ABSTRACT
Inappropriate platelet aggregation creates a cardiovascular risk that is largely managed with thienopyridines and aspirin. Although effective, these drugs carry risks of increased bleeding and drug 'resistance', underpinning a drive for new antiplatelet agents. To discover such drugs, one strategy is to identify a suitable druggable target and then find small molecules that modulate it. A good and unexploited target is the platelet collagen receptor, GPVI, which promotes thrombus formation. To identify inhibitors of GPVI that are safe and bioavailable, we docked a FDA-approved drug library into the GPVI collagen-binding site in silico. We now report that losartan and cinanserin inhibit GPVI-mediated platelet activation in a selective, competitive and dose-dependent manner. This mechanism of action likely underpins the cardioprotective effects of losartan that could not be ascribed to its antihypertensive effects. We have, therefore, identified small molecule inhibitors of GPVI-mediated platelet activation, and also demonstrated the utility of structure-based repurposing.
Subject(s)
Molecular Docking Simulation , Platelet Membrane Glycoproteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Cardiotonic Agents/chemistry , Cardiotonic Agents/pharmacology , Cinanserin/chemistry , Cinanserin/pharmacology , Humans , Losartan/chemistry , Losartan/pharmacology , Molecular Sequence Data , Platelet Activation , Platelet Membrane Glycoproteins/chemistry , Platelet Membrane Glycoproteins/metabolism , Protein Binding , Small Molecule Libraries/chemistrySubject(s)
Cinanserin/pharmacology , Coronavirus 229E, Human/enzymology , Enzyme Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Animals , Cell Line , Cinanserin/chemistry , Cricetinae , DNA, Complementary/metabolism , Drug Design , Humans , Models, Biological , RNA/chemistry , Serotonin Antagonists/pharmacologyABSTRACT
The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.
