Fig 1.
Fluorescence and radioactivity assays for human nSMase2.
(A) nSMase2-catalyzed reaction in the direct confirmatory assay with 14C-labeled SM (labeled atoms shown in magenta), which is hydrolyzed to generate labeled product. (B) Fluorescence assay for human nSMase2 consisting of three coupled reactions (in blue) and Amplex red dye to generate a red fluorescent product, resorufin (in red).
Fig 2.
Table showing the most well-known nSMase inhibitors, their potency, the species of nSMase in which the compounds were first identified and their physicochemical properties.
Fig 3.
Expression and characterization of recombinant human nSMase2.
(A) Western blot showing expression of recombinant human nSMase2-Flag tagged construct in stably transfected HEK293 cells. Positive control corresponds to GST-tagged human nSMase2 construct commercially available. (B) Human nSMase2 activity dependence with respect to the presence of EDTA and DMSO. (C) Modulation of enzymatic activity by known nSMase inhibitors (GW4869 and altenusin) and by a prototype aSMase specific inhibitor (zoledronic acid). Concentration of GW4869 is a nominal concentration based on dilutions from the stock solution; due to the low aqueous solubility of GW4869, the actual concentration is likely lower than 150 μM.
Fig 4.
Characterization of fluorescence based activity assay for human nSMase2.
Plots show the dependence of enzymatic activity with respect to (A) protein concentration in the presence of 250 μM SM for 1 h; (B) substrate concentration for 0.6 μg of protein from human nSMase2-containing cell lysate for 1 h; and (C) time in the presence of 0.6 μg of protein from nSMase2 expressing cell lysate and 250 μM SM. Data for activity vs. substrate concentration were fitted by non-linear least squares fitting to the Michaelis-Menten equation to determine Km and Vmax.
Fig 5.
Inhibition of human nSMase2 by cambinol and closely related analogs.
(A) Cambinol (compound 1) structure. (B) Dose response curve for inhibition of human nSMase2 by increasing concentrations of cambinol ([14C]-SM assay). (C) Non-linear least squares fit of the Michaelis-Menten equation for the activity of human nSMase2 vs. SM concentration in the presence of different concentrations of cambinol. Km and Vmax values decrease with increasing concentrations of inhibitor. (D) Lineweaver-Burk plot for data shown in (C). (E) Dose response curve for inhibition of human nSMase2 by cambinol analog compound 2. (F) Dose response curve for inhibition of human nSMase2 by cambinol analog compound 6.
Fig 6.
Structure-activity relationship study for cambinol and human nSMase2.
Table illustrating the IC50 values for various analogs of cambinol in the direct radioactive assay using 14C-labeled substrate.
Fig 7.
Effect of cambinol on TNF-α-induced changes in ceramide profiles of rat primary neurons.
(A) Heat-map represents ceramide profiles of rat primary neurons after treatment with TNF-α, cambinol or cambinol + TNF-α. Columns represent control, 100 ng/ml TNF-α for 2 min, 10 μM cambinol and cambinol + TNF-α. Drug was added to cells 15 min prior to TNF-α stimulation. Heat maps were generated based on normalization of the ceramide values using z transformations. Color scale illustrates quantitation, red color indicates increase of ceramide abundance and green color indicates depletion with respect to control treatment. (B) Quantitative analysis representation of ceramide levels for the four treatments shown in (A).
Fig 8.
Effect of cambinol on TNF-α or IL-1β-induced loss of viability in neurons.
(A) Effects of cambinol (0.1–25 μM), inactive analog compound 2 (10–25 μM) or zoledronic acid (10–25 μM) on the survival of rat primary neurons treated with TNF-α or IL-1β. (100 ng/ml for 18 h) (B) Effects of SIRT1/2 inhibitors CHIC-35 (200 nM) and sirtinol (40 μM) on TNF-α induced neuronal cell death. Compounds were added 15 min prior to cytokine. Error bars correspond to S.E.M. of at least 3 independent determinations. *** p < 0.001.
Fig 9.
Effect of cambinol on TNF-α-induced decrease in dendrite length of rat primary hippocampal neurons.
Cells were treated with cambinol (0.1–30 μM) and TNF-α (100 ng/ml) for 18 h. Dendrite length was assessed by imaging of MAP-2 stained cells using Neurolucida software. Results shown are the average of 5–6 experiments. Error bars correspond to S.E.M. * p < 0.05 and ** p < 0.01.