Journal: Nature Communications

Article Title: A therapeutic approach to pantothenate kinase associated neurodegeneration

doi: 10.1038/s41467-018-06703-2

Figure Lengend Snippet: Activation of PanK by PZ-2891. a Kinetic model illustrating how PZ-2891 binding activates PANK3. PANK3 is a highly-cooperative enzyme where both active sites exist in either the active or inactive conformation. The inactive ‘open’ conformation (red rectangles) is stabilized by the binding of acetyl-CoA, and the active ‘closed’ conformation (green or curved shape) is stabilized by ATP binding. In the normal catalytic cycle, PANK3 returns to a ligand-free state that allows it to bind acetyl-CoA and switch to the inactive conformation. PZ-2891 binds tightly to the active, ATP-bound PANK3 conformation. When a fraction of PANK3 is occupied by PZ-2891, the catalytic cycle is operating on only one protomer of the dimer. In this catalytic cycle, the active monomer of PANK3 empties, but remains locked in the active conformation by ATP•Mg 2+ •PZ-2891 binding to the other protomer. The drug-induced catalytic cycle prevents PANK3 from adopting the open, inactive conformation making PANK3 refractory to feedback regulation by acetyl-CoA. Pan pantothenate, P-Pan phosphopantothenate. b PZ-2891 renders PANK3 refractory to acetyl-CoA inhibition. PANK3 (1 μg/assay) activity at different concentrations of acetyl-CoA was determined with ATP (1 mM) in the presence (open red circles) and absence (filled circles) of 2.5 μM PZ-2891. c The time course assays contained PANK3 (1 μg/assay), ATP (1 mM) and acetyl-CoA (100 μM) such that PANK3 was inhibited about 95% by acetyl-CoA in the absence of pantazine. The addition of 2.5 μM PZ-2891 to the assay increased the PANK3 rate in a pantothenate-dependent manner. Filled black circles are 45 μM pantothenate, filled blue squares are 90 μM pantothenate, red open circles are 45 μM pantothenate plus 2.5 μM PZ-2891, and green open squares are 90 μM pantothenate plus 2.5 μM PZ-2891. Data in b and c are from two independent experiments that were performed in duplicate. d C3A cells were radiolabeled with [ 3 H]pantothenate in the presence (red trace) or absence (black trace) of 10 μM PZ-2891 for 24 h. The cells were extracted, and the labeled metabolites were analyzed by thin-layer chromatography and imaged with a Bioscan detector. Representative chromatograms from two independent experiments in duplicate are shown

Article Snippet: The distribution of radioactivity on the plate was quantified using a Bioscan Imager (Bioscan, Inc.) together with quantification of the total cellular radioactivity determined by a Liquid Scintillation Analyzer (PerkinElmer Tri-Carb 2910 TR).

Techniques: Activation Assay, Binding Assay, Inhibition, Activity Assay, Labeling, Thin Layer Chromatography