Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.
The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.
*NOXXON Pharma AG, Berlin, Germany.
Werner G
WG
Purschke
*NOXXON Pharma AG, Berlin, Germany.
Kai
K
Hoehlig
*NOXXON Pharma AG, Berlin, Germany.
Klaus
K
Buchner
*NOXXON Pharma AG, Berlin, Germany.
Dirk
D
Zboralski
*NOXXON Pharma AG, Berlin, Germany.
Frank
F
Schwoebel
*NOXXON Pharma AG, Berlin, Germany.
Axel
A
Vater
*NOXXON Pharma AG, Berlin, Germany.
Sven
S
Klussmann
D020533:Angiogenesis Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D000818:Animals; D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003412:Cricetulus; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors; D008246:Lysophospholipids / Q000037:antagonists & inhibitors*; D009389:Neovascularization, Pathologic; D049349:Receptors, Lysosphingolipid / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors; D000071557:beta-Arrestins
D020533:Angiogenesis Inhibitors; D052157:Aptamers, Nucleotide; D019390:Arrestins; D008246:Lysophospholipids; C000591924:NOX-S93; D049349:Receptors, Lysosphingolipid; D042461:Vascular Endothelial Growth Factor A; D000071557:beta-Arrestins; D016222:Fibroblast Growth Factor 2; C060506:sphingosine 1-phosphate; D007334:Insulin-Like Growth Factor I; D013110:Sphingosine; D002118:Calcium