p2x1 Receptor

NF449 inhibits the binding rate of GTP[γS] to rGsα-s while having little effect on binding to rGiα-1 (IC50=140 nM), inhibits stimulation of adenylate cyclase activity in S49 cyclized membranes (lacking endogenous Gsα) by exogenously added Gsα-s, and blocks coupling of β-adrenergic receptors to Gs (EC50=7.9 μM)[2].

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P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or alphabeta-methyleneadenosine 5'-trisphosphate-induced inward currents of homomeric rat P2X(1)-P2X(4) receptors or heteromeric P2X(1 + 5) and P2X(2+3) receptors, respectively. NF449 most potently blocked P2X(1) and P2X(1 + 5) receptors with IC(50) values of 0.3 nM and 0.7 nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X(3) or heteromeric P2X(2 + 3) receptors (IC(50) 1.8 and 0.3 microM, respectively). NF449 was least potent at homomeric P2X(2) receptors (IC(50) 47 microM) and homomeric P2X(4) receptors (IC(50) > 300 microM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X(1) subunit such as the P2X(1) homomer and the P2X(1 + 5) heteromer.[1]

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The aim was to determine whether the newly described P2X1 antagonist NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt] could selectively antagonize the platelet P2X1 receptor and how it affected platelet function. NF449 inhibited alpha,beta-methyleneadenosine 5'-triphosphate-induced shape change (IC50 = 83 +/- 13 nM; n = 3) and calcium influx (pA2 = 7.2 +/- 0.1; n = 3) (pIC50 = 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X1 receptor. NF449 also antagonized the calcium rise mediated by the P2Y1 receptor, but with lower potency (IC50 = 5.8 +/- 2.2 microM; n = 3). In contrast, it was a very weak antagonist of the P2Y12-mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X1 receptor with NF449 led to reduced collagen-induced aggregation, confirming a role of this receptor in platelet activation induced by collagen.

At a dose of 10 mg/kg, NF449 inhibited in vitro aggregation triggered by 5 g/mL collagen in WT mouse platelets, without affecting aggregation induced by 5 μM ADP. At a higher dose (50 mg/kg), NF449 inhibited in vitro platelet aggregation not only in response to 10 g/mL collagen but also in response to 5 M ADP, indicating non-selective inhibition of P2Y1 and/or P2Y12 receptors[3].

Binding Assays. [2]
The binding of [35S]GTP[γS] to rGsα-s and rGiα-1 (2–4 pmol/assay) was carried out as described. β-Adrenergic receptors were labeled with the antagonist [125I]CYP; rat cardiac membranes (8–12 μg/assay) or S49 cyc− membranes (3–6 μg/assay) were incubated in TEMA buffer (in mM: 50 Tris⋅HCl, pH 7.5, 5 MgCl2, 1 EDTA, 1 ascorbic acid) and the concentrations of [125I]CYP, isoproterenol, suramin analogues, and GTP[γS] indicated in the figure legends. High-affinity agonist binding was reconstituted in S49 cyc− membranes with oligomeric Gs (a combination of 3 pmol of rGsα-s and 10 pmol of purified βγ dimers/reaction) as outlined in ref. 20. Nonspecific binding was determined in the presence of 100 μM isoproterenol (<15% of total binding). A1-adenosine receptors were labeled with the agonist [125I]HPIA. The binding reaction was carried out in 50 μl containing TEMA buffer, 8 units/ml adenosine deaminase, human brain membranes (∼6–9 μg), or membranes from stably transfected 293 cells (12–15 μg), 1 nM [125I]HPIA in the absence and presence of increasing concentrations of suramin analogues. Nonspecific binding (<10% of total binding) was determined in the presence of 1 μM CPA (N6-cyclopentyladenosine). Angiotensin II type 1 receptors were labeled with the antagonist [125I]saralasin II or the agonist [125I]angiotensin II. The binding assay was carried out in 30 μl containing TEMA buffer, 12.5 μg/ml truncated human ACTH11–24, 100 μM bacitracin, glomerular membranes (5 μg), 0.5 nM [125I]saralasin II, or [125I]angiotensin II in the absence and presence of increasing concentrations of GTP[γS] and of suramin analogues. Nonspecific binding (≤20% of total binding) was determined in the presence of 1 μM unlabeled saralasin ([125I]angiotensin II) or angiotensin II ([125I]saralasin). After 60 min at 30°C, the binding reactions were stopped by filtration over glass-fiber filters (presoaked in 1% BSA for angiotensin receptor binding).

