P2Y11 (pKi = 7.35); P2Y11 (IC50 = 463 nM); P2Y11 (Ki = 44.3 nM); P2Y1 (Ki = 187 µM); P2Y2 (Ki = 28.9 µM)

NF157 is not as selective as P2X1, but it is more selective for P2Y11 than P2Y1 (>650-fold), P2Y2 (>650-fold), P2X2 (3-fold), P2X3 (8-fold), P2X4 (>22-fold), and P2X7 (>67-fold)[1]. In a dose-dependent manner, NF157 (30 and 60 µM; 24 hours) dramatically decreased the breakdown of type II collagen. Type II collagen is nearly entirely restored from TNF-α (10 ng/mL)-induced degradation when 60 µM NF157 is applied [2]. NF157 (30 and 60 µM; 24 hours) dramatically decreased NF-κB luciferase activity while nearly fully restoring TNF-α (10 ng/mL)-triggered p65 nuclear translocation [2].

Electrophysiological Evaluation of 8f/NF157 at Recombinant P2X Receptors. [1]
The inhibitory potency of NF157 at P2X receptors was evaluated on X. laevis oocytes recombinantly expressing various rat (r) and human (h) P2X subtypes (rP2X1, hP2X1, rPX2, rP2X3, hP2X3, rP2X4, hP2X4, rP2X7) using previously described protocols. Concentration−inhibition curves and IC50 values were derived from nonlinear least-squares fits of the Hill equation to the pooled data. The nondesensitizing properties of the rP2X2 receptor allowed quantifying inward current inhibition under steady-state conditions by coapplying NF157 during continued stimulation with ATP. Current inhibition occurred almost instantaneously as inferred from the immediate decrease of the current amplitude upon coapplication of an effective NF157 concentration. The classical Cheng−Prusoff equation could be applied to calculate the Ki value of NF157 for the rP2X2 receptor. The inhibitory potency of NF157 at desensitizing P2X receptors was determined from peak current measurements. As detailed previously, an accurate assessment of IC50 values for desensitizing receptors can usually not be achieved by coapplying agonist and antagonist, as the agonist-induced current will start to decline by desensitization before a binding equilibrium between the two compounds is reached. To account for this problem, oocytes expressing desensitizing P2X receptors were preequilibrated with NF157 for 15 s before being challenged with ATP in the continued presence of NF157. We have previously shown that suramin derivatives block P2X receptors competitively. Accordingly, if NF157 does not dissociate significantly from the receptor during the time needed to reach the peak current response, ATP can only bind to receptors unoccupied by NF157, leading to a pseudo-irreversible type of inhibition. Under these conditions, Ki and IC50 values will be equal. We therefore assume that the IC50 values deduced from peak current measurements are close or equal to the Ki values. In any case, Ki values deviate from IC50 values maximally by a factor of 2, as ATP was applied at a concentration close to its EC50 value. All results are presented as means ± SEM from at least three experiments.

Western Blot Analysis[2]
Cell Types: SW1353 Cell
Tested Concentrations: 30 and 60 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished TNF-α-induced NF-κB activation.

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NF-κB promoter-luciferase assay [2]
The transcriptional activity of NF-κB was assessed by measuring the NF-κB promoter-luciferase activity. The NF-κB promoter-luciferase and β-galactosidase plasmids were transfected into SW1353 cells with the Lipofectamine 2000. Human chondrocytic SW1353 cells were treated with 10 ng/ml TNF-α in the presence or absence of NF157 at the concentrations of 30 and 60 μM for 24 h. Cell lysates were prepared and used for determining luciferase activity and β-galactosidase activity using a Secrete-PairTM Dual luminescence assay kit on a luminometer. Luciferase activity was normalized to β-galactosidase activity.

[1]. Synthesis and structure-activity relationships of suramin-derived P2Y11 receptor antagonists with nanomolar potency. J Med Chem. 2005 Nov 3;48(22):7040-8.

[2]. Inhibition of P2Y11R ameliorated TNF-α-induced degradation of extracellular matrix in human chondrocytic SW1353 cells. Am J Transl Res. 2019 Apr 15;11(4):2108-2116.

