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

Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2-25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.

Activity-Based Imaging of Acid Ceramidase in Living Cells.

Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

Discovery of deoxyceramide analogs as highly selective ACER3 inhibitors in live cells.

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.