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Determination of Adenylyl Cyclase Activity. [2]
Adenylyl cyclase activity was reconstituted to S49 cyc− membranes by addition of rGsα-s as described with minor modifications; rGsα-s (0.1 mg/ml) was preactivated in buffer (in mM: Hepes⋅NaOH, pH 7.6, 1 EDTA, 1 DTT, 0.01 GTP[γS], 10 MgSO4, 0.1% Lubrol) for 30 min at 30°C and diluted to give the appropriate amounts of rGsα-s. Alternatively, inactive rGsα-s was diluted in buffer lacking GTP[γS] and MgSO4. S49 cyc− membranes (12.5 μg) were preincubated with rGsα-s in 20 μl for 20 min on ice; the reaction was started by adding 30 μl of substrate solution to yield (in mM) 50 Hepes⋅NaOH, pH 7.6, 1 EDTA, 0.1 DTT, 0.05 [α-32P]ATP (∼400 cpm/pmol), 9 MgCl2, 1 MgSO4, 1 μM GTP[γS] or 10 μM GTP in the absence and presence of 10 μM NF449 or of 10 μM NF503. The incubation lasted for 30 min at 20°C. Experiments were carried out in duplicate; if not otherwise indicated, representative experiments are shown, which were repeated at least twice.

Electrophysiological current recordings of recombinant P2X receptors in Xenopus laevis oocytes [1]
P2X receptor cRNAs were synthesized and injected at concentrations of 5 ng/μl (rP2X2) or 0.5 μg/μl (rP2X1, rP2X3, rP2X4, rP2X5) in 50-nl volumes into collagenase defolliculated Xenopus oocytes prepared as described in detail previously (Schmalzing et al., 1991). For the expression of hetero-oligomeric rP2X1+5 and rP2X2+3 receptors, cRNAs ratios of 1:1 were used, 0.5 μg/μl each. Two-electrode voltage-clamp recordings of whole-cell currents were performed 2–6 days after RNA injection in nominally Ca2+-free frog Ringer solution at ambient temperature (22–24 °C) and a holding potential of −60 mV as described previously (Rettinger and Schmalzing, 2003, Rettinger and Schmalzing, 2004). For the analysis of the NF449-mediated inhibition of the fast desensitizing rP2X1 and rP2X3 receptor currents, the receptors were activated 3- to 5-times for 10 s at 4 min intervals with 1 μM ATP, a concentration close to the respective EC50 value (Rettinger and Schmalzing, 2003, Rettinger and Schmalzing, 2004), to obtain constant and reproducible current responses. After this equilibration period, rP2X1 or rP2X3 receptor-expressing oocytes were pre-incubated with the desired concentration of NF449 for the indicated period of time before ATP was co-applied with NF449. At the non-desensitizing or slowly desensitizing rP2X2 and rP2X4 receptors, NF449 was usually co-applied together with 10 μM ATP. For activation of rP2X1+5 and rP2X2+3 receptors αβmeATP (1 μM) was used as an agonist. After each washout of agonist or agonist and NF449, control responses were elicited by the appropriate agonist to monitor run-down or run-up artifacts and to check for complete reversibility of antagonism. For determination of IC50 values, responses were only used when the difference between control responses recorded before and after the NF449 application was less than 10%. The remaining error was corrected by assuming a linear characteristic of run-down or run-up. The concentration-inhibition curves and the resulting IC50 values were derived from non-linear least-square fits of the Hill equation to the pooled data points:(1) where Imax denotes the peak current response in absence of NF449, I the peak current response at the respective NF449 concentration, and nH the Hill coefficient. Results are presented as means ± SEM from n experiments.

