Design, synthesis and characterization of a novel GCP derivative as a potential 14-3-3 sigma homodimer stabilizer

This study reports the design and characterization of a novel dimer stabilizer targeting the 14-3-3σ protein, a signaling protein with purported tumor-suppressive roles. Using an in silico design strategy based on the derivatization of GCP-Lys-OMe, a known stabilizer, a series of analogues was evaluated, leading to the identification of 2-N,5-N-dicarbamimidoyl-1H-pyrrole-2,5-dicarboxamide (Compound 14) as the most promising candidate. Furthermore, molecular docking studies and three independent molecular dynamics simulations suggested that the charged state of the guanidinocarbonyl-pyrrole moiety substantially contributed to binding at the dimer interface. Compound 14 was synthesized via a novel route and characterized by NMR and LC-MS/MS, and its binding to recombinantly expressed 14-3-3σ was validated using multiple orthogonal biophysical techniques. ¹H CPMG NMR indicated interaction at the dimer interface and enhanced the binding of 14-3-3σ to an exoenzyme S-derived peptide. Meanwhile, UV-Vis titrations yielded an estimated dissociation constant of 1.21 ± 0.15 μM, while thermal-ramp dynamic light scattering showed an increase in aggregation temperature consistent with dimer stabilization. Together, these results identify Compound 14 as a new 14-3-3σ dimer stabilizer and provide a basis for future analogue development.