A protein-fragment complementation assay reveals that celastrol and gambogic acid suppress ERα mutants in breast cancer

Somatic gain-of-function mutations within estrogen receptor alpha (ERα) are highly associated with hormone therapy resistance in breast cancer. However, current understanding of abnormal activity of ERα mutants and their relevant targeted intervention is still very limited. Herein, we developed a new, real-time, and reliably Gaussia luciferase-based protein-fragment complementation assay (GLPCA) for evaluating ERα mutants activities. We found that, compared with ER WT, ERα mutants (Y537S/N and D538G) exhibit high ligand-independent activity, suggesting the gain-of-function phenotype of these ERα mutants. Notably, Y537S, the most common ERα mutant type, has the highest intrinsic activation. We then collected and screened a natural product library for potential ERα antagonists via GLPCA and identified celastrol and gambogic acid as new antagonists of the ERα Y537S mutant. Moreover, interactions between these two compounds and the ERα Y537S mutant were confirmed by molecular docking and cellular thermal shift assay. Importantly, we further demonstrated that celastrol and gambogic acid exhibit synergistic antiproliferative and pro-apoptotic effects when combined with an approved CDK4/6 inhibitor abemaciclib in breast cancer cells expressing ERα Y537S. In summary, GLPCA provides a powerful platform for exploring innovative functional biology and drug discovery of antagonists targeting ERα mutants.