Intravenous injection of 10 mg/kg NF449 into mice resulted in selective inhibition of the P2X1 receptor and decreased intravascular platelet aggregation in a model of systemic thromboembolism (35 +/- 4 versus 51 +/- 3%) (P = 0.0061; n = 10) but without prolongation of the bleeding time (106 +/- 16 versus 78 +/- 7 s; n = 10) (N.S.; P = 0.1209). At a higher dose (50 mg/kg), NF449 inhibited the three platelet P2 receptors. This led to a further reduction in platelet consumption compared with mice injected with saline (13 +/- 4 versus 42 +/- 3%) (P = 0.0002; n = 5). NF449 also reduced dose-dependently the size of thrombi formed after laser-induced injury of mesenteric arterioles. Overall, our results indicate that NF449 constitutes a new tool to investigate the functions of the P2X1 receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors. [3]

[1]. Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X1 receptor antagonist. Neuropharmacology. 2005;48(3):461-468.

[2]. Gsalpha-selective G protein antagonists. Proc Natl Acad Sci U S A. 1998;95(1):346-351.

[3]. Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt]. J Pharmacol Exp Ther. 2005;314(1):232-243.

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the Galpha subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for Gsalpha. Two compounds have been identified: NF449 (4,4',4",4'"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4, 4'-[carbonylbis[imino-3,1-phenylene-(2, 5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (i) suppress the association rate of guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[gammaS]) binding to Gsalpha-s but not to Gialpha-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc- membranes (deficient in endogenous Gsalpha) by exogenously added Gsalpha-s, and (iii) block the coupling of beta-adrenergic receptors to Gs with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A1-adenosine receptor with its cognate G proteins (Gi/Go) at concentrations that are >30-fold higher than those required for uncoupling of beta-adrenergic receptor/Gs tandems; similarly, the angiotensin II type-1 receptor (a prototypical Gq-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for Gsalpha-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition. [2]

C41H32N6O29S8

1329.24

1503.75349

C, 32.72; H, 1.61; N, 5.58; Na, 12.22; O, 30.83; S, 17.04

389142-38-5

627034-85-9

Typically exists as solids at room temperature

[Na]OS(C1=CC=C(NC(C2C=C(NC(NC3=CC(C(NC4=CC=C(S(O[Na])(=O)=O)C=C4S(O[Na])(=O)=O)=O)=CC(C(NC4=CC=C(S(O[Na])(=O)=O)C=C4S(O[Na])(=O)=O)=O)=C3)=O)C=C(C(NC3=CC=C(S(O[Na])(=O)=O)C=C3S(O[Na])(=O)=O)=O)C=2)=O)C(S(O[Na])(=O)=O)=C1)(=O)=O

KCBZSNWCUJBMHF-UHFFFAOYSA-F

InChI=1S/C41H32N6O29S8.8Na/c48-37(44-29-5-1-25(77(53,54)55)15-33(29)81(65,66)67)19-9-20(38(49)45-30-6-2-26(78(56,57)58)16-34(30)82(68,69)70)12-23(11-19)42-41(52)43-24-13-21(39(50)46-31-7-3-27(79(59,60)61)17-35(31)83(71,72)73)10-22(14-24)40(51)47-32-8-4-28(80(62,63)64)18-36(32)84(74,75)76;;;;;;;;/h1-18H,(H,44,48)(H,45,49)(H,46,50)(H,47,51)(H2,42,43,52)(H,53,54,55)(H,56,57,58)(H,59,60,61)(H,62,63,64)(H,65,66,67)(H,68,69,70)(H,71,72,73)(H,74,75,76);;;;;;;;/q;8*+1/p-8

octasodium;4-[[3-[[3,5-bis[(2,4-disulfonatophenyl)carbamoyl]phenyl]carbamoylamino]-5-[(2,4-disulfonatophenyl)carbamoyl]benzoyl]amino]benzene-1,3-disulfonate

nf449; 627034-85-9; NF449 octasodium salt; 389142-38-5; 4,4',4'',4'''-[Carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid, octasodium salt; octasodium;4-[[3-[[3,5-bis[(2,4-disulfonatophenyl)carbamoyl]phenyl]carbamoylamino]-5-[(2,4-disulfonatophenyl)carbamoyl]benzoyl]amino]benzene-1,3-disulfonate; Lopac-N-4784; CHEMBL1253351;

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)