Chloride channels (specifically, outwardly rectifying chloride channel (ORCC) and voltage-gated chloride channel 3 (CLC-3) in glioma cells) NPPB blocks these channels. [1][2]

The channel opening probability is decreased by NPPB (0.1 mM) from 0.89±0.06 to 0.11±0.04[1]. The concentration of NPPB (IC50=125 μM) chloride depends on the current. With a GI50 of roughly 500 μM, NPPB itself likewise demonstrates cytotoxicity to neuroastrological tumor cells [2].

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NPPB blocks chloride currents in a dose-dependent manner in U373MG human glioma cells, with an IC50 of 125 μM for inhibiting the peak current at +120 mV. It functions as a non-specific chloride channel blocker. [2]
In acutely dissociated rat hippocampal CA1 pyramidal neurons, NPPB (0.1 mM) applied to the bath solution reduced the open probability of the outwardly rectifying chloride channel (ORCC) from 0.89 ± 0.06 to 0.11 ± 0.04 (n=5, P<0.01) in inside-out patch clamp recordings. [1]
NPPB itself shows cytotoxicity against human glioma cells (U373MG, U87MG, LN18) and mouse microglial BV-2 cells. The GI50 values for U373MG, U87MG, LN18, and BV-2 cells are approximately 542 μM, 653 μM, 571 μM, and 423 μM, respectively. [2]
NPPB suppresses the migration and invasion of U373MG human glioma cells. In wound healing assays, it decreased cell migration rates. In Matrigel invasion assays, it significantly reduced the number of invading cells. [2]
NPPB does not induce DNA double-strand breaks on its own, as measured by the comet assay, showing no significant increase in percentage tail DNA or tail moment. [2]
NPPB alone (287 μM, 48h) induced cell death in a smaller percentage of U373MG cells compared to TMZ-NPPB, primarily in early and late apoptosis stages as measured by flow cytometry (FITC-annexin V/PI staining). [2]

Covalent DNA modification assay: To test if NPPB alkylates DNA, DNA fragments were treated with 1 mM NPPB for 10 hours. The treated DNA was then used in a linear amplification reaction with a FAM-labeled primer. The resulting DNA samples were separated on a denaturing urea polyacrylamide gel. The fragmentation pattern for NPPB-treated DNA was compared to control and TMZ-treated samples. Unlike TMZ, NPPB did not produce the characteristic fragmentation pattern indicative of DNA methylation. [2]

Electrophysiology (chloride current recording): U373MG cells were used. Chloride currents were recorded using the whole-cell patch-clamp technique. The holding potential was -40 mV, and voltage pulses were applied from -60 mV to +120 mV in 20-mV increments. The external solution contained 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10.55 mM glucose, and 32.5 mM HEPES (pH 7.4). The internal solution contained 140 mM CsCl, 2 mM MgCl2, 4.9 mM CaCl2, 10 mM EGTA, and 10 mM HEPES (pH 7.2). For inhibition experiments, NPPB (125 or 300 μM) was applied to the recording chamber. Percent inhibition was calculated using the maximum outward currents at +120 mV. [2]
Cell Proliferation (MTT) Assay: Cells (U373MG, U87MG, LN18, BV-2) were seeded in 96-well plates at 5x10^3 cells per well and incubated for 24h. The culture medium was then replaced with fresh media containing various concentrations of NPPB and incubated for 24h. MTT solution (0.5 mg/mL) was added and incubated for 1h. The formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. Concentration-response curves were fitted to a Hill equation to obtain GI50 values. [2]
Flow Cytometry (Apoptosis) Assay: U373MG cells were treated with 287 μM NPPB for 48h. Untreated cells served as control. Cells were harvested, washed, and resuspended in Annexin V Binding Buffer. They were stained with FITC-labeled Annexin V and propidium iodide (PI) for 15 min at room temperature in the dark. Stained cells were immediately analyzed using flow cytometry to quantify the percentage of cells in live, early apoptotic, late apoptotic, and necrotic stages. [2]
Comet Assay (Single Cell Gel Electrophoresis): U373MG cells were treated with 287 μM NPPB for 24h. After incubation, cells were harvested and mixed with LM agarose, then pipetted onto CometSlide. The slides were placed in alkaline lysis solution (200 mM NaOH, 1 mM EDTA, pH >13) and then washed with Tris-borate EDTA buffer. Electrophoresis was carried out at 45 V for 5 min. Slides were washed, immersed in 70% ethanol, and stained with SYBR green. Images were captured at 10x magnification using fluorescence microscopy. Percentage tail DNA and tail moment were calculated with 30 comets per sample using CASP image analysis software. [2]
Wound Healing Assay (Migration): U373MG cells were seeded in poly-lysine coated 6-well plates at 5x10^5 cells per well and incubated overnight. Cell monolayers were scratched with a pipette tip to create a wound. After washing away detached cells, cells were incubated with NPPB at its GI50 (approx. 542 μM) for 24h. Images of the scratched regions were taken before and after incubation. Migration rates were calculated from migration areas determined using Image J software. [2]
Matrigel Invasion Assay: Matrigel invasion chambers (8.0 μm pore size) were rehydrated. U373MG cell suspension (1x10^5 cells per well) in medium containing NPPB at its GI50 (approx. 542 μM) was added to the chamber. After 24h incubation, cells were fixed with formaldehyde and methanol and stained with trypan blue. Cells remaining on the upper surface of the membrane were removed, and the number of invading cells was counted. [2]

[1]. Enhancement of an outwardly rectifying chloride channel in hippocampal pyramidal neurons after cerebral ischemia. Brain Res. 2016 Aug 1;1644:107-17.

[2]. Double Blockade of Glioma Cell Proliferation and Migration by Temozolomide Conjugated withNPPB, a Chloride Channel Blocker. ACS Chem Neurosci. 2016 Mar 16;7(3):275-85.

5-Nitro-2-(3-Phenylacetylamino)benzoic acid is a nitrobenzene.

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NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) is a non-specific chloride channel blocker. It has been shown to suppress glioma cell migration and invasion by blocking voltage-gated chloride channels (like CLC-3), which are crucial for cell shape and volume changes during migration. [2]
In hippocampal neurons, NPPB blocks an outwardly rectifying chloride channel (ORCC) that is involved in apoptosis and cell volume regulation. [1]
NPPB has a negatively charged carboxylic group that is important for its channel-blocking activity, blocking the channel pore from the intracellular side via electrostatic interactions. [2]
The GI50 of NPPB against BV-2 mouse microglial cells was found to be 423.5 ± 30.3 μM. The ratio of GI50 for glioma cells (U373MG) to GI50 for microglia cells for NPPB is 1.28, indicating relative selectivity. [2]

C16H16N2O4

300.3092

300.111

107254-86-4

4549

Light yellow to yellow solid powder

1.323 g/cm3

523.5ºC at 760 mmHg

178-179ºC

8.73E-12mmHg at 25°C

1.653

3.933

2

5

6

22

377

0

WBSMIPAMAXNXFS-UHFFFAOYSA-N

InChI=1S/C16H16N2O4/c19-16(20)14-11-13(18(21)22)8-9-15(14)17-10-4-7-12-5-2-1-3-6-12/h1-3,5-6,8-9,11,17H,4,7,10H2,(H,19,20)

5-nitro-2-(3-phenylpropylamino)benzoic acid

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)

DMSO : ~100 mg/mL (~332.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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