Kinase experiment:

Pure tubulin is obtained from sheep brain by 2 cycles of assembly and disassembly in vitro. Immediately prior to use the protein is centrifuged at 130000 g for 30 min to remove any aggregates. It is used at a protein concentration of 0-2 mg/mL in 0.025 M Pipes buffer, 0-5 mM EGTA, 0-25 mM Mg2SOsup>4, 0.1 mM GTP. Drug binding is determined by equilibrium dialysis using concentrations of parbendazole between 0.1 μM and 4 μM, and 2% (v/v) DMF (dimethyl formamide) as a carrier. Equilibrium is achieved by constant stirring for 2 h at 26℃, bovine serum albumin being used as a standard. 200 μL aliquots are counted in PCS in a 25-200B liquid scintillation counter[2].

Cell experiment:

Vero cells, an established cell line derived from monkey kidney are seeded in DMEM supplemented with 10% (v/v) foetal calf serum onto glass coverslips in multiwell dishes. They are allowed to settle, and spread for 2-5 h in a humid atmosphere at 37℃. After this time the medium is changed to DMEM containing 2, 10 or 20 μM parbendazole and 1% (v/v) DMSO controls contained 1 % (v/v) DMSO or had no additions[2].

Parbendazole is a potent inhibitor of microtubule assembly, destabilizes tubulin, with an EC50 of 8.79 nM, and exhibits a broad-spectrum anthelmintic activity.

Parbendazole is a tubulin destabilizer, with an EC50 of 8.79 nM, and can induce DNA damage[1]. Parbendazole (2-10 μM) inhibits the assembly of microtubules dose-dependently, with an IC50 of 3 μM. Parbendazole (2-20 μM)-treated cells show an complete absence of microtubules in Vero cells[2]. Parbendazole (up to 10 μM) inhibits the growth of CLd-AXE myxamoebae. Parbendazole (2-5 μM) potently inhibits tubulin purified from the wild-type myxamoebae[3].

[1]. Lo YC, et al. Computational Cell Cycle Profiling of Cancer Cells for Prioritizing FDA-Approved Drugs with Repurposing Potential. Sci Rep. 2017 Sep 12;7(1):11261. [2]. Lo YC, et al. Computational Cell Cycle Profiling of Cancer Cells for Prioritizing FDA-Approved Drugs with Repurposing Potential. Sci Rep. 2017 Sep 12;7(1):11261. [3]. Havercroft JC, et al. Binding of parbendazole to tubulin and its influence on microtubules in tissue-culture cells as revealed by immunofluorescence microscopy. J Cell Sci. 1981 Jun;49:195-204. [4]. Havercroft JC, et al. Binding of parbendazole to tubulin and its influence on microtubules in tissue-culture cells as revealed by immunofluorescence microscopy. J Cell Sci. 1981 Jun;49:195-204. [5]. Foster KE, et al. A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro. Eur J Biochem. 1987 Mar 16;163(3):449-55. [6]. Foster KE, et al. A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro. Eur J Biochem. 1987 Mar 16;163(3):449-55.