Methylene Blue Is a Nonspecific Protein–Protein Interaction Inhibitor with Potential for Repurposing as an Antiviral for COVID-19
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Abstract
We have previously identified methylene blue, a tricyclic phenothiazine dye approved for clinical use for the treatment of methemoglobinemia and for other medical applications as a small-molecule inhibitor of the protein–protein interaction (PPI) between the spike protein of the SARS-CoV-2 coronavirus and ACE2, the first critical step of the attachment and entry of this coronavirus responsible for the COVID-19 pandemic. Here, we show that methylene blue concentration dependently inhibits this PPI for the spike protein of the original strain as well as for those of variants of concern such as the D614G mutant and delta (B.1.617.2) with IC50 in the low micromolar range (1–5 μM). Methylene blue also showed promiscuous activity and inhibited several other PPIs of viral proteins (e.g., HCoV-NL63–ACE2, hepatitis C virus E–CD81) as well as others (e.g., IL-2–IL-2Rα) with similar potency. This nonspecificity notwithstanding, methylene blue inhibited the entry of pseudoviruses bearing the spike protein of SARS-CoV-2 in hACE2-expressing host cells, both for the original strain and the delta variant. It also blocked SARS-CoV-2 (B.1.5) virus replication in Vero E6 cells with an IC50 in the low micromolar range (1.7 μM) when assayed using quantitative PCR of the viral RNA. Thus, while it seems to be a promiscuous PPI inhibitor with low micromolar activity and has a relatively narrow therapeutic index, methylene blue inhibits entry and replication of SARS-CoV-2, including several of its mutant variants, and has potential as a possible inexpensive, broad-spectrum, orally bioactive small-molecule antiviral for the prevention and treatment of COVID-19.
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SciScore for 10.1101/2022.03.22.485299: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Briefly, HEK293T cells (CRL-3216; ATCC, Manassas, VA, USA) were seeded onto 96-well plates at a density of 5×104 cells per well in 100 μL complete medium (DMEM supplemented with 10% fetal bovine serum). HEK293Tsuggested: NoneVero-E6 cells (African Green Monkey renal epithelial cells; ATCC cat. no. CRL-1586) engineered to overexpress hACE2/Furin were seeded in 24-well plates to obtain a confluence of 80%. Vero-E6suggested: NoneBriefly, Vero E6 cells (European … SciScore for 10.1101/2022.03.22.485299: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Briefly, HEK293T cells (CRL-3216; ATCC, Manassas, VA, USA) were seeded onto 96-well plates at a density of 5×104 cells per well in 100 μL complete medium (DMEM supplemented with 10% fetal bovine serum). HEK293Tsuggested: NoneVero-E6 cells (African Green Monkey renal epithelial cells; ATCC cat. no. CRL-1586) engineered to overexpress hACE2/Furin were seeded in 24-well plates to obtain a confluence of 80%. Vero-E6suggested: NoneBriefly, Vero E6 cells (European Collection of Authenticated Cell Cultures) were seeded onto 96-well plates at a density of 3×104 cells per well in 100 μL cell culture media that consisted of DMEM (Lonza, Basel, Switzerland), 1% penicillin–streptomycin (Lonza, Basel, Switzerland) and 2% heat-inactivated fetal bovine serum (Gibco, Waltham, MA, USA) the day before the experiment. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Cell fluorescence was detected using an EVOS FL microscope (Life Technologies, Carlsbad, CA, USA) and was quantified in ImageJ (US National Institutes of Health, Bethesda, MD, USA) [26] using the Analyze Particles tool after thresholding for the corresponding colors. ImageJsuggested: (ImageJ, RRID:SCR_003070)IC50 values were determined using non-linear regression analysis (four-parameter logistic model; GraphPad Prism 8). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)As before [12, 23], binding data were converted to percent inhibition and fitted with standard log inhibitor vs. normalized response models [30] using nonlinear regression in GraphPad Prism (GraphPad, La Jolla, CA, USA) to establish half-maximal inhibitory concentrations (IC50). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04619290 Recruiting Outpatient Treatment With CoVid-19 With Prexablu NCT04635605 Recruiting Methylene Blue Treatment of COVID-19 NCT04370288 Recruiting Clinical Application of Methylene Blue for Treatment of Covi… Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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