Ki: 36 nM (P2X3 Receptor)

In the human tumor cell line panel, NF110 exhibits activity with an IC30 of 362.3 μM[2]. With an IC50 of 0.87 μM, NF110 inhibits HMGA2-DNA interactions[3].

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NF110 only carries 4 negative charges. However, it strongly inhibits HMGA2-DNA interactions with an IC50 of 0.87 ± 0.04 μM. In contrast, although NF023 has 6 negative charges, its inhibition IC50 was determined to be 10.63 ± 0.46 μM, tenfold higher than that of NF110. These results suggest that both the charge and structure are very important for the inhibition of HMGA2-DNA interactions by these related compounds.

NF110 exhibits efficacy with a peak amplitude of 675 pA at endogenous P2X receptors in rat DRG neurons[1].

HMGA2 AlphaScreen ultra HTS assay [3]
Using a Labcyte Echo 555 acoustic dispenser 5 nL of DMSO were added to columns 1–4 and 45–48 of a white 1536 well plate, while 5 nL of 2 mM compounds in DMSO were added to columns 5–44. Using a Beckman BioRAPTR FRD bulk reagent dispenser 1 µL of assay buffer (30 mM Citrate, 300 mM NaCl, and 0.005% Tween 20) was added to columns 1 and 2. Next 1 µL of assay buffer containing 125 nM HMGA2 was added to columns 3–48 with a Beckman BioRAPTR FRD bulk reagent dispenser. Finally, 1 µL of assay buffer containing 25 nM FL814 was added to every well of the plate using a Beckman BioRAPTR FRD bulk reagent dispenser. The plate was then centrifuged at 200 × g for 1 min. After 30 min at room temperature, 2 µL of bead buffer (10 mM HEPES, 300 mM NaCl and 0.005% Tween 20) containing 20 µg/mL anti-6xHIS acceptor beads and 20 µg/mL streptavidin donor beads were dispensed into every well using a Beckman BioRAPTR FRD bulk reagent dispenser. The plates were then centrifuged at 200 × g for 1 min. After 1 h at room temperature the plates were read on a Perkin Elmer Envision multimode plate reader in AlphaScreen mode.

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HMGA2 Lance assay [3]
Using a Labcyte Echo 555 acoustic dispenser 5 nL of DMSO were added to columns 1–4 and 45–48 of a white 1536 well plate, while 5 nL of 2 mM compounds in DMSO were added to columns 5–44. Using a Beckman BioRAPTR FRD bulk reagent dispenser 1 µL of assay buffer (10 mM Tris, 300 mM NaCl and 0.005% Tween 20) was added to columns 1 and 2. Next, 1 µL of assay buffer containing 125 nM HMGA2 was added to columns 3–48 with a Beckman BioRAPTR FRD bulk reagent dispenser. Finally, 1 µL of assay buffer containing 25 nM FL814 was added to every well of the plate using a Beckman BioRAPTR FRD bulk reagent dispenser. The plate was then centrifuged at 200 × g for 1 min. After 30 min at room temperature 1 µL of assay buffer containing 250 nM LANCE Ultra ULight-anti-6xHIS and 1 µL of assay buffer containing 12 nM LANCE Eu-W1024 Streptavidin was dispensed into every well using a Beckman BioRAPTR FRD bulk reagent dispenser. The plates were then centrifuged at 200 × g for 1 min. After 1 h at room temperature the plates were read on a Perkin Elmer Envision multimode plate reader in TR-FRET mode (excitation @340 nm, first emission at @665 nm, second emission at 615 nm).

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BRD4 AlphaScreen assay [3]
Using a Labcyte Echo 555 acoustic dispenser 5 nL of DMSO were added to columns 1–4 and 45–48 of a white 1536 well plate, while 5 nL of 2 mM compounds in DMSO were added to columns 5–44. Using a Beckman BioRAPTR FRD bulk reagent dispenser 1 µL of assay buffer (50 mM HEPES, 100 mM NaCl, 0.1% BSA and 0.0005% CHAPS) was added to columns 1 and 2. Next, 1 µL of assay buffer containing 50 nM BRD4 was added to columns 3–48 with a Beckman BioRAPTR FRD bulk reagent dispenser. Finally, µL of assay buffer containing 50 nM peptide was added to every well of the plate using a Beckman BioRAPTR FRD bulk reagent dispenser. The plate was then centrifuged at 200 × g for 1 min. After 60 min at room temperature 2 µL of assay buffer containing 20 µg/mL anti-6xHis acceptor beads and 20 µg/mL streptavidin donor beads was dispensed into every well using a Beckman BioRAPTR FRD bulk reagent dispenser. The plates were then centrifuged at 200 × g for 1 min. After an overnight incubation at room temperature the plates were read on a Perkin Elmer Envision multimode plate reader in AlphaScreen mode.

In the patient tumour cells, the analogues, with the exception of FCE 26644, were mostly less potent than suramin (Table 3). To get an indication of the similarity of the analogues in their mode of action compared with suramin, correlation coefficients between SI values at 200 μg/ml were calculated. Correlation to suramin activity patterns in the cell line panel was highest for NF037 and low to moderate for the remaining analogues (Table 4). In patients' cells, high correlation coefficients were obtained for FCE 26644, NF110, NF031 and NF037, indicating a similar mode of action (Table 4). The lowest correlations in both cell systems were obtained for the two asymmetrical molecules (NF033 and NF036) and for NF067, which contains two diphosphatylated phenyl rings instead of the polysulphonated napthalenes [2].

