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Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

Purschke WG, Hoehlig K, Buchner K, Zboralski D, Schwoebel F, Vater A, Klussmann S - Biochem. J. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: *NOXXON Pharma AG, Berlin, Germany.

ABSTRACT
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.

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Inhibition of S1P signalling by NOX-S93 in cell-based assays(A) PathHunter™ eXpress growth-arrested EDG-1 CHO-K1 β-arrestin cells were stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. The activity of the reporter enzyme, β-galactosidase, was measured and plotted against the Spiegelmer® concentration as a percentage activity with the largest value set to 100%. In this representative experiment NOX-S93 inhibited the S1PR1 mediated effects of S1P with an IC50 of 10.3 nM (means±S.D. of triplicate assays). (B) Intracellular calcium signalling in stably transfected CHO cells expressing S1PR3 and Gα15 was stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. Increase in intracellular calcium was measured and plotted against the Spiegelmer® concentration as a percentage with the largest value set to 100%. NOX-S93 (●) inhibited the S1P-induced calcium increase with an IC50 of 5.45±0.89 nM (n=4). The graph is representative of the result of one experiment (means±S.D. of triplicate assays). The control Spiegelmer® revNOX-S93 (■) showed no inhibition (n=3).
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Figure 3: Inhibition of S1P signalling by NOX-S93 in cell-based assays(A) PathHunter™ eXpress growth-arrested EDG-1 CHO-K1 β-arrestin cells were stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. The activity of the reporter enzyme, β-galactosidase, was measured and plotted against the Spiegelmer® concentration as a percentage activity with the largest value set to 100%. In this representative experiment NOX-S93 inhibited the S1PR1 mediated effects of S1P with an IC50 of 10.3 nM (means±S.D. of triplicate assays). (B) Intracellular calcium signalling in stably transfected CHO cells expressing S1PR3 and Gα15 was stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. Increase in intracellular calcium was measured and plotted against the Spiegelmer® concentration as a percentage with the largest value set to 100%. NOX-S93 (●) inhibited the S1P-induced calcium increase with an IC50 of 5.45±0.89 nM (n=4). The graph is representative of the result of one experiment (means±S.D. of triplicate assays). The control Spiegelmer® revNOX-S93 (■) showed no inhibition (n=3).

Mentions: To confirm that high-affinity binding to S1P also translates into inhibition, the activity of Spiegelmer® NOX-S93 was tested in two cell-based assays employing two different cell lines expressing the human receptors S1PR1 and S1PR3. Signalling in both cell lines was induced by S1P in the same concentration range with EC50 values at 10 nM. As shown in Figure 3(A), NOX-S93 inhibited activation of S1PR1 with a low nanomolar IC50 (n=2). Calcium signalling in the S1PR3-expressing cell line was inhibited by NOX-S93 with an IC50 of 5.5 nM (n=4) (Figure 3B). The non-functional Spiegelmer® revNOX-S93 with the reverse sequence, tested in the S1PR3-based cell assay, showed no inhibitory effect and confirmed the specificity of the NOX-S93 effect (Figure 3B).


Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

Purschke WG, Hoehlig K, Buchner K, Zboralski D, Schwoebel F, Vater A, Klussmann S - Biochem. J. (2014)

Inhibition of S1P signalling by NOX-S93 in cell-based assays(A) PathHunter™ eXpress growth-arrested EDG-1 CHO-K1 β-arrestin cells were stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. The activity of the reporter enzyme, β-galactosidase, was measured and plotted against the Spiegelmer® concentration as a percentage activity with the largest value set to 100%. In this representative experiment NOX-S93 inhibited the S1PR1 mediated effects of S1P with an IC50 of 10.3 nM (means±S.D. of triplicate assays). (B) Intracellular calcium signalling in stably transfected CHO cells expressing S1PR3 and Gα15 was stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. Increase in intracellular calcium was measured and plotted against the Spiegelmer® concentration as a percentage with the largest value set to 100%. NOX-S93 (●) inhibited the S1P-induced calcium increase with an IC50 of 5.45±0.89 nM (n=4). The graph is representative of the result of one experiment (means±S.D. of triplicate assays). The control Spiegelmer® revNOX-S93 (■) showed no inhibition (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Inhibition of S1P signalling by NOX-S93 in cell-based assays(A) PathHunter™ eXpress growth-arrested EDG-1 CHO-K1 β-arrestin cells were stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. The activity of the reporter enzyme, β-galactosidase, was measured and plotted against the Spiegelmer® concentration as a percentage activity with the largest value set to 100%. In this representative experiment NOX-S93 inhibited the S1PR1 mediated effects of S1P with an IC50 of 10.3 nM (means±S.D. of triplicate assays). (B) Intracellular calcium signalling in stably transfected CHO cells expressing S1PR3 and Gα15 was stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. Increase in intracellular calcium was measured and plotted against the Spiegelmer® concentration as a percentage with the largest value set to 100%. NOX-S93 (●) inhibited the S1P-induced calcium increase with an IC50 of 5.45±0.89 nM (n=4). The graph is representative of the result of one experiment (means±S.D. of triplicate assays). The control Spiegelmer® revNOX-S93 (■) showed no inhibition (n=3).
Mentions: To confirm that high-affinity binding to S1P also translates into inhibition, the activity of Spiegelmer® NOX-S93 was tested in two cell-based assays employing two different cell lines expressing the human receptors S1PR1 and S1PR3. Signalling in both cell lines was induced by S1P in the same concentration range with EC50 values at 10 nM. As shown in Figure 3(A), NOX-S93 inhibited activation of S1PR1 with a low nanomolar IC50 (n=2). Calcium signalling in the S1PR3-expressing cell line was inhibited by NOX-S93 with an IC50 of 5.5 nM (n=4) (Figure 3B). The non-functional Spiegelmer® revNOX-S93 with the reverse sequence, tested in the S1PR3-based cell assay, showed no inhibitory effect and confirmed the specificity of the NOX-S93 effect (Figure 3B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: *NOXXON Pharma AG, Berlin, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus