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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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Comparison of equilibrium dissociation constants to S1P of the non-PEGylated Spiegelmers 215-F9–002 compared with 215-F9-002-4xD (NOX-S93001) and the 5′-PEGylated Spiegelmers NOX-S93 compared with revNOX-S93The affinities were determined in a competitive pull-down assay using labelled 215-F9–002 as a reference. (A) 215-F9–002 without modification (●) and with deoxyribonucleotide modifications (■) (215-F9-002-4xD=NOX-S93001). (B) NOX-S93 (◆) and the reverse sequence revNOX-S93 (○). The absence of any binding of revNOX-S93 confirmed the specificity of NOX-S93.
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Figure 2: Comparison of equilibrium dissociation constants to S1P of the non-PEGylated Spiegelmers 215-F9–002 compared with 215-F9-002-4xD (NOX-S93001) and the 5′-PEGylated Spiegelmers NOX-S93 compared with revNOX-S93The affinities were determined in a competitive pull-down assay using labelled 215-F9–002 as a reference. (A) 215-F9–002 without modification (●) and with deoxyribonucleotide modifications (■) (215-F9-002-4xD=NOX-S93001). (B) NOX-S93 (◆) and the reverse sequence revNOX-S93 (○). The absence of any binding of revNOX-S93 confirmed the specificity of NOX-S93.

Mentions: An in vitro selection process was started with a theoretical library size of more than 3×1015 different RNA sequences and concentrations of 10 μM of both library and target S1P [L-e-S1P(bio)]. A relatively high concentration of 4 mg/ml serum albumin was added to the selection buffer to ensure the solubility of S1P. During the first six rounds, no noticeable affinity of the library to the target over background was detectable. Only starting with round seven the ratio of bound RNA compared with the applied concentration of L-e-S1P(bio) slowly increased, indicating emerging affinity in the library (Supplementary Figure S1A at http://www.biochemj.org/bj/462/bj4620153add.htm). In order to select for the best binding aptamers, the stringency of the selection process was gradually increased by decreasing the concentration of both target and library to a final concentration of 40 pM and 2.5 nM respectively, in round 16. The affinity of the enriched library was determined by a competitive pull-down assay [Kd(comp)=88 nM; Supplementary Figure S1B). The bound RNA of round 16 was reverse transcribed, amplified by PCR, cloned and 47 clones were sequenced. The alignment of the clones revealed essentially one sequence with few point mutations (Supplementary Figure S1C). Truncation by omitting the primer binding sites and ranking resulted in the 46mer 215-F9-001. By deleting the terminal A/U base pair of 215-F9-001, a length of 44 nucleotides was defined as the minimal binder (215-F9-002; Supplementary Figure S1C) that displayed a dissociation constant of 53 nM (Supplementary Figure S1D). Further truncations were not possible without loss of affinity. The sequence 215-F9-002 was then synthesized in its L-configuration as a Spiegelmer®. In a competitive pull-down assay the dissociation constant [Kd(comp)] of Spiegelmer® 215-F9-002 to naturally occurring D-e-S1P was determined to be 27 nM (Figure 2A). Variants of 215-F9-002 in which the individual ribonucleotides at positions 1, 11, 19, 21 or 32 are exchanged for the corresponding deoxyribonucleotide revealed improved binding affinity (Figure 1A). The highest affinity increase resulted in a change of positions 21 [Kd(comp)=11 nM] or 19 [Kd(comp)=16 nM]. Combinations of several substitutions were additive and led to 215-F9-002-4xD with four exchanged ribonucleotides and a dissociation constant Kd(comp) of 5.7 nM (Figure 2A). This 44mer variant constitutes the final candidate sequence (Figure 1A; deoxyribonucleotide positions are shown shaded in grey) named NOX-S93001. A secondary structure prediction using the software mfold is depicted in Figure 1(B) [25]. According to this software the whole molecule seems to form a typical hairpin structure with several bulge regions. The respective five terminal nucleotides at each end are self-complementary and thus presumably form a helix.


