Pharmacological inhibition of COP9 by ZnDTC phenocopy CSN5 genetic disruption.

(A) Illustration of zinc-ditiocarb (ZnDTC) formation from disulfiram and zinc. Dose response curve showing amebicidal effect of ZnDTC and metronidazole after 48 hours of incubation at indicated doses. EC50 for ZnDTC (11.21 ± 6.39 nM) and metronidazole (10.26 ± 6.06 μM) was calculated from nonlinear robust regression fit of the dose response curves. (B) Target validation using viability assay of vector control, CSN5 knockdown and CSN5 overexpression parasites incubated with increasing doses of ZnDTC. CSN5 overexpression reduces sensitivity to ZnDTC while CSN5 knockdown increases sensitivity to ZnDTC. (C) Immunoblot analysis for neddylated cullin1 with anti-E. histolytica cullin1 and Nedd8 antibodies on ZnDTC treated cells. (D) Quantification of live cells accumulating GFP, in ZnDTC treated cells with representative confocal micrographs. (E) Fluorometric assay of GFP degradation. ZnDTC inhibits GFP degradation in a dose dependent manner. Data represent mean ± SD of quintuples from one experiment and are representative of three independent experiments. ***P < .001, two-tailed t test.