A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.
The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 μM in the SPA that induced dose-dependent lymphopenia in mice.
1 Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
Mohammed A
MA
Kashem
Chunling
C
Wa
John P
JP
Wolak
Nicholas S
NS
Grafos
Kelli R
KR
Ryan
Mary L
ML
Sanville-Ross
Kylie E
KE
Fogarty
Irina V
IV
Rybina
Alycia
A
Shoultz
Teresa
T
Molinaro
Sudha N
SN
Desai
Anusha
A
Rajan
John D
JD
Huber
Richard M
RM
Nelson
D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D008231:Lymphopenia / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000378:metabolism; D012588:Scintillation Counting; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase)