Arecoline as a Novel Scaffold Targeting the ATAD2 Bromodomain for Cell Cycle Modulation

<b>Background/Objectives</b>: ATPase family AAA domain-containing protein 2 (ATAD2) is an oncogenic chromatin regulator that amplifies E2F/MYC transcriptional programs, yet direct modulators remain scarce. Arecoline (ARE), the primary alkaloid of the areca nut, is a known carcinogen but paradoxically exhibits context-dependent anti-proliferative activities. In this study, we resolve this paradox by defining ARE's anti-cancer mechanism. <b>Methods</b>: Breast cancer cell proliferation and colony formation assays were performed to evaluate the anti-proliferative effects of ARE. Cell-cycle distribution was analyzed to determine phase-specific effects. Transcriptomic profiling was conducted to identify affected gene networks. An unbiased Cellular Thermal Shift Assay-Mass Spectrometry (CETSA-MS) screening was used to identify direct protein targets, followed by CETSA-Western blotting for validation. Finally, in silico structure-based design was applied to generate novel derivatives with improved predicted properties. <b>Results</b>: ARE suppressed breast cancer cell proliferation and colony formation by inducing G1/S phase arrest. Transcriptomic analysis revealed that this phenotype was driven by profound suppression of the E2F/Cell Cycle gene network. CETSA-MS identified ATAD2 through multi-omics convergence, as the 67 direct targets were collectively most significantly enriched in the E2F pathway. CETSA-Western blotting confirmed that ARE binds and thermally stabilizes ATAD2. Mechanistically, ARE engagement of ATAD2 led to downregulation of key downstream proteins, including MYC and Cyclin D1, directly linking target modulation to G1/S arrest. Structure-based design further yielded novel derivatives with predicted enhanced ATAD2 binding and substantially reduced toxicity. <b>Conclusions</b>: Together, these findings uncover ATAD2 as a druggable target of ARE, establish proof-of-concept for repurposing this scaffold, and provide a rational framework for developing safer ATAD2-targeted therapies.