Patient samples [2]
A total of 19 patient tumour samples from the different diagnoses were used to determine the activity of suramin analogues. The tumour samples were obtained by bone marrow/peripheral blood sampling, routine surgery or diagnostic biopsy, and this sampling has been approved by the local ethical committee at the Uppsala University Hospital. Leukaemic cells were isolated from bone marrow or peripheral blood by 1.077 g/ml Ficoll-Paque density gradient centrifugation. Tumour tissue from solid tumour samples was minced into small pieces and the cells were then isolated by collagenase dispersion followed by Percoll density gradient centrifugation. Cell viability was determined by trypan blue exclusion test and the proportion of tumour cells in the preparation was judged by inspection of May–Grünwald–Giemsa stained cytospin preparations by a cytopathologist. In some cases, cells were cryopreserved in 10% dimethylsulphoxide in inactivated fetal calf serum by initial freezing for 24 h at −70°C, followed by storage in liquid nitrogen. Cryopreservation in this way does not affect drug sensitivity.

[1]. The suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF110) potently blocks P2X3 receptors: subtype selectivity is determined by location of sulfonic acid groups. Mol.

[2]. Antitumour activity of suramin analogues in human tumour cell lines and primary cultures of tumour cells from patients. Eur J Cancer. 2000;36(6):803-809.

[3]. Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays. Sci Rep. 2020;10(1):18850. Published 2020 Nov 2.

We previously identified the suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-phenyltriylbis(carbonylimino)))tetrabenzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of the recombinant P2X(1) receptor. In this paper, we characterized three suramin isomer analogs using a two-electrode voltage-clamp electrophysiological technique, which were named para-4,4',4'',4''''-(carbonylbis(imino-5,1,3-phenyltriylbis(carbonylimino)))tetrabenzenesulfonic acid (NF110) and meta-(3,3',3'',3''''-(carbonylbis(imino-5,1,3-phenyltriylbis(carbonylimino)))tetrabenzenesulfonic acid (NF448). And ortho-(2,2',2'',2''''-(carbonylbis(imino-5,1,3-benzentriylbis(carbonylimino)))tetrabenzenesulfonic acid (MK3), and their efficacy in antagonizing rat P2X receptor-mediated inward current in Xenopus laevis oocytes was investigated. Meta, para, and ortho refer to the position of a single sulfonic acid group relative to the amide bond connecting four symmetrically oriented benzenesulfonic acid moieties to the centrally unchanged suramin core. NF448, NF110, and MK3 were more than 200 times less effective at blocking P2X(1) receptors than NF449, and their structural difference from NF449 was only one sulfonic acid residue on each benzene ring instead of two. Although the meta and ortho isomers retained selectivity for P2X(1) receptors, the para isomer NF110 showed significantly enhanced activity against P2X(3) receptors (K(i) approximately 36). nM), and exhibited the following unique selectivity spectrum in suramin derivatives: P2X(2+3) = P2X(3) > P2X(1) > P2X(2) >> P2X(4) > P2X(7). The application value of NF110 as a P2X(3) receptor antagonist in natural tissues can be demonstrated by the following experiments: NF110 can block αβ-methylene-ATP-induced currents in rat dorsal root ganglion neurons with similar potency to recombinant rat P2X(3) receptors. These data collectively highlight the importance of the number and exact location of negatively charged groups for the potency and selectivity of P2X subtype receptors. [1]

C41H28N6NA4O17S4

1096.91

1096

111150-22-2

16066783

White to off-white solid powder

8.717

6

17

10

72

1910

0

AQJHZNCSXLBXMY-UHFFFAOYSA-J

InChI=1S/C41H32N6O17S4.4Na/c48-37(42-27-1-9-33(10-2-27)65(53,54)55)23-17-24(38(49)43-28-3-11-34(12-4-28)66(56,57)58)20-31(19-23)46-41(52)47-32-21-25(39(50)44-29-5-13-35(14-6-29)67(59,60)61)18-26(22-32)40(51)45-30-7-15-36(16-8-30)68(62,63)64;;;;/h1-22H,(H,42,48)(H,43,49)(H,44,50)(H,45,51)(H2,46,47,52)(H,53,54,55)(H,56,57,58)(H,59,60,61)(H,62,63,64);;;;/q;4*+1/p-4

tetrasodium;4-[[3-[[3,5-bis[(4-sulfonatophenyl)carbamoyl]phenyl]carbamoylamino]-5-[(4-sulfonatophenyl)carbamoyl]benzoyl]amino]benzenesulfonate

NF 110; 111150-22-2; CHEMBL4160315; tetrasodium;4-[[3-[[3,5-bis[(4-sulfonatophenyl)carbamoyl]phenyl]carbamoylamino]-5-[(4-sulfonatophenyl)carbamoyl]benzoyl]amino]benzenesulfonate; Benzenesulfonic acid, 4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriylbis(carbonylimino)]]tetrakis-, tetrasodium salt;

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)

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.)