Methylene Blue Inhibits the SARS-CoV-2 Spike–ACE2 Protein-Protein Interaction–a Mechanism that can Contribute to its Antiviral Activity Against COVID-19

Due to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC ₅₀ = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction. Methylene blue inhibited the entry of a SARS-CoV-2 spike bearing pseudovirus into ACE2-expressing cells with similar IC ₅₀ (3.5 μM). Hence, this PPI inhibitory activity could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.