Selective and potent P2Y(11) receptor antagonists have yet to be developed, thus impeding an evaluation of this G protein-coupled receptor mainly expressed on immune cells. Taking suramin with moderate inhibitory potency as a template, 18 ureas with variations of the methyl groups of suramin and their precursors were functionally tested at P2Y(11), P2Y(1), and P2Y(2) receptors. Fluorine substitution of the methyl groups of suramin led to the first nanomolar P2Y(11) antagonist (8f, NF157, pK(i): 7.35). For selectivity, 8f was also tested at various P2X receptors. 8f displayed selectivity for P2Y(11) over P2Y(1) (>650-fold), P2Y(2) (>650-fold), P2X(2) (3-fold), P2X(3) (8-fold), P2X(4) (>22-fold), and P2X(7) (>67-fold) but no selectivity over P2X(1). QSAR studies confirm that residues with favored resonance and size parameters in the aromatic linker region can indeed lead to an increased potency as is the case for 8f. A symmetric structure linking two anionic clusters seems to be required for bioactivity. 8f may be helpful for studies evaluating the physiological role of P2Y(11) receptors. [1]
This study presents the synthesis and structure−activity relationships of a series of suramin analogues and led to the discovery of the first nanomolar potency P2Y11 receptor antagonist 8f with at least 650-fold selectivity for P2Y11 over P2Y1 and P2Y2, and a 3- to >67-fold selectivity over P2X2,3,4,7 receptors. 8f is, however, approximately equipotent at P2Y11 and P2X1 receptors. A symmetric structure linking two anionic clusters seems to be required for bioactivity. QSAR studies reveal that a substitution with favored values for resonance (R), size (B5), and partial charges of the amide group oxygen in ortho-position of the residue R (Q(Oortho)) in the aromatic linker region can indeed lead to an increased potency as is the case for the fluorine derivative 8f. The QSAR results may guide the directed synthesis and development of further potent and selective (also against P2X1) P2Y11 ligands. Thus, this study and the novel ligand 8f may be helpful to obtain a deeper insight into the physiological and pathophysiological role of P2Y11 receptors. [1]

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Osteoarthritis is a major global health burden. Joint destruction resulting from excessive degradation of type II collagen and aggrecan in the articular extracellular matrix by metalloproteinases and aggrecanases, respectively, is a major pathological hallmark of osteoarthritis. However, the exact mechanisms remain poorly understood. Currently, there are few non-invasive therapies capable of slowing or halting the progression of the disease. In the present study, we investigated the involvement of the P2Y11 purinergic protein and its receptor P2Y11R in TNF-α-mediated degradation of the extracellular matrix in SW1353 cell line chondrocytes using the novel P2Y11R antagonist NF157. To our knowledge, this is the first study to explore the effects of NF157 in OA. Our results indicate that P2Y11R may indeed play a role in TNF-α-induced degradation of extracellular matrix in OA as treatment with NF157 significantly reduced expression of metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5, and ameliorated degradation of type II collagen and aggrecan in SW1353 chondrocytes in a dose-dependent manner. Furthermore, we show that treatment with NF157 significantly reduced nuclear translocation of p65 and subsequent activation of nuclear factor κB (NF-κB). [2]
NF-κB is well-recognized as a key pro-inflammatory transcription factor that promotes the development and progression of OA as well as many other inflammatory diseases. A recent study from Japan demonstrated that activation of the P2Y11 receptor via γ-radiation-induced release of ATP activated NF-κB through the p38/mitogen-activated protein kinase (MAPK) pathway. In a related study, the authors showed that treatment with NF157 inhibited activation of NF-κB Another study demonstrated that ATP selectively targeted p65 to activate NF-κB through the P2Z purinoceptor, which like P2Y11R, has a high affinity for ATP. In the present study, we showed that TNF-α-induced nuclear translocation of p65 could be suppressed by blockage of P2Y11R with 30 and 60 µM NF157 in a dose-dependent manner. Additionally, we confirmed that antagonism of P2Y11R with NF157 significantly decreased NF-κB luciferase activity in a dose-dependent manner. The findings of our study demonstrate for the first time that blockage of the P2Y11 purinoceptor using the selective P2Y11 antagonist NF157 can significantly prevent degradation of the components of the articular ECM by downregulating TNF-α-induced expression of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, subsequent degradation of type II collagen and aggrecan, and activation of NF-κB. These positive results suggest that NF157 may have potential as a targeted therapeutic agent for the treatment and prevention of excessive degradation of type II collagen and aggrecan in OA. Further study is required to better understand the complex mechanisms through which these results were achieved. [2]

C49H34F2N6O23S6

1305.19

1435.89

C, 45.09; H, 2.63; F, 2.91; N, 6.44; O, 28.19; S, 14.74

104869-26-3

Off-white to pink solid powder

11.271

UDVIAMRWOLIUAE-UHFFFAOYSA-N

InChI=1S/C49H34F2N6O23S6/c50-33-9-7-25(47(60)54-35-11-13-39(83(69,70)71)31-19-29(81(63,64)65)21-41(43(31)35)85(75,76)77)17-37(33)56-45(58)23-3-1-5-27(15-23)52-49(62)53-28-6-2-4-24(16-28)46(59)57-38-18-26(8-10-34(38)51)48(61)55-36-12-14-40(84(72,73)74)32-20-30(82(66,67)68)22-42(44(32)36)86(78,79)80/h1-22H,(H,54,60)(H,55,61)(H,56,58)(H,57,59)(H2,52,53,62)(H,63,64,65)(H,66,67,68)(H,69,70,71)(H,72,73,74)(H,75,76,77)(H,78,79,80)

8-[[4-fluoro-3-[[3-[[3-[[2-fluoro-5-[(4,6,8-trisulfonaphthalen-1-yl)carbamoyl]phenyl]carbamoyl]phenyl]carbamoylamino]benzoyl]amino]benzoyl]amino]naphthalene-1,3,5-trisulfonic acid

NF-157; NF 157; NF157

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : ~1 mg/mL (~0.70 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)