Study of the interaction between nucleolin and 6,19-dihydroxy-ent-trachiloban-17-oic acid

The characterization of protein/plant molecule interactions in drug discovery, medicinal chemistry, biology and pharmacology is one of the new fields of interest in the last decades. 1 A target identification of bioactive plant molecules by a multidisciplinary approach represents an important goal of our research group. In particular, our research group are focused on the study of the interaction between plant molecules and proteins mainly involved in cancer and inflammation processes. One of our protein target is nucleolin (NCL). NCL is a multilocalized protein, found in nucleolus, nucleoplasm, cytoplasm as well as on the cell membrane.2 It is implicated in many physiological processes such as remodelling of chromatin structure, ribosome biogenesis, DNA transcription, and in cancer, inflammation and viral diseases.3 In order to study plant small molecules able to modulate nucleolin activity, a library of diterpenes was screened through the Celluar Thermal Shift Assay (CETSA). It allows us to monitor and quantify the extent to which a drug candidate reaches and directly binds to a protein target of interest within a cell. CETSA relies on the principle of thermodynamic stabilization inferred to the protein as a result of ligand binding.4 The screening, performed on HeLa cells, led us to obtain the diterpene 6,19-dihydroxy-ent-trachiloban-17-oic acid as main ligand of NCL. This molecule has been extracted from Psiadia punctulata (DC) Vatke (Asteraceae). In order to confirm the interaction, the apparent melting curve of nucleolin (Tm: 55°C), and the EC50 (5µM) of the complex has been determined by CETSA. Subsequently, a chemical proteomic cell-based approach of Drug Affinity Responsive Target Stability (DARTS) has been performed to validate the interaction.5 The DARTS results analized by Mass Spectrometry and Western Blot analysis confirmed the diterpene/NCL interaction