Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties

This work was supported by National Institute of Mental Health grants: P30 MH075673-06 to BSS, CR, NJH and JCM; R01 MH096636 and R01 MH077542 to NJH (http://projectreporter.nih.gov/reporter.​cfm); and The Johns Hopkins Brain Science Institute to BSS.Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (Ki = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.Publisher PDFPeer reviewe

Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (Ki = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.</p

Characterization of fluorescence based activity assay for human nSMase2.

<p>Plots show the dependence of enzymatic activity with respect to <b>(A)</b> protein concentration in the presence of 250 μM SM for 1 h; <b>(B)</b> substrate concentration for 0.6 μg of protein from human nSMase2-containing cell lysate for 1 h; and <b>(C)</b> time in the presence of 0.6 μg of protein from nSMase2 expressing cell lysate and 250 μM SM. Data for activity <i>vs</i>. substrate concentration were fitted by non-linear least squares fitting to the Michaelis-Menten equation to determine <i>K</i>_m and <i>V</i>_max.</p

Effect of cambinol on TNF-α-induced changes in ceramide profiles of rat primary neurons.

<p><b>(A)</b> 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>(B)</b> Quantitative analysis representation of ceramide levels for the four treatments shown in (A).</p

Effect of cambinol on TNF-α-induced decrease in dendrite length of rat primary hippocampal neurons.

<p>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.</p

Inhibition of human nSMase2 by cambinol and closely related analogs.

<p><b>(A)</b> Cambinol (compound 1) structure. <b>(B)</b> Dose response curve for inhibition of human nSMase2 by increasing concentrations of cambinol ([¹⁴C]-SM assay). <b>(C)</b> Non-linear least squares fit of the Michaelis-Menten equation for the activity of human nSMase2 <i>vs</i>. SM concentration in the presence of different concentrations of cambinol. <i>K</i>_m and <i>V</i>_max values decrease with increasing concentrations of inhibitor. <b>(D)</b> Lineweaver-Burk plot for data shown in (C). <b>(E)</b> Dose response curve for inhibition of human nSMase2 by cambinol analog compound 2. <b>(F)</b> Dose response curve for inhibition of human nSMase2 by cambinol analog compound 6.</p