S1P2 (Sphingosine-1-Phosphate 2; EDG-5); human S1P2 (IC50 = 17 nM); rat S1P2 (IC50 = 22 nM)

JTE-013 lowers cell viability at 50-200 μM for 1-3 days[1].
JTE-013 prevents S1P-induced ERK activation and reverses S1P-induced Akt inhibition[1].
JTE-013 exhibits a 4.2% inhibition of S1P3 and, at concentrations up to 10 μM, does not agitate S1P1[1].

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AB1 versus JTE-013 on GB Cell Migration. [1]
A well described biologic consequence of S1P2 signal transduction is inhibition of cell migration (Lepley et al., 2005). To compare the efficiency of JTE-013 and AB1 as S1P2 antagonists, cell migration assays were performed. GB cell lines U118 and U87 were used since it is well known that S1P inhibits cell migration in S1P2 high-expressing U118 cells, whereas S1P induces cell migration in S1P2 low-expressing U87 cells (Lepley et al., 2005; Fig. 3, A and C). AB1 treatment was moderately more potent than JTE-013 in reversing S1P-mediated cell migration inhibition in U118 cells and enhancing S1P-stimulated cell migration in U87 cells via blocking S1P2 signaling (Fig. 3, B and D).

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AB1 versus JTE-013 on the Levels of Molecules Downstream of S1P2 in SK-N-AS Cells.[1]
S1P2 exerts diverse cellular functions by regulating different downstream effector molecules. Our prior studies as well as studies performed by others have shown that such molecules include intracellular signaling mediators (p-Akt, p-ERK), as well as growth and differentiation modulators such as CTGF (Sanchez et al., 2007; Li et al., 2008a). To further compare the efficiency of JTE-013 and AB1 in vitro, Western blot analysis was performed. Similar to JTE-013, AB1 was able to reverse S1P-induced Akt inhibition and inhibit S1P-induced ERK activation at concentrations between 100 nM and 1 μM (Fig. 4A). The quantitative real-time polymerase chain reaction further showed that AB1 was relatively more effective than JTE-013 at inhibiting S1P-induced CTGF mRNA expression (Fig. 4B).

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AB1 versus JTE-013 on Cell Viability in SK-N-AS Cells.[1]
To investigate the potential mechanisms of AB1’s tumor inhibitory effect, cell viability was assessed in SK-N-AS cells treated with JTE-013 or AB1. MTT assays showed that AB1 is less potent than JTE-013 in terms of reduced cell viability at concentrations higher than 50 μM in SK-N-AS cells, whereas it had similar potency at lower concentrations (Fig. 6), suggesting that the improved inhibitory effect elicited by AB1 is not caused by direct inhibition of cell survival on cancer cells.

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In vitro activity: JTE-013 reverses the inhibitory effects of S1P2 signaling on cell migration of vascular ECs and smooth muscle cells. It also regulates endothelial tight junctions and barrier function in vitro. Blockage of S1P2 signaling by JTE-013 significantly enhances the effects of S1P on the increase of TEER, an in vitro measurement of endothelial integrity, as well as the formation of TJs in senescent ECs Kinase Assay: JTE 013 is a novel potent and selective S1P2 (sphingosine-1-phosphate 2) antagonist with IC50 of 17.6 nM. It binds to the human and rat receptors with IC50 values of 17 and 22 nM, respectively, and with IC50 values >10 µM for human S1P1 and S1P3. It reverses the inhibitory effects of S1P2 signaling on cell migration of vascular ECs and smooth muscle cells. It also regulates endothelial tight junctions and barrier function in vitro. Blockage of S1P2 signaling by JTE-013 significantly enhances the effects of S1P on the increase of TEER, an in vitro measurement of endothelial integrity, as well as the formation of TJs in senescent ECs. Cell Assay: HUVECs preincubated with various concentrations of JTE-013 for 10 min are allowed to migrate for 4 h toward the lower chamber where the indicated concentrations of Sph-1-P are placed. The migrated cells on the lower side of the filter are fixed, stained, and counted.[2]

JTE-013 (gavage; 30 mg/kg daily for 14 days in a row) decreases tumor weight and size[1].

