Closantel

Protein sample buffer and bicinchoninic acid assay reagent were from Thermo Scientific (Rockford, IL). Closantel was from Sigma (St. Louis, MO) and WNK463 was from MedChemExpress (Princeton, NJ). STOCK1S-50699 and STOCK1S-14279 were from InterBioScreen (Chernogolovka, Russian Federation). Horseradish peroxidase (HRP)-conjugated anti-rabbit Ig was from Molecular Probes (Eugene, OR). RIPA buffer and enhanced chemiluminescence agent (ECL) reagent were from Pierce (Rockford, IL).

In vitro antibacterial activity was tested using the broth microdilution assay⁵³ . Experiments were carried out in triplicate using Müller-Hinton broth (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 10% FBS in 96-well plates (BD Biosciences) at a total assay volume of 100 μL. Antimicrobials used in the pilot study was selected from our previous HTS assay^{14 (link),21 (link),54} and tested against H. pylori. Anthelmentics (niclosamide, oxyclozanide, closantel, rafoxanide) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Two-fold serial dilutions were prepared between the concentration range 0.01–64 μg/mL. An initial bacterial inoculum was adjusted to OD₆₀₀ = 0.06 and incubated with test compounds at 37 °C for 3 days in humidified incubators under the 5% CO₂ atmosphere. OD₆₀₀ was measured and the lowest concentration of compound that inhibited bacterial growth was reported as the MIC⁵⁵ . The broth microdilution assay was used to demonstrate the stability of the niclosamide using MHB supplemented with 10% FBS. The pH was adjusted to acidic condition with 1 N HCl. The minimal bactericidal concentration (MBC) was determined by plating 5 μL of broth culture from the MIC assay onto Müller-Hinton agar (BD Biosciences) supplemented with 10% FBS. After 72 h, the lowest concentration at which colonies were not observed was reported as the MBC.

DIOA (Sigma, D129) was used at 50 μM, bumetanide (Sigma, B3023) at 100 μM, torin1 (Merck, 47599) at 50 nM, FCCP at 0.3 μM, and closantel (Sigma, 34093) at 30 μM. Rapamycin (J62473, Alfa Aesar, UK) was dissolved at 50 mg/ml in ethanol, then diluted 1:1000 in water. Rapamycin solutions were kept shielded from light using tin foil and changed weekly.