ABSTRACT
The outbreak of the COVID-19 pandemic in late 2019 has so far caused more than 6 million deaths worldwide according to WHO. Currently, there is only one FDA-approved novel antiviral drug therapy (Paxlovid, Pfizer) for treatment of critically ill patients from COVID-19 infection. However, there is a need for a great diversity of antiviral therapies because of the constant mutations of the virus and the possibility of other similar viruses causing similar symptoms in patients. By utilizing natural products from medicinal plants, it is possible to provide a drug, or drug leads for a future novel antiviral therapy. Currently, we are testing isolated and characterized natural products (some novel and some already elucidated) from water lily (Nuphar lutea) and eagle fern (Pteridium aquilinum) on the SARS-CoV-2 virus where we are working on developing a good pipeline to test these compounds in vitro. So far, we have tested the effect of the compounds on inhibition of cell entry using a pseudovirus system and HEK293-FT cells transiently transfected with SARS-CoV-2 receptors ACE-2 and TMPRSS2. Some of the compounds showed a slightly inhibitory effect. Next steps will be to establish a cell line with stable expression of ACE-2 and TMPRSS2 and confirm the results in cells with endogenous expression of these receptors. We will proceed working with different variants of the hospital isolates of the virus within the next months, where we will further analyse the potential effect of these compounds on viral replication. The goal is to determine and understand the mechanism of inhibitory action of the natural products on the virus. In conclusion, we are at an early stage of researching the abundant possibilities of antiviral effects of these natural products on SARS-CoV-2.
ABSTRACT
The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.