PMID- 32734807
OWN - NLM
STAT- MEDLINE
DCOM- 20220217
LR  - 20220830
IS  - 2472-5560 (Electronic)
IS  - 2472-5552 (Print)
IS  - 2472-5552 (Linking)
VI  - 26
IP  - 1
DP  - 2021 Jan
TI  - Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via 
      Target-Based High-Throughput Screening.
PG  - 113-121
LID - 10.1177/2472555220945283 [doi]
AB  - There is interest in developing inhibitors of human neutral ceramidase (nCDase) 
      because this enzyme plays a critical role in colon cancer. There are currently no 
      potent or clinically effective inhibitors for nCDase reported to date, so we 
      adapted a fluorescence-based enzyme activity method to a high-throughput 
      screening format. We opted to use an assay whereby nCDase hydrolyzes the 
      substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases 
      umbelliferone, resulting in a fluorescent signal. As designed, test compounds 
      that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, 
      thereby decreasing fluorescence. This assay uses a 1536-well plate format with 
      excitation in the blue spectrum of light energy, which could be a liability, so 
      we incorporated a counterscreen that allows for rapid selection against 
      fluorescence artifacts to minimize false-positive hits. The high-throughput 
      screen of >650,000 small molecules found several lead series of hits. Multiple 
      rounds of chemical optimization ensued with improved potency in terms of IC(50) 
      and selectivity over counterscreen assays. This study describes the first 
      large-scale high-throughput optical screening assay for nCDase inhibitors that 
      has resulted in leads that are now being pursued in crystal docking studies and 
      in vitro drug metabolism and pharmacokinetics (DMPK).
FAU - Otsuka, Yuka
AU  - Otsuka Y
AD  - Department of Molecular Medicine, Scripps Research, The Scripps Research 
      Molecular Screening Center, Jupiter, FL, USA.
FAU - Airola, Michael V
AU  - Airola MV
AD  - Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, 
      NY, USA.
FAU - Choi, Yong-Mi
AU  - Choi YM
AD  - Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, 
      NY, USA.
FAU - Coant, Nicolas
AU  - Coant N
AUID- ORCID: 0000-0001-9237-2132
AD  - Stony Brook University Cancer Center, Stony Brook, NY, USA.
FAU - Snider, Justin
AU  - Snider J
AD  - Stony Brook University Cancer Center, Stony Brook, NY, USA.
FAU - Cariello, Chris
AU  - Cariello C
AD  - Department of Pathology, Stony Brook Renaissance School of Medicine, Stony Brook, 
      NY, USA.
FAU - Saied, Essa M
AU  - Saied EM
AUID- ORCID: 0000-0002-7342-6200
AD  - Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.
FAU - Arenz, Christoph
AU  - Arenz C
AUID- ORCID: 0000-0001-7613-9437
AD  - Institute for Chemistry, Humboldt Universitat zu Berlin, Berlin, Germany.
FAU - Bannister, Thomas
AU  - Bannister T
AD  - Department of Molecular Medicine, Scripps Research, The Scripps Research 
      Molecular Screening Center, Jupiter, FL, USA.
FAU - Rahaim, Ron Jr
AU  - Rahaim R Jr
AD  - Department of Molecular Medicine, Scripps Research, The Scripps Research 
      Molecular Screening Center, Jupiter, FL, USA.
FAU - Hannun, Yusuf A
AU  - Hannun YA
AD  - Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, 
      NY, USA.
AD  - Stony Brook University Cancer Center, Stony Brook, NY, USA.
FAU - Shumate, Justin
AU  - Shumate J
AD  - Department of Molecular Medicine, Scripps Research, The Scripps Research 
      Molecular Screening Center, Jupiter, FL, USA.
FAU - Scampavia, Louis
AU  - Scampavia L
AD  - Department of Molecular Medicine, Scripps Research, The Scripps Research 
      Molecular Screening Center, Jupiter, FL, USA.
FAU - Haley, John D
AU  - Haley JD
AD  - Stony Brook University Cancer Center, Stony Brook, NY, USA.
AD  - Department of Pathology, Stony Brook Renaissance School of Medicine, Stony Brook, 
      NY, USA.
FAU - Spicer, Timothy P
AU  - Spicer TP
AUID- ORCID: 0000-0002-9080-4226
AD  - Department of Molecular Medicine, Scripps Research, The Scripps Research 
      Molecular Screening Center, Jupiter, FL, USA.
LA  - eng
GR  - R01 CA221948/CA/NCI NIH HHS/United States
GR  - S10 OD025279/OD/NIH HHS/United States
GR  - S10 OD026857/OD/NIH HHS/United States
GR  - S10 OD025282/OD/NIH HHS/United States
GR  - R35 GM118128/GM/NIGMS NIH HHS/United States
GR  - P01 CA097132/CA/NCI NIH HHS/United States
GR  - R35 GM128666/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20200731
PL  - United States
TA  - SLAS Discov
JT  - SLAS discovery : advancing life sciences R & D
JID - 101697563
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Small Molecule Libraries)
RN  - EC 3.5.1.23 (Neutral Ceramidase)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Dose-Response Relationship, Drug
MH  - Drug Discovery/*methods
MH  - Drug Screening Assays, Antitumor/methods
MH  - Enzyme Activation/drug effects
MH  - Enzyme Inhibitors/*chemistry/*pharmacology
MH  - *High-Throughput Screening Assays/methods
MH  - Humans
MH  - Neutral Ceramidase/*antagonists & inhibitors/*chemistry
MH  - Small Molecule Libraries
PMC - PMC7749003
MID - NIHMS1648683
OTO - NOTNLM
OT  - HTS
OT  - colon cancer
OT  - fluorescence
OT  - neutral ceramidase
OT  - pharmacological inhibitors
COIS- Declaration of Conflicting Interests The authors declared no potential conflicts 
      of interest with respect to the research, authorship, and/or publication of this 
      article.
EDAT- 2020/08/01 06:00
MHDA- 2022/02/19 06:00
PMCR- 2021/01/01
CRDT- 2020/08/01 06:00
PHST- 2020/08/01 06:00 [pubmed]
PHST- 2022/02/19 06:00 [medline]
PHST- 2020/08/01 06:00 [entrez]
PHST- 2021/01/01 00:00 [pmc-release]
AID - S2472-5552(22)06659-X [pii]
AID - 10.1177/2472555220945283 [doi]
PST - ppublish
SO  - SLAS Discov. 2021 Jan;26(1):113-121. doi: 10.1177/2472555220945283. Epub 2020 Jul 
      31.