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AB1 versus JTE-013 on a SK-N-AS Cell–Based NB Xenograft Model. [1]
In prior study, JTE-013 was demonstrated to significantly inhibit the growth of NB xenografts (Li et al., 2011). Here it was found that AB1 was again somewhat more potent than JTE-013 in inhibiting the growth of NB xenografts by both tumor size (Fig. 5A) and tumor weight at 14 days after treatment (Fig. 5B). Taken together, the above data strongly suggest that AB1 may have enhanced in vivo antitumor activity compared with JTE-013.

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AB1 versus JTE-013 on CCL2 Expression and Tumor-Associated Macrophage Infiltration in NB Xenografts.[1]
To elucidate the mechanism of AB1’s enhanced antitumor effect, researchers quantified effects on the gene expression levels of several S1P2 downstream molecules in treated NB xenografts. CCL2 is one of these genes (Li et al., 2014). Expression of CCL2 has been shown to be positively correlated with tumor-associated macrophage (TAM) infiltration (Zhang et al., 2010). In our prior study, blockade of S1P2 signaling by JTE-013 not only inhibited the growth of NB xenografts (Li et al., 2011), but it also reduced CCL2 expression and the subsequent TAM infiltration (Li et al., 2014), suggesting that inhibition of CCL2 is beneficial to anticancer therapy. As expected, CCL2 expression tended to be decreased at both mRNA and protein levels in JTE-013–treated or AB1-treated NB xenografts (Fig. 7, A and B). Furthermore, immunohistochemical staining for the murine macrophage marker F4/80 showed that both JTE-013 and AB1 significantly inhibited the TAM infiltration. However, AB1 did not display any improved inhibitory effects (Fig. 7C), indicating that the enhanced antitumor effect of AB1 was not attributable to effects on CCL2 expression and subsequent TAM infiltration.

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AB1 versus JTE-013 on Tumor Fibrosis and Apoptosis in NB Xenografts.[1]
CTGF is a central mediator of fibrosis (Lipson et al., 2012). Interestingly, AB1 inhibited CTGF expression to a greater extent than JTE-013 at both the mRNA and protein levels (Fig. 8), suggesting that the improved antitumor effect of AB1 may be partially a result of its beneficial effects on tumor fibrosis. Histologic studies utilizing Ki67 staining did not find any significant difference in the number of Ki67-positive proliferating cells among the three groups (Supplemental Fig. 2). However, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining (Fig. 9A) and cleaved caspase-3 detection (Fig. 9B) showed that AB1 was more potent than JTE-013 at inducing tumor cell apoptosis on these NB xenografts. Taken together, our data suggest that the improved antitumor effect elicited by AB1 may be attributed to effects on tumor fibrosis and tumor apoptosis in NB xenografts.

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JTE-013 inhibition of S1P2 significantly inhibits microvascular permeability in an in vivo animal model. It modulates the responses of brain endothelium by inhibiting cerebrovascular permeability, the development of intracerebral heamorrhage, and neurovascular injury in an experimental model of stroke. JTE-013 reduced mast cell activation, airway infiltration, and the serum levels of histamine and several cytokines in vitro and in vivo studies. In a murine model, JTE-013 suppresses streptozotocin-induced blood glucose increases, pancreatic b cell apoptosis, and the incidence of diabetes. In a New Zealand obese diabetic mouse model under high-fat diet conditions, it protected pancreatic b cells. Treatment with JTE-013 also reduces plasma levels of IL-1b and IL-18 (endotoxin-induced inflammatory cytokines) in ApoE−/− mice and S1P2 gene deficiency reduces atherosclerosis. The compound offers a novel means of treating inflammatory disorders, such as, atherosclerosis and sepsis.[2]

