Target-based study to identify new bioactive diterpenes
Creators: name: Fiengo, Lorenzo
Publisher: name: Universita degli studi di Salerno
Year: 2019
Subjects: subject: BIO/15 BIOLOGIA FARMACEUTICA
subject: Drug discovery
subject: Diterpenes
subject: Tumor markers

description: 2017 - 2018
description: Target identification and mechanism of action studies of plant-derived compounds play a critical role in drug discovery. The knowledge of the bioactivity of natural compounds can lead to a number of advantages: first of all, it is possible to understand their full therapeutic potential; in addition, it can allow the further identification of their side-effects, their toxicity and also structure-activity relationships studies. This research project is focused on a Reverse Chemical Genetics approach, which relies on the screening of libraries of plant small molecules (provided by the Department of Pharmacy (DIFARMA)-Bioactive Natural Products, University of Salerno (UNISA), Fisciano, Italy) able to bind specific target proteins and on the validation of the ligand/protein interaction. In my PhD project I focused on three protein targets, over-expressed in cancer and identified as potential markers in several tumor cell lines: Nucleolin, Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90). Nucleolin (NCL) is a multifunctional protein involved in many process such as DNA transcription, ribosome biogenesis and regulation of mRNAs of anti-apoptotic and antiproliferative proteins such as AKT1, Bcl2, p53. Firstly, a screening of ent-kaurane and ent-trachilobane library by Cellular Thermal Shift Assay (CETSA) on Jurkat (leukemia T cells) and HeLa (cervical carcinoma) was performed, obtaining as main ligand of Nucleolin the 6,19-dihydroxy-ent-trachiloban-17-oic acid (12) from Psiadia punctulata ((Vatke) Asteraceae). Full length Nucleolin/12 interaction was validated in HeLa (cervical carcinoma) cells by CETSA and Drug Affinity Responsive Target Stability (DARTS). Nucleolin RNA Binding Domains 1-2/12 interaction was investigated by Saturation Transfer Difference NMR (STD-NMR), WaterLOGSY and Surface Plasmon Resonance (SPR): no interaction was observed with these two domains of the protein. The mechanism of action of the selected diterpene was studied by Flow Cytometry (sub G0/G1 cell cycle arrest), WB analysis (reduction of intracellular AKT1 and Bcl2 levels and pNCL levels on the cell membrane), RTq-PCR (reduction of AKT1 and Bcl2 mRNAs), MTT (IC50: 20 ± 1 μM), Protein Synthesis and Wound Healing assays in HeLa cells (reduction of 20% of migration). Therefore, the 6,19-dihydroxy-ent-trachiloban-17-oic acid (12) may be considered as a new promising modulator of Nucleolin. The second target protein was the molecular chaperon Heat Shock Protein 70 (Hsp70). A diterpene library was screened by SPR assay, in order to select putative Hsp70 ligands. SPR results showed that the ent-7β-acetoxy,18-hydroxy-15α,16α-epoxikaurane (epoxysiderol or compound 27) from Sideritis spp (Lamiaceae) interacts with Hsp70 (KD: 54 ± 1.2 nM). Epoxysiderol ability to modulate Hsp70 activity was assessed through MS (no covalent binding), DARTS and WB experiments. Moreover, epoxysiderol was tested on HeLa cells by MTT (IC50: 20 ± 0.9 μM), Flow Cytometry (G2/M and subG0/G1 cell cycle arrest), WB for its effect on the intracellular levels of Hsp70, Hsp90, and Hsp70 client proteins (reduction of pAKT1, p-p38 and p-JNK1) in HeLa cells, and by WB also for Hsp70 cytosolic and cell membrane levels (reduction of Hsp70 levels). Finally, ATPase assay (50% of reduction in dose-dependent manner) and molecular docking studies (interaction with the Hsp70 Nucleolide Binding Domain) were carried out. Therefore, in this study epoxysiderol was identified as a new Hsp70 inhibitor through cell-free and cell-based assays. Another target object of study in this PhD project was the Heat Shock Protein 90 (Hsp90). Fusicoccane diterpenes from Hypoestes forsskaolii ((Vahl) Acanthaceae), abietane diterpenes form Zhumeria majdae ((Rech.f. &amp; Wendelbo) Lamiaceae) and from different Salvia spp (Lamiaceae) were screened against Hsp90 by SPR and by MTT in HeLa, Jurkat and MCF7 cells, selecting the 18-hydroxyhypoestenone (6) and lanugon Q (20) as Hsp90 ligands. Subsequently, MTT assay was performed to investigate their cytotoxic and anti-proliferative activity: 18-hydroxyhypoestenone was the most cytotoxic in HeLa cells (IC50: 18 ±1 μM), whereas lanugon Q showed higher activity towards MCF7 (IC50: 20 ± 2 μM). In addition, Flow Cytometry and WB analyses were carried out: G2/M cell cycle arrest and reduction of p-Cdc2, pAKT1 and pERK1 levels were observed in HeLa cells after treatment with 6 (10 μM and 20 μM for 48h); Decrease of pERK, pAKT, cyclin A was observed in MCF7 after 48h of treatment with 20 (18 μM). Selected diterpenes were also tested against Hsp90 by ATPase activity assay: dose-dependent reduction (40%) of hydrolysis was observed with compound 6 (1,5, 10 μM), while no inhibition was induced by 20. Furthermore, molecular docking studies were implemented with compound 6, and the computational analysis of the Hsp90/6 interaction suggested a C-terminal domain. In conclusion, in this study 18-hydroxyhypoestenone and lanugon Q were identified as new Hsp90 interactors, able to modulate its activity and its client proteins levels. [edited by Author]