Flubendazole

Adult female B. malayi 12–24 weeks of age were isolated from susceptible
IL-4Rα^–/–IL-5^–/– immunodeficient mice, washed in PBS, and added to lymphatic endothelial
cell cocultures (HMVECdly; LEC; Lonza) at a density of two parasites
per well. Successful test compounds from the mf assay were diluted
to 10 μM and added to the trans-wells in 6 mL endothelial basal
media with supplements (EGM-2 MV; Lonza). Twelve replicates (n = 6 wells) were set up per group with flubendazole (10
μM; Sigma) and DMSO (0.5% v/v) added as controls. Plates were
incubated for 14 days at 37 °C, 5% CO₂ with daily
motility scoring, as above. Individual parasites were taken for MTT
analysis at day 14.

The Screen-Well FDA-approved drug library was purchased from Enzo Life Sciences (catalogue no. BML-2841; Farmingdale, NY, USA). The 640 compounds in this library were provided at 2 mg/mL in dimethyl sulfoxide (DMSO). Flubendazole (FLB), mebendazole (MEB), benomyl (BEN), nisoldipine (NIS), nifedipine (NIF), felodipine (FEL), niguldipine (NIG), amphotericin B (AMB), itraconazole (ITZ), voriconazole (VRZ) and 5-flucytosine (5FC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fluconazole (FLC) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of amphotericin B, itraconazole and voriconazole were prepared at 1.6 mg/mL in water; 5-flucytosine was made at 6.4 mg/mL in water; fluconazole was made at 12.8 mg/mL in water and all remaining drugs were dissolved in DMSO at 2 mg/mL. All compounds were stored at −80 °C.

Praziquantel (PZQ), 4-phenyl-1,2,5-oxadiazole-3-carbonile-2-oxide (OXA), N-acetylcysteine (NAC), flavona (Flav), flubendazole (FBZ), and propidium iodide (PI) were purchased from Sigma-Aldrich (Lisboa, Portugal), and resveratrol (Resv) from Santa Cruz Biotechnology (Heidelberg, Germany), artesunate (AS), vandetanib (VDT), curcumin (Curc), kaempferol (kaempf), and melatonin (Mel) from Cayman Chemical (Ann Arbor, MI, USA) and the dipeptide H-L-tryptophan-L-serine-OH (H-Trp-Ser-OH, DiPept) from Bachem (Bubendorf, Switzerland). The culture media M199, Hank’s Balanced Salt Solution (HBSS) and supplements as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (1M), penicillin (10,000 U/mL)/streptomycin (10 mg/mL), and amphotericin B were purchases from Sigma-Aldrich and heat inactivated fetal bovine serum (iFBS) from Lonza (Basel, Switzerland). For in vitro assays, stock solutions of (2–4 mg/mL) were freshly prepared in 100% dimethylsulfoxide (DMSO) (Sigma-Aldrich) and stored at 4 °C.