Journal: ACS Chemical Neuroscience

Article Title: Ester Prodrug NLRP3 Inflammasome Inhibitor NT-0796 is Brain Active due to Activation by Local Expression of Carboxylesterase‑1

doi: 10.1021/acschemneuro.5c00843

Figure Lengend Snippet: CES1 immunodepletion diminishes NT-0796 metabolism to NDT-19795 in human brain homogenates. Human brain tissue was homogenized and subjected to centrifugation (20,000 g ) to generate a membrane pellet and supernatant fraction (A) or to high-speed centrifugation (80,000 g ) to generate a soluble fraction (B, C). (A) Supernatant and membrane fractions were incubated with 3 μM NT-0796 at 37 °C, and aliquots of the reaction mixtures were quenched at the indicated time points. CES1 supersomes and buffer-only controls were included as controls. The human brain supernatant fraction was also incubated with NT-0796 in the presence of 1 mM BNPP. Quenched samples were subsequently analyzed by LC-MS/MS; NDT-19795 levels are shown as a function of treatment time. (B) Human brain soluble fraction (1.2 mg) was incubated with 4 μg of anti-CES1 (or isotype control IgG), after which immune complexes were depleted by Protein G-agarose capture. Aliquots of the resulting postimmunoprecipitate supernatants (or untreated soluble fraction) were incubated with NT-0796 at 37 °C, and the formation of NDT-19795 was assessed by LC-MS/MS. NT-0796 incubated in buffer only was included as a control. (C) Western blot of Protein G-agarose captured immunoprecipitates following treatment of human soluble brain fraction with (lane 2) anti-CES1, (lane 3) isotype control IgG, or detergent-solubilized CES1 supersomes treated with (lane 6) anti-CES1 or (lane 7) isotype control IgG. Lanes 4 and 5 were blank. Lane 1 contains MW markers (size indicated on the left in kDa). The blot was probed with anti-CES1, followed by Dylight 800-conjugated goat-antirabbit secondary antibody.

Article Snippet: Finally, the nitrocellulose blots were probed with a goat antirabbit secondary antibody conjugated to Dylight 800 (Rockland, 611-145-002; diluted 1:10,000 in PBS) for 1 h, followed by multiple washes with PBS.

Techniques: Immunodepletion, Centrifugation, Membrane, Incubation, Liquid Chromatography with Mass Spectroscopy, Control, Western Blot