Fenbendazole

The following small-molecule drugs showed high-affinity constants against HELZ2, and were purchased from Sigma-Aldrich Co, Japan; amifostine trihydrate, brompheniramine maleatem, clemastine fumarate, fenbendazole, fulvestrant, Ga, loperamide hydrochloride, pirenperone, ritodrine hydrochloride, rivastigmine tartrate, salbutamol, sibutramine hydrochloride, sulbactam, voriconazole, and zoxazolamine.

Individual chemicals, i.e., albendazole, alexidine, azelastine, bosutinib, carmofur, cladribine, digoxigenin, dronedarone, emetine, miconazole, pentamidine, pitavastatin, sertraline, solifenacin, sulcanozole, tadalafil, terfenadine, thioridazine, trifluorothymidine, and toremifene were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Additionally, 10-deacetylbaccatin, allyl-thiourea, aminoacridine, bazedoxifene, cephalomannine, cetrimonium bromide, ciclopirox, clomifene, cytarabine, fenbendazole, ouabain, oxethazaine, penfluridol, pyrithione zinc, tioconazole, thioguanine, and vinorelbine were purchased from Sigma-Aldrich. Flubendazole was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA), and gemcitabine and niclosamide were purchased from AdipoGen^® Life Science (Liestal, Switzerland), whereas thonzonium bromide and pralatrexate were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Peripheral blood mononuclear cells [PBMC] were isolated from buffy coats obtained from healthy blood bank donors, using Ficoll density gradient centrifugation. PBMC of two individual donors were cultured in a 1:1 ratio for 48 h. Subsequently, anti-TNF [infliximab] or control IgG [Sigma Aldrich, Zwijndrecht, The Netherlands] was added, to a final concentration of 10 µg/ml. Where appropriate, additional compounds were added at the concentrations indicated. Albendazole [#A4673], mebendazole [#46404], docetaxel [#01885], paclitaxel [#T7402], budesonide [#PHR1178], fenbendazole [#35032], pyrythione zinc [#H6377], ciclopirox olamine [#C0415], pyrimethamine [#46706], quinacrine [#Q3251], mycophenolic acid [#M3536], adapalene [#A7486], amitryptiline [#A8404], duloxetine [#SML0474], triamterene [#T4143], and colchicine [#C9754] were all obtained from Sigma Aldrich. AMPK activator A769662 was obtained from SelleckChem [#S2697, Munich, Germany] and Compound C from Calbiochem [#171260–1, San Diego, CA]. Cultures were incubated for another 4–5 days and analysed by flow cytometry.
For analysis of effects on isolated macrophages, mixed lymphocyte reactions (MLR) were cultured for 48 h. CD14-expressing cells were isolated using magnetic cell sorting [CD14-microbeads, Miltenyi, Bergisch Gladbach, Germany] and cultured for an additional 72 h in the presence of anti-TNF and/or Albendazole.

Mebendazole (MBZ), albendazole (ABZ), fenbendazole (FBZ), lipopolysaccharide (LPS), interferon gamma (IFNγ), IL4, IL13 and phorbol myristate acetate (PMA) were purchased from Sigma (St Louis, MO, USA). The ERK/MEK/Raf inhibitors used were purchased from Selleckchem (Houston, TX, USA). The compounds were kept as 10 mM stock solutions in dimethyl sulphoxide (DMSO, Sigma) or sterile water, and further diluted with culture medium (Sigma or ATCC) as needed.

Fenbendazole, ALB and LEV were purchased from Sigma-Aldrich (Schnelldorf, Germany), and stock solutions of the drugs (100.000 µM) were prepared in 100% dimethylsulfoxid (DMSO) (Sigma-Aldrich, Schnelldorf, Germany) and stored at 5°C until use within 1 week.