(2R,3R) 1-deoxyceramides provide a scaffold to obtain ACER3 inhibitors.
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ACER3 inhibition by (2R,3R) 1-deoxyceramides depend on the N-acyl moiety.
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ACER3 inhibitors are inactive on acid and neutral ceramidase (NC) in live cells.
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Recombinant NC is not appropriate to screen libraries for NC inhibitors.
Abstract
Acid (AC), neutral (NC) and alkaline ceramidase 3 (ACER3) are the most ubiquitous ceramidases and their therapeutic interest as targets in cancer diseases has been well sustained. This supports the importance of discovering potent and specific inhibitors for further use in combination therapies. Although several ceramidase inhibitors have been reported, most of them target AC and a few focus on NC. In contrast, well characterized ACER3 inhibitors are lacking. Here we report on the synthesis and screening of two series of 1-deoxy(dihydro)ceramide analogs on the three enzymes. Activity was determined using fluorogenic substrates in recombinant human NC (rhNC) and both lysates and intact cells enriched in each enzyme. None of the molecules elicited a remarkable AC inhibitory activity in either experimental setup, while using rhNC, several compounds of both series were active as non-competitive inhibitors with Ki values between 1 and 5 μM. However, a dramatic loss of potency occurred in NC-enriched cell lysates and no activity was elicited in intact cells. Interestingly, several compounds of Series 2 inhibited ACER3 dose-dependently in both cell lysates and intact cells with IC50’s around 20 μM. In agreement with their activity in live cells, they provoked a significant increase in the amounts of ceramides. Overall, this study identifies highly selective ACER3 activity blockers in intact cells, opening the door to further medicinal chemistry efforts aimed at developing more potent and specific compounds.