Fluorescent Imaging Plate Reader Assay.[1]
The calcium flux assay on a FLIPRTETRA instrument [fluorescent imaging plate reader (FLIPR) assay] was performed by a CRO company to profile test compounds for dose-dependent agonist and antagonist activities on S1P1–5. Briefly, the agonist assay was conducted on a FLIPRTETRA instrument, in which the test compounds, vehicle controls, and the reference agonist S1P were added to the assay plate after a fluorescence baseline was established. A duration of 180 seconds was used to assess each compound’s ability to activate each S1PR. Upon completion of the agonist assay, the assay plate was removed from the FLIPRTETRA instrument and incubated at 25°C for 7 minutes. After that, the assay plate was placed back in the FLIPRTETRA instrument and the antagonist assay was initiated. Using EC80 potency values determined during the agonist assay, all preincubated sample compound wells were challenged with EC80 concentration of the reference agonist S1P after establishment of a fluorescence baseline. Another duration of 180 seconds was used to assess each compound’s ability to inhibit each S1PR. All assay plate data were subjected to appropriate baseline corrections. After baseline corrections were applied, maximum fluorescence values were exported and data were processed to calculate the percentage of activation (relative to Emax reference agonist S1P and vehicle control values) and the percentage of inhibition (relative to EC80 and vehicle control values). All dose-response curves were generated using GraphPad Prism software

Cell Line: SK-N-AS cells
Concentration: 50, 100, 150, 200 μM
Incubation Time: 1-3 days
Result: Reduced cell viability.

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Migration Assay. [1]
The migration assay was performed in a 96-well chemotaxis microchamber), as described previously (Li et al., 2009b). Briefly, a polycarbonate filter (8-µm pore size) was coated with 50 µg/ml fibronectin. S1P was diluted and added into the lower chamber at 85 µl per well. GB cells were serum starved for 2 hours prior to trypsinization and were pretreated with or without JTE-013 and AB1 for 10 minutes. They were then placed in the upper compartment at 5 × 104 cells per well in 0.39 ml medium and allowed to migrate 5 hours at 37°C. The filter was then fixed overnight at 4°C and the nonmigrated cells were removed with a cotton swab. Attached cells were stained with 0.1% crystal violet and eluted with 10% acetic acid in 96-well plates. The absorbance was measured at 595 nm.

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Methylthiazolyldiphenyl-Tetrazolium Bromide Assay.[1]
The viability of SK-N-AS cells treated with JTE-013 or AB1 was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, as previously described (Li et al., 2013). Briefly, SK-N-AS cells were seeded in 96-well plates and treated with different concentrations of JTE-013 or AB1 for different times, followed by incubation of MTT at 37°C for 2 hours. The insoluble formazan formed in viable cells were dissolved by dimethylsulfoxide and the absorbance was measured at 595 nm by using a Bio-Rad Microplate Reader. Results are presented as the percentage of cell viability relative to the nondrug-treated controls.

Six-week-old female athymic NCr-nu/nu nude mice
30 mg/kg
Gavage; daily for 14 consecutive days

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Subcutaneous NB Tumor Model. [1]
Animal experiments were conducted according to our institution’s and the National Research Council’s guide for the care and use of laboratory animals. Six-week-old female athymic NCr-nu/nu nude mice (National Cancer Institute, Frederick, MD) were used in this study. Briefly, each mouse received a subcutaneous flank injection containing 1 × 107 SK-N-AS cells in 0.1 ml phosphate-buffered saline. After the tumor size reached approximately 100 mm3, the mice were randomized into three groups: vehicle control, JTE-013, and AB1. Both JTE-013 and AB1 were dissolved in dimethylsulfoxide first, diluted with 2% (2-hydroxypropyl)-β-cyclodextrin in phosphate-buffered saline, and given by gavage at 30 mg/kg daily for 14 consecutive days. Tumors were measured every other day with a caliper, and tumor volumes were calculated using the following formula: tumor volume = length × width2 × 0.52. Two weeks later, the mice were euthanized and tumor masses were collected for different assays. Intravenous Pharmacokinetic Analysis. [1]
Male CD-1 mice were used in this study. Briefly, a catheter was implanted in the carotid artery of each mouse to facilitate the subsequent repeated blood draws. Then the tested compounds were given intravenously at 1 mg/kg. A small amount of blood (30 μl) was taken from the catheter at different time points and the drug concentrations in the blood were determined by high-performance liquid chromatography analysis/mass spectrometry.

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1.2 mg/kg; i.p

Mice(C57BL/6×129Sv genetic background)

[1]. Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma. J Pharmacol Exp Ther. 2015 Sep;354(3):261-8.

[2]. Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature. Am J Physiol Heart Circ Physiol. 2009 Jan;296(1):H33-42.

Bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play crucial roles in various pathological processes, including cancer. The S1P axis has become an important target for cancer therapy. The known S1P2 antagonist JTE-013 [N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinoamide] exhibits poor in vivo stability. Therefore, developing structurally modified, more potent, and more stable S1P2 inhibitors would be an ideal pharmacological tool. One of the derivatives of JTE-013, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridin-6-yl)-amino-N'-(2,6-dichloropyridin-4-yl)urea], exhibits stronger S1P2 antagonistic activity compared to JTE-013. Intravenous pharmacokinetic studies showed that AB1 had enhanced stability or slower clearance in vivo. Glioblastoma cell migration assays indicated that AB1 was slightly superior to JTE-013 in blocking S1P2-mediated cell migration inhibition. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 was at least as potent as JTE-013 in affecting downstream S1P2 signaling molecules. Similarly, AB1 also showed stronger inhibitory effects on the growth of SK-N-AS xenograft tumors compared to JTE-013. Cell viability assays ruled out the possibility that the enhancement effect of AB1 was due to the inhibition of cancer cell survival. Both JTE-013 and AB1 showed a tendency to inhibit the expression of (CC motif) ligand 2 and significantly inhibited subsequent tumor-associated macrophage infiltration in NB xenografts. Notably, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) and cleaved caspase-3 assays further confirmed that AB1-treated NB xenografts showed increased apoptosis compared to JTE-013. In conclusion, the modified product AB1 of JTE-013 improved its potency, intravenous pharmacokinetics, cellular activity and antitumor activity, and may enhance its clinical and experimental value. [1] In summary, we report here a novel modified product AB1 of the S1P2 antagonist JTE-013. AB1 showed moderately improved potency and intravenous pharmacokinetics and better stability. In the context of neuroblastoma (NB), AB1 also appears to have better cellular activity and antitumor activity. Based on these findings, we conclude that AB1 may have greater clinical and experimental value, overcoming some of the shortcomings of JTE-013. [1]

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Sphingosine-1-phosphate (S1P) regulates a variety of molecular and cellular events in cultured endothelial cells, such as cytoskeleton remodeling, cell-extracellular matrix interactions, and intercellular junction interactions. We investigated the role of S1P signaling in the regulation of microvascular permeability using a venous leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats. S1P signaling is mediated by S1P family G protein-coupled receptors (S1P(1-5) receptors). S1P(1) and S1P(2) receptors transmit stimulatory and inhibitory signals, respectively, and are expressed in cremaster muscle vascular endothelial cells. In Sprague-Dawley rats, injection of S1P alone via the carotid artery did not protect the cremaster muscle vascular bed from histamine-induced venous leakage. However, activation of S1P(1)-mediated signaling using two S1P(1) agonists, SEW2871 and FTY720, significantly inhibited histamine-induced microvascular leakage. Antagonism of S1P(1)-regulated signaling using VPC 23019 significantly enhanced histamine-induced venous leakage. Inhibition of the S1P(2) signaling pathway using the S1P(2)-specific antagonist JTE-013 enabled S1P to protect microvascular permeability in vivo. Furthermore, in cultured endothelial cells, tight junctions and barrier function were synergistically regulated by S1P(1) and S1P(2)-mediated signaling pathways. These data suggest that the balance between the S1P(1) and S1P(2) signaling pathways regulates the homeostasis of microvascular permeability in the peripheral circulation, which may affect total peripheral vascular resistance. [2]

C17H19CL2N7O

408.29

407.102

C, 50.01; H, 4.69; Cl, 17.37; N, 24.01; O, 3.92

547756-93-4

547756-93-4

25168534

Typically exists as White to off-white solid at room temperature

1.5±0.1 g/cm3

1.697

4.42

3

5

5

27

504

0

CN1C2=NC(NNC(NC3C(Cl)=CN=CC=3Cl)=O)=CC(CCC)=C2C(C)=N1

GDFXUTXWCNQTEF-UHFFFAOYSA-N

InChI=1S/C17H19Cl2N7O/c1-4-5-10-6-13(21-16-14(10)9(2)25-26(16)3)23-24-17(27)22-15-11(18)7-20-8-12(15)19/h6-8H,4-5H2,1-3H3,(H,21,23)(H2,20,22,24,27)

1-(3,5-dichloropyridin-4-yl)-3-[(1,3-dimethyl-4-propylpyrazolo[3,4-b]pyridin-6-yl)amino]urea

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)