Hydroxychloroquine sulfate

Chloroquine diphosphate (C6628), hydroxychloroquine sulfate (90527), amodiaquine dihydrochloride dihydrate (A2799) and primaquine bisphosphate (160393) were purchased from Sigma-Aldrich and solubilized in distilled water. Quinine (22620), mefloquine hydrochloride (M2319) and pamaquine naphthoate (S509515) were purchased from Sigma-Aldrich and solubilized in dimethyl sulfoxide. G418 was purchased from MicroCombiChem. Mefloquine stereoisomers (+)-anti-mefloquine hydrochloride [(+)-1] and (-)-syn-mefloquine hydrochloride [(-)-2] were synthesized and purified as described [16 (link),17 (link)]. Mefloquine stereoisomers (-)-anti-mefloquine hydrochloride [(-)-1] and (+)-syn-mefloquine hydrochloride [(+)-2] were synthesized in an analogous manner (Fig 1). Briefly, compound 5 was prepared via coupling of 3 and 4. Using known synthetic procedures, 5 was converted to diol 6 by treatment with ADmix α, which was then converted to (+)-1 using a three-step sequence. (–)-2 was also generated from 6 in three steps, but using a different synthetic sequence. (–)-1 and (+)-2, the enantiomeric forms of (+)-1 and (–)-2, respectively, were prepared in an analogous way, but using Admix β.

Methylene blue (methylthioninium chloride; Proveblue^®) was provided by Provepharm SAS (Marseille, France). Hydroxychloroquine sulfate (Sigma Aldrich, St Quentin Fallavier, France) and remdesivir (Apollo Scientific, Manchester, UK) were used as comparators. Stock solutions of methylene blue and hydroxychloroquine were prepared in water and remdesivir in DMSO/water 10%. All the stock solutions were then diluted in Minimum Essential Media (MEM, Gibco, ThermoFischer, Waltham, MA, USA) in order to have 7 final concentrations ranging from 0.1 µM to 100 µM. Two clinically-isolated SARS-CoV-2 strains (IHUMI-3 and IHUMI-6), collected in hospitalized patients during the first COVID-19 outbreak in March 2020 in Marseille [29 (link)], were maintained in production in Vero E6 cells (American type culture collection ATCC^® CRL-1586™) in MEM with 4% of fetal bovine serum and 1% of glutamine (complete medium). Vero E6 cells are one of the most used cells for the culture of SARS-CoV-2 due to the presence of high expression of angiotensin converting enzyme 2 (ACE2) receptors, essential for SARS-CoV-2 cellular entry [30 (link),31 (link)]. Vero E6 cells were found to be relevant for antiviral drug screening models [31 (link),32 (link)].

Parental COS-7 cells and HEK293 cells were purchased from ATCC (Manassas, VA, USA). Hydroxychloroquine Sulfate and Chloroquine Disulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin, streptavidin agarose, protein G-agarose beads, and horseradish peroxidase-conjugated anti-mouse antibodies were obtained from Pierce Biotechnology (Rockford, IL, USA). We purchased [³H]-labeled estrone sulfate from PerkinElmer (Waltham, MA, USA). Mouse anti-myc antibody (9E10) was purchased from Roche (Indianapolis, IN, USA). Mouse anti-E-cadherin antibody was purchased from Abcam (Cambridge, MA, USA). Mouse anti-ubiquitin antibody, mouse anti-β-actin antibody and normal mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). A 20S proteasome assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Normal mouse lgG, mouse monoclonal anti-ubiquitin and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA).