{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T15:51:56Z","timestamp":1791474716207,"version":"4.3.4"},"posted":{"date-parts":[[2026,10,2]]},"group-title":"Biochemistry","reference-count":36,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T00:00:00Z","timestamp":1790899200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R01CA273263"],"award-info":[{"award-number":["R01CA273263"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P01CA097132"],"award-info":[{"award-number":["P01CA097132"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,10,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor.\n                  <jats:italic>In vitro<\/jats:italic>\n                  enzymatic assays showed that KPB82 inhibited ACER2 with an IC\n                  <jats:sub>50<\/jats:sub>\n                  of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC\n                  <jats:sub>50<\/jats:sub>\n                  &gt; 100 \u00b5M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn\u00b2\u207a-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.\n                <\/jats:p>","DOI":"10.64898\/2026.09.29.754712","type":"posted-content","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T18:40:19Z","timestamp":1790966419000},"source":"Crossref","is-referenced-by-count":0,"title":["KPB82 potently and selectively inhibits ACER2 and protects tumor and primary cells from doxorubicin-induced cytotoxicity"],"prefix":"10.64898","author":[{"given":"Arpita","family":"Devi","sequence":"first","affiliation":[{"name":"Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York 11794, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruijuan","family":"Xu","sequence":"additional","affiliation":[{"name":"Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York 11794, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zdzislaw","family":"Szulc","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ian","family":"Kim","sequence":"additional","affiliation":[{"name":"Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York 11794, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed","family":"Salama","sequence":"additional","affiliation":[{"name":"Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York 11794, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Krishna P.","family":"Bhabak","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Guwahati, Guwahati, 781039 Assam, 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