002/cbin.12021 · Cited by 10 · see citing works
Involvement of RFC3 in tamoxifen resistance in ER-positive breast cancer through the cell cycle
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“The methodology of organoid culture should be provided.”
The ubiquitin ligase RNF2 stabilizes ERα and modulates breast cancer progression
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Identification of a Novel Substrate for eEF2K and the AURKA‐SOX8 as the Related Pathway in TNBC
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“- Previous studies have demonstrated that lower concentrations of bufalin that the ones used in the present study have an inhibitory effect on the steroid receptor coactivators SRC as well as on the viability in MCF-7 breast cancer cells.”
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Self-citation
Flagged first, then by citations
Cited by
Sorted by citations
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“These data suggested that the anti-cancer effects of bufalin are related to its ability to overcome tumor endocrine resistance (Jiang et al., 2025).”
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“…protein-binding ligand) by introducing a minimal E3 ligase-recruiting fragment [32]—such as the glutarimide moiety for CRBN or the α -pyrrole fragment for ZFP91 (as seen in natural products like bufalin)—at specific positions, thereby conferring molecular glue functionality [138,139] (Figure 10).”
Target discovery and drug design in the era of artificial intelligence
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Self-citationParthenolide inhibits methamphetamine-induced depressive-like behavior by targeting ADORA2A
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Molecular Glue Degraders Redefining Targeted Therapies From Discovery to Therapeutic Applications
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“Among them, bufalin (BFL) and cinobufotalin (CBF) are representative bufadienolides frequently associated with antitumor potential (Jiang et al., 2025; Wang J. et al., 2022; Wu S. et al., 2024).”
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“[6] Activity-based protein profiling (ABPP) can be utilized for candidate target identification and combined with DARTS-MS to improve the reliability of target screening.”
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“In oestrogen receptor-positive (ER-positive) breast cancer, bufalin was identified as an ER α -directed molecular glue that directly binds ER α and promotes its interaction with the E3 ubiquitin ligase STUB1, resulting in ER α ubiquitination and proteasomal degradation [29].”
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- Authors
- Jiang, S., Liu, K., Jiang, T., Li, H., Wei, X., Wan, X., Zhong, C., Gong, R., Chen, Z., Zou, C., Zhang, Q., Cheng, Y., & Cao, D.
- Affiliations
- Central South University (CN)
- Sichuan University (CN)
- Corresponding author
- Cheng, Yan
- Cao, Dongsheng
- Venue
- Nature Communications
- Published
- 2025-08-22
- Volume
- 16
- Issue
- 1
- Pages
- 7854-7854
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC12373995
- OpenAlex
- W4413426186
- Cited by
- 23
- References
- 73
- Citations by year
- 2026: 20 · 2025: 3
- FWCI
- 6.00
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nature Communications (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Natural Science Foundation of China (22173118)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
- Keywords
- bufalin, estrogen receptor alpha, molecular glues, STUB1, proteasomal degradation, tamoxifen resistance, endocrine therapy resistance, breast cancer, natural products, E3 ubiquitin ligase, drug target identification
- MeSH terms
- Animals, Antineoplastic Agents, Artificial Intelligence, Breast Neoplasms, Bufanolides, Female, Humans, Protein Binding, Tamoxifen, Drug Resistance, Neoplasm, Xenograft Model Antitumor Assays, Cell Line, Tumor, Estrogen Receptor alpha, Mice, Molecular Dynamics Simulation, MCF-7 Cells, Molecular Docking Simulation
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