Geraniin

Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with no target, and chemotherapy remains the only treatment in the clinic. Lactate dehydrogenase A (LDHA), a critical enzyme in glycolysis, was reported to be upregulated in TNBC. Only machilin A and berberine were reported as natural LDHA inhibitors with moderate activity at micromolar levels. The aim of this study is to discover novel potent natural LDHA inhibitors, evaluate their anti‐TNBC activity, and explore the mechanisms. LDH activity assay was developed to discover LDHA inhibitors. Biolayer interferometry, molecular docking, and molecular dynamics were applied to explore the mechanisms at the protein level. CCK8, three‐dimensional spheroid formation, wound healing assay, and flow cytometry were applied to detect proliferation, migration, cell cycle, reactive oxygen species, and mitochondrial membrane potential. Quantitative real‐time PCR and western blotting were used for detecting mRNA and protein levels. Five novel nanomolar natural LDHA inhibitors were found. Geraniin exhibited the most potent LDHA inhibition with an IC 50 value of 26.7 ± 7.3 nM. Geraniin showed anti‐TNBC activity both in two‐dimensional and three‐dimensional cell models. Mechanistically, geraniin directly binds to LDHA at the allosteric pocket, downregulates the key enzymes and transcription factors in glycolysis and lipid de novo, arrests the cell cycle in the S phase, suppresses cell migration, raises reactive oxygen species, and disrupts mitochondrial membrane potential. Geraniin was a potent natural LDHA allosteric inhibitor. Geraniin could be a potential candidate for anti‐TNBC treatment.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) are widely regarded as the most promising strategy to treat non-small cell lung cancer (NSCLC). While the existing development model cannot meet the requirements.

Here, we developed a drug prediction and screening platform based on state-of-the-art deep learning (DL) algorithms and cell membrane chromatography (CMC). Using this platform, we discovered several novel EGFR antagonists from massive molecules. The interactions between target compounds and EGFR were investigated using frontal analysis, Surface plasmon resonance analysis, cellular thermal shift assay, and molecule docking. Xenografts models were established to study the anti-tumor activity of most promising compound. Proteomics, immunofluorescence, and Western blots were conducted to explore its mechanism.

The deep neural network had good performance, with an area under the curve of 0.950 ± 0.012. Among the top 100 molecules predicted by the model, 25 compounds had retention on SNAP-tagged (ST)-EGFR/CMC, of which 12 molecules exhibited significant anti-tumor activity. Interaction analysis revealed that Geraniin, Brazilin, and 7-Epitaxol would directly bind to EGFR and inhibit its activation. 7-Epitaxol exhibited significant anti-tumor activity in vivo and in vitro. 7-Epitaxol combined with EGFR and inhibited its phosphorylation, blocked PI3K/AKT pathways, thereby exerting its anti-tumor activity by promoting autophagy in A549 cells.

The results provide a novel and powerful platform for drug discovery and development in NSCLC research. By using this platform, Geraniin, Brazilin, and 7-epitaxol were identified as novel EGFR antagonists. We also innovatively demonstrated that 7-epitaxol promotes autophagy in NSCLC.