Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

Abstract

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 (IC50 = 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 with an activity similar, possibly slightly higher, than those found for it for other PPIs. This PPI inhibitory activity of methylene blue 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.

Methylene Blue has a potent antiviral activity against SARS-CoV-2 and H1N1 influenza virus in the absence of UV-activation in vitro

“…Another report describes inhibition by MB in vitro of the SARS-CoV-2 Spike -ACE2 protein-protein interaction. (15). Finally, a recent study compared the in vitro median Effective Concentration (EC50) of 3 drugs against SARS-COV-2, when administered 4h post infection.…”

Chemoinformatic Analysis of Psychotropic and Antihistaminic Drugs in the Light of Experimental Anti-SARS-CoV-2 Activities

“…This molecule is however interesting mechanistically as in vitro it was found to block (low micromolar in a protein-based ELISA setup) the Spike-ACE2 protein-protein. 49 Such a compound is also likely to interact with cell membranes and seems to be a phospholipidosis inducer. 50 Nitazoxanide was, for example, found promising in its ability to hinder the replication of a SARS-CoV-2 isolate 51 and is now in several clinical trials either alone (eg, see 52 ), against placebo, or in combination with for instance Ivermectin (a broad-spectrum anti-parasite medication that was not found to be active in the CPE assays that we investigated here and that was highly cytotoxic in the Vero E6 cell counterscreen, not mentioning potential risks due to interactions or the lack of interactions with some drug transporters in some patients 53 ).…”

Small-MoleculeIn VitroInhibitors of the Coronavirus Spike – ACE2 Protein-Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

“…As such, they are of no value here being nonspecific; they were included as positive controls. This screening also identified methylene blue (MeBlu, 6), a phenothiazine dye approved by the FDA for the treatment of methemoglobinemia and also used for several other therapeutic applications in the developed world (69-71) and with additional potential for certain developing world applications such as malaria (72), as showing promising inhibitory activity for the SARS-CoV-2-S-hACE2 PPI, likely contributing to its anti-CoV activity (73); this has been discussed separately (60).…”

“…were coated overnight at 4 °C with 100 μL/well of Fc-conjugated ACE2 receptor diluted in PBS pH 7.2. This was followed by blocking with 200 μL/well of SuperBlock (PBS) (Thermo Fisher Scientific) for 1 h at RT (60). Then, plates were washed twice using washing solution (PBS pH 7.4, 0.05% Tween-20) and tapped dry before the addition of the tagged ligand (SARS-CoV-2 S1 or RBD) and test compounds diluted in binding buffer (100 mM HEPES, pH 7.2) to give a total volume of 100 μL/well.…”

“…Since previously we found that starting from organic dyes one can identify SMIs for cosignaling PPIs as potential immunomodulatory agents (48,(53)(54)(55)(56)(57)(58)(59), we initiated a screen of such compounds for their ability to inhibit the SARS-CoV-2-S-ACE2 PPI. This led to the identification of several organic dyes (1-5, Figure 1) that show promising inhibitory activity of this PPI in vitro, including methylene blue (6), a phenothiazine dye approved by the FDA for the treatment of methemoglobinemia, which we have described separately (60). More importantly, it also led to the identification of new and more potent SMIs (7-13) that are more drug-like and no longer contain color-causing chromophores as summarized below.…”