Herein we describe a combined experimental and in silico study of the interaction of a series of pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-one derivatives (PBTs) with parallel G-quadruplex (GQ) DNA aimed at correlating their previously reported anticancer activities and the stabilizing effects observed by us on c-myc oncogene promoter GQ structure. Circular dichroism (CD) melting experiments were performed to characterize the effect of the studied PBTs on the GQ thermal stability. CD measurements indicate that two out of the eight compounds under investigation induced a slight stabilizing effect (2–4 °C) on GQ depending on the nature and position of the substituents. Molecular docking results allowed us to verify the modes of interaction of the ligands with the GQ and estimate the binding affinities. The highest binding affinity was observed for ligands with the experimental melting temperatures (T_ms). However, both stabilizing and destabilizing ligands showed similar scores, whilst Molecular Dynamics (MD) simulations, performed across a wide range of temperatures on the GQ in water solution, either unliganded or complexed with two model PBT ligands with the opposite effect on the T_ms, consistently confirmed their stabilizing or destabilizing ability ascertained by CD. Clues about a relation between the reported anticancer activity of some PBTs and their ability to stabilize the GQ structure of c-myc emerged from our study. Furthermore, Molecular Dynamics simulations at high temperatures are herein proposed for the first time as a means to verify the stabilizing or destabilizing effect of ligands on the GQ, also disclosing predictive potential in GQ-targeting drug discovery.

The development of molecules that interact with G-quadruplex (G4) sequences requires effective evaluation methods. Several techniques are currently available, including nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS), fluorescence using FRET-melting, G4-fluorescent intercalator displacement assay (G4-FID) and affinity chromatography. Among these, CD spectroscopy is gaining prominence due to its lower material requirements, faster experimentation and quicker data processing. However, conventional CD methods have limitations, such as higher sample volume required and the inability to handle high-throughput analysis efficiently. The use of synchrotron radiation in high-throughput analysis methods (HT-SRCD) has further advanced the investigation of small-molecule interactions with DNA G4 structures in the presence of various monovalent cations. HT-SRCD offers the capability to analyze multiple samples simultaneously, overcoming the limitations of conventional CD methods. To validate this approach, three biologically relevant G4 sequences—HTelo1, G3T3 and T95-2T—were investigated. Their interactions with a library of small tetrazole-based molecules, synthesized via a four-component Ugi reaction, and with a peptide sequence deriving from RHAU helicases (Rhau25), were evaluated. The results demonstrate that this method not only effectively discriminates between different ligands but also provides valuable insights into the selectivity and the modes of interaction of these ligands with the G4 sequences.

The natural-like spiroketal, 2-hydroxy-8-methyl-1,7-dioxaspiro[5.5]undec-3-en-5-one (1) has been synthesised and has shown a potent antitumor activity against human tumor cells of different nature and histotype. We have now performed studies to verify its in vivo activity on a murine melanoma model and in vitro studies to shed some light on the mechanism of action. Spiroketal 1 have shown a potent dose-dependent antitumor efficacy in vivo in a syngenic murine model (C57Black mice) of melanoma (B16). The compound suppressed the tumor growth by an average of 90% at a dose of 5 mg/kg by intra-peritoneum administration at alternate days for 15 days. It also displayed high antitumor activity in vitro in the B16 cells with nanomolar IC₅₀ value. In addition to the proapoptotic and telomerase inhibition activities, compound 1 has shown to inhibit cell migration and to strongly reduce the HIF1alfa expression, that is considered a regulator of multiple cellular functions related to the progression from primary to metastatic cancer disease. Therefore, although the full mechanism of action of this molecule has yet to be completely elucidated, spiroketal 1 is a promising antitumor drug candidate for the clinical treatment of melanoma and various cancers. References: Fuggetta, M. P.; De Mico, A.; Cottarelli, A.; Morelli, F.; Zonfrillo, M.; Ulgheri, F.; Peluso, P.; Mannu, A.; Deligia, F.; Marchetti, M.; Roviello, G.; Reyes Romero, A.; Dömling, A.; Spanu, P. Synthesis and Enantiomeric Separation of a Novel Spiroketal Derivative: A Potent Human Telomerase Inhibitor with High in Vitro Anticancer Activity. J. Med. Chem. 2016, 59, 9140−9149 and references quoted therein