Alkaline ceramidase 2 (ACER2) catalyzes ceramide hydrolysis to sphingosine and free fatty acids and has roles in cell proliferation, survival, and the DNA damage response. The study identifies N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) as a potent and selective ACER2 inhibitor. In vitro enzymatic assays, kinetic characterization, structural modeling, molecular docking, cellular cytotoxicity assays, and lipidomic profiling were used to define KPB82 activity and biological effects. These results are reported in a preprint and have not been peer reviewed.
ACER2 hydrolyzes ceramides to generate sphingosine and free fatty acids, biochemical conversions that influence multiple cellular processes. The enzyme has been implicated in regulation of cell proliferation, cell survival, and responses to DNA damage. Because pharmacological modulators of ACER2 were lacking, the authors sought a small-molecule tool to interrogate ACER2 catalytic and signaling functions.
The compound N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide, termed KPB82, was identified and tested in enzymatic assays against recombinant ACER2. KPB82 inhibited ACER2 with an IC50 of 190 nM, indicating submicromolar potency in vitro. The authors present this value as evidence that KPB82 is a potent biochemical inhibitor of ACER2.
Selectivity testing showed that KPB82 displayed minimal activity against acid and neutral ceramidases, with reported IC50 values greater than 100 µM for those enzymes. Additionally, KPB82 produced no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3 in the assays described. These data indicate a wide selectivity window favoring inhibition of ACER2 over other ceramidase family members.
Enzymatic kinetic studies indicated that KPB82 behaves as a competitive inhibitor of ACER2, consistent with binding at or near the substrate-binding site.
To support a structural basis for inhibition, the three-dimensional structure of ACER2 was modeled using the AlphaFold3 server. The predicted structure revealed Zn2+-coordinating histidine residues and a substrate-binding pocket suitable for docking studies. Molecular docking placed KPB82 inside this pocket, where the compound was predicted to form hydrogen bonds with catalytically important residues Asp93 and His215, in addition to hydrophobic interactions with nearby residues. These modeling results provide a plausible molecular explanation for competitive inhibition, though the work relies on in silico modeling rather than experimentally solved ACER2 structures.
Cell-based assays assessed whether biochemical inhibition of ACER2 by KPB82 translates to altered cellular responses to DNA damage. Lactate dehydrogenase (LDH) release assays were used as a measure of cytotoxicity. KPB82 significantly reduced LDH release (i.e., reduced cytotoxicity) in multiple contexts:
These cellular results indicate that KPB82 can mitigate ACER2-dependent cytotoxic responses and reduce cell death induced by a DNA-damaging agent across both tumor-derived cell lines and primary endothelial cells.
Lipidomic analysis reported that KPB82 inhibited sphingosine production driven by either ACER2 overexpression or by doxorubicin treatment. Because ACER2 enzymatic activity converts ceramide to sphingosine, reduced sphingosine levels after KPB82 treatment are consistent with on-target biochemical inhibition in cells and provide mechanistic linkage between enzyme inhibition and the observed protection from cytotoxicity.
The combined biochemical, structural modeling, cellular, and lipidomic data support KPB82 as a potent and selective tool compound for probing ACER2 biology and ACER2-mediated signaling pathways. KPB82’s selectivity versus other ceramidases and its ability to reduce doxorubicin-induced cytotoxicity in multiple cell types highlight potential utility for experimental modulation of ceramide metabolism.
Limitations noted by the source: the structural evidence is based on an AlphaFold3 model and molecular docking rather than experimentally determined ACER2–inhibitor co-crystal structures. The report is a preprint and has not undergone peer review; additional validation, including in vivo studies, pharmacokinetics, off-target profiling beyond the ceramidase family, and structural confirmation, were not reported in the source.
KPB82 is presented as a selective, competitive inhibitor of ACER2 (IC50 190 nM) that reduces sphingosine production and protects tumor and primary cells from doxorubicin-induced cytotoxicity in vitro. The molecule provides a pharmacological starting point to study ACER2 function and ceramide metabolism; further validation and peer review are required to confirm these findings and define translational potential.