From Traditional Ethnopharmacology to Modern Natural Drug Discovery: A Methodology Discussion and Specific Examples.

Ethnopharmacology, through the description of the beneficial effects of plants, has provided an early framework for the therapeutic use of natural compounds. Natural products, either in their native form or after crude extraction of their active ingredients, have long been used by different populations and explored as invaluable sources for drug design. The transition from traditional ethnopharmacology to drug discovery has followed a straightforward path, assisted by the evolution of isolation and characterization methods, the increase in computational power, and the development of specific chemoinformatic methods. The deriving extensive exploitation of the natural product chemical space has led to the discovery of novel compounds with pharmaceutical properties, although this was not followed by an analogous increase in novel drugs. In this work, we discuss the evolution of ideas and methods, from traditional ethnopharmacology to in silico drug discovery, applied to natural products. We point out that, in the past, the starting point was the plant itself, identified by sustained ethnopharmacological research, with the active compound deriving after extensive analysis and testing. In contrast, in recent years, the active substance has been pinpointed by computational methods (in silico docking and molecular dynamics, network pharmacology), followed by the identification of the plant(s) containing the active ingredient, identified by existing or putative ethnopharmacological information. We further stress the potential pitfalls of recent in silico methods and discuss the absolute need for in vitro and in vivo validation as an absolute requirement. Finally, we present our contribution to natural products' drug discovery by discussing specific examples, applying the whole continuum of this rapidly evolving field. In detail, we report the isolation of novel antiviral compounds, based on natural products active against influenza and SARS-CoV-2 and novel substances active on a specific GPCR, OXER1.

Natural Polyphenols Inhibit the Dimerization of the SARS-CoV-2 Main Protease: The Case of Fortunellin and Its Structural Analogs.

3CL-Pro is the SARS-CoV-2 main protease (MPro). It acts as a homodimer to cleave the large polyprotein 1ab transcript into proteins that are necessary for viral growth and replication. 3CL-Pro has been one of the most studied SARS-CoV-2 proteins and a main target of therapeutics. A number of drug candidates have been reported, including natural products. Here, we employ elaborate computational methods to explore the dimerization of the 3CL-Pro protein, and we formulate a computational context to identify potential inhibitors of this process. We report that fortunellin (acacetin 7-O-neohesperidoside), a natural flavonoid O-glycoside, and its structural analogs are potent inhibitors of 3CL-Pro dimerization, inhibiting viral plaque formation in vitro. We thus propose a novel basis for the search of pharmaceuticals as well as dietary supplements in the fight against SARS-CoV-2 and COVID-19.

p-cymene impairs SARS-CoV-2 and Influenza A (H1N1) viral replication: In silico predicted interaction with SARS-CoV-2 nucleocapsid protein and H1N1 nucleoprotein.

Therapeutic regimens for the COVID-19 pandemics remain unmet. In this line, repurposing of existing drugs against known or predicted SARS-CoV-2 protein actions have been advanced, while natural products have also been tested. Here, we propose that p-cymene, a natural monoterpene, can act as a potential novel agent for the treatment of SARS-CoV-2-induced COVID-19 and other RNA-virus-induced diseases (influenza, rabies, Ebola). We show by extensive molecular simulations that SARS-CoV-2 C-terminal structured domain contains a nuclear localization signal (NLS), like SARS-CoV, on which p-cymene binds with low micromolar affinity, impairing nuclear translocation of this protein and inhibiting viral replication, as verified by preliminary in vitro experiments. A similar mechanism may occur in other RNA-viruses (influenza, rabies and Ebola), also verified in vitro for influenza, by interaction of p-cymene with viral nucleoproteins, and structural modification of their NLS site, weakening its interaction with importin A. This common mechanism of action renders therefore p-cymene as a possible antiviral, alone, or in combination with other agents, in a broad spectrum of RNA viruses, from SARS-CoV-2 to influenza A infections.