New fluorogenic probes for neutral and alkaline ceramidases

Abstract

New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.

A fluorogenic substrate for the detection of lipid amidases in intact cells

“…This compound was synthesized, but it was not hydrolyzed by AC, indicating that the C1OH function was required in RBM14-C12 for AC hydrolysis. Since the RBM14 vinylogs RBM15 were fluorogenic substrates of ceramidases ( 29 ), compound RBM1-151, a vinylog of DoxRBM15 ( Scheme 1 ), was anticipated as a putatively specific AC substrate. The double bond was located at the C5 position because β-elimination of aldehyde 22 to produce umbelliferone occurs with higher efficiency under milder conditions than from its isomer at C2 ( 36 ).…”

“…All in vitro assays were conducted in 96-well plates at a final volume of 100 μl/well using reported procedures for AC ( 29 ), NAAA ( 30 ), and FAAH ( 31 ). The specific enzyme sources and reaction conditions are summarized in supplemental Table S1 .…”

“…Enzyme sources were AC, lysates from A375/AC (20 μg/well); NC, rhNC (5 ng/well); ACER3, lysates of ASAH2 (−/−) MEFs (140 μg/well); ACER1 and ACER2, microsomes of HeLa T-Rex cells stably overexpressing mACER1 and hACER2 ( 32 ) (100 μg/well); FAAH, lysates (25 μg/well) or microsomes (50 μg/well) of LNCaP cells or ASAH2 (−/−) MEFs, respectively; NAAA, lysates of HEK293 cells transiently overexpressing human NAAA (5–10 μg/well). Cell lysates and microsomes were prepared as reported ( 29 ). Reaction buffers were AC and NAAA, 25 mM acetic/acetate buffer (pH 4.5); NC, 50 mM Hepes, 150 mM NaCl, 1% sodium cholate (pH 7.4); ACERs, 50 mM Hepes, 1 mM CaCl 2 (pH 9.0); FAAH, 50 mM Hepes, 1 mM EDTA, 0.1% BSA (pH 7.4).…”

High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase

“…Thus, to discard a selectivity issue, the activity of nine detected hits against neutral ceramidase was next explored at 50 µM. Activity over NC was tested using recombinant human NC (rhNC) and a specific NC substrate bearing a nervonic acid amide (RBM14-C24:1) 30 . Remarkably, none of the molecules elicited a significant inhibitory activity, thereby confirming the selective inhibition of AC over NC ( Supplemental Table 1 ).…”