SignificanceA thorough characterization of the binding interaction between a drug and its molecular target is fundamental to successfully lead drug design. We demonstrate that this characterization is also possible using the recently developed method of funnel-metadynamics (FM), here applied to investigate the binding of berberine to DNA G-quadruplex. We computed a quantitatively well-characterized free-energy landscape that allows identifying two low-energy ligand binding modes and the presence of higher energy prebinding states. We validated the accuracy of our calculations by steady-state fluorescence experiments. The good agreement between the theoretical and experimental binding free-energy value demonstrates that FM is a most reliable method to study ligand/DNA interaction.

The coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the severe acute respiratory syndrome coronavirus (SARS)-CoV-2. In light of the urgent need to identify novel approaches to be used in the emergency phase, we have embarked on an exploratory campaign aimed at repurposing natural substances and clinically available drugs as potential anti-SARS-CoV2-2 agents by targeting viral proteins. Here we report on a strategy based on the virtual screening of druggable pockets located in the central β-sheet core of the SARS-CoV-2 Spike's protein receptor binding domain (RBD). By combining an in silico approach and molecular in vitro testing we have been able to identify several triterpenoid/steroidal agents that inhibit interaction of the Spike RBD with the carboxypeptidase domain of the Angiotensin Converting Enzyme (ACE2). In detail, we provide evidence that potential binding sites exist in the RBD of the SARS CoV-2 Spike protein and that occupancy of these pockets reduces the ability of the RBD to bind to the ACE2 consensus in vitro. Naturally occurring and clinically available triterpenoids such as glycyrrhetinic and oleanolic acids, as well as primary and secondary bile acids and their amidated derivatives such as glyco-ursodeoxycholic acid and semi-synthetic derivatives such as obeticholic acid reduces the RBD/ACE2 binding. In aggregate, these results might help to define novel approaches to COVID-19 based on SARS-CoV-2 entry inhibitors.

Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety.

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