A Natural LDHA Allosteric Inhibitor Geraniin Suppresses Triple‐Negative Breast Cancer Cell Growth

ABSTRACT 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.