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 (ER alpha) are highly associated with hormone therapy resistance in breast cancer. However, current understanding of abnormal activity of ER alpha 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 ER alpha mutants activities. We found that, compared with ER WT, ER alpha mutants (Y537S/N and D538G) exhibit high ligand-independent activity, suggesting the gain-of-function phenotype of these ER alpha mutants. Notably, Y537S, the most common ER alpha mutant type, has the highest intrinsic activation. We then collected and screened a natural product library for potential ER alpha antagonists via GLPCA and identified celastrol and gambogic acid as new antagonists of the ER alpha Y537S mutant. Moreover, interactions between these two compounds and the ER alpha 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 ER alpha Y537S. In summary, GLPCA provides a powerful platform for exploring innovative functional biology and drug discovery of antagonists targeting ER alpha mutants.

Automated summary

Gain-of-function mutations in estrogen receptor alpha (ER alpha) are associated with hormone therapy resistance in breast cancer. A new GLPCA method was developed to evaluate ER alpha mutants, identifying natural products celastrol and gambogic acid as antagonists, which showed synergistic anti-cancer effects when combined with CDK4/6 inhibitor abemaciclib.