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. Author manuscript; available in PMC: 2018 May 31.
Published in final edited form as: J Am Chem Soc. 2017 May 19;139(21):7264–7272. doi: 10.1021/jacs.7b01084

Figure 4. Copper complexes protect human iPSC-derived neuronal cells from BoNT/A-induced SNAP-25 cleavage.

Figure 4

(A) Structures and names of active copper ligands and complexes in BoNT/A cellular assay. (B) Dose-responsive anti-BoNT/A activity of Cu(PBT2)2. +/− Tx C = Control lane with or without toxin. HiPSC-derived neurons were exposed to 200 U/well of BoNT/A1 and were treated at 1.5 post-toxin exposure with 2:1 PBT2/CuCl2 (preformed in DMSO at 100X concentration). Cells were harvested 7.5 h post-toxin and cell lysates were analyzed by Western blot for SNAP-25 cleavage. Morphology changes were graded on a scale of 0 (−) through 3 (+++) to indicate severity of cell rounding. Percent cytotoxicity was evaluated by LDH release from cells relative to a maximum LDH release control. Experiments were run in duplicate with one representative gel shown in the figure. (C) Densiometric representation of panel B gel for EC50 determination of Cu(PBT2)2. (D) Reduction of copper complex releases copper to inhibit BoNT/A LC. Sodium ascorbate (ASC) was added to BoNT/A LC in the presence of DCOQ-Cu complex and enzyme activity was measured by SNAPtide asssay. [DCOQ ligand] = 20 μM, [CuCl2] = 10 μM. ASC alone had no effect on activity while Cu alone gave <1% activity. For panels C/D, data represent mean ± SEM of two replicates.