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)

Comparison of equilibrium dissociation constants to S1P of the non-PEGylated Spiegelmers 215-F9–002 compared with 215-F9-002-4xD (NOX-S93001) and the 5′-PEGylated Spiegelmers NOX-S93 compared with revNOX-S93The affinities were determined in a competitive pull-down assay using labelled 215-F9–002 as a reference. (A) 215-F9–002 without modification (●) and with deoxyribonucleotide modifications (■) (215-F9-002-4xD=NOX-S93001). (B) NOX-S93 (◆) and the reverse sequence revNOX-S93 (○). The absence of any binding of revNOX-S93 confirmed the specificity of NOX-S93.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4109837&req=5

Figure 2: Comparison of equilibrium dissociation constants to S1P of the non-PEGylated Spiegelmers 215-F9–002 compared with 215-F9-002-4xD (NOX-S93001) and the 5′-PEGylated Spiegelmers NOX-S93 compared with revNOX-S93The affinities were determined in a competitive pull-down assay using labelled 215-F9–002 as a reference. (A) 215-F9–002 without modification (●) and with deoxyribonucleotide modifications (■) (215-F9-002-4xD=NOX-S93001). (B) NOX-S93 (◆) and the reverse sequence revNOX-S93 (○). The absence of any binding of revNOX-S93 confirmed the specificity of NOX-S93.
Mentions: An in vitro selection process was started with a theoretical library size of more than 3×1015 different RNA sequences and concentrations of 10 μM of both library and target S1P [L-e-S1P(bio)]. A relatively high concentration of 4 mg/ml serum albumin was added to the selection buffer to ensure the solubility of S1P. During the first six rounds, no noticeable affinity of the library to the target over background was detectable. Only starting with round seven the ratio of bound RNA compared with the applied concentration of L-e-S1P(bio) slowly increased, indicating emerging affinity in the library (Supplementary Figure S1A at http://www.biochemj.org/bj/462/bj4620153add.htm). In order to select for the best binding aptamers, the stringency of the selection process was gradually increased by decreasing the concentration of both target and library to a final concentration of 40 pM and 2.5 nM respectively, in round 16. The affinity of the enriched library was determined by a competitive pull-down assay [Kd(comp)=88 nM; Supplementary Figure S1B). The bound RNA of round 16 was reverse transcribed, amplified by PCR, cloned and 47 clones were sequenced. The alignment of the clones revealed essentially one sequence with few point mutations (Supplementary Figure S1C). Truncation by omitting the primer binding sites and ranking resulted in the 46mer 215-F9-001. By deleting the terminal A/U base pair of 215-F9-001, a length of 44 nucleotides was defined as the minimal binder (215-F9-002; Supplementary Figure S1C) that displayed a dissociation constant of 53 nM (Supplementary Figure S1D). Further truncations were not possible without loss of affinity. The sequence 215-F9-002 was then synthesized in its L-configuration as a Spiegelmer®. In a competitive pull-down assay the dissociation constant [Kd(comp)] of Spiegelmer® 215-F9-002 to naturally occurring D-e-S1P was determined to be 27 nM (Figure 2A). Variants of 215-F9-002 in which the individual ribonucleotides at positions 1, 11, 19, 21 or 32 are exchanged for the corresponding deoxyribonucleotide revealed improved binding affinity (Figure 1A). The highest affinity increase resulted in a change of positions 21 [Kd(comp)=11 nM] or 19 [Kd(comp)=16 nM]. Combinations of several substitutions were additive and led to 215-F9-002-4xD with four exchanged ribonucleotides and a dissociation constant Kd(comp) of 5.7 nM (Figure 2A). This 44mer variant constitutes the final candidate sequence (Figure 1A; deoxyribonucleotide positions are shown shaded in grey) named NOX-S93001. A secondary structure prediction using the software mfold is depicted in Figure 1(B) [25]. According to this software the whole molecule seems to form a typical hairpin structure with several bulge regions. The respective five terminal nucleotides at each end are self-complementary and thus presumably form a helix.

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