The intrinsically disordered protein (IDP) c-Myc plays a crucial role in regulating cell growth, proliferation, and apoptosis, making it a significant target for cancer therapy. Herein, we report an integrated approach combining computational and biological methods to uncover key c-Myc structural aspects aimed at identifying modulators of this protein. A virtual screening campaign starting from commercially available libraries was performed, leading to the selection of 11 promising c-Myc binders. Preliminary biophysical and biological analysis, including Surface Plasmon Resonance (SPR) and MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)] assays, highlighted three new potential c-Myc/Max heterodimerization inhibitors. Among them, 11 emerged as the most active one, displaying promising dissociation costant (KD) values on c-Myc (~ 60 μM), only slightly higher than the known binder 10058-F4 (with a KD ~ 40 μM) and on Max (with a KD ~ 80 μM). Notably, 11 showed significant antiproliferative effects on three different malignant cell lines (i.e., U937, HCT-116, and HT-29, as models of acute myeloid leukemia and colorectal cancer cell lines, respectively) in a dose- and cell-dependent manner. Importantly, a significant decrease in c-Myc levels was observed, especially in U937 leukemia cells with an 85 % reduction. Finally, molecular dynamics simulations and proteomic studies were performed to identify its binding site and key interacting residues. In summary, this work presents a robust research strategy and offers valuable insights for identifying new c-Myc/Max disruptors.
