A chloride channel blocker and GPR35 agonist
NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, is a potent inhibitor of chloride channel with IC50 of 80 nM for the short circuit current.[1]
Chloride channel blockers possess several sites of interaction, including the negatively charged carboxylate group, the secondary amine group which probably carries a positive partial charge, and for the very potent agents like NPPB an additional negative partial charge at the -NO2 substituent. In addition, an apolar interaction with a cycloaryl residue is necessary, and this site of interaction has a specific spacing from the secondary amino nitrogen.[1]
NPPB was evaluated for the activity on the equivalent short circuit current, corresponding to the secondary active transport of Cl- and measurements of the voltage across the basolateral membrane. The result revealed that NPPB possessed a good potency with IC50 of 80 nM for inhibiting the short circuit current. Furthermore, NPPB was also tested for its activity on various anion channels. Adopting freshly-isolated cells from the rat portal vein, the effects of NPPB were investigated on evoked and spontaneous currents by use of conventional whole-cell recording and perforated-patch techniques. At a holding potential of -60 mV in potassium-free, caesium-containing solutions, NPPB (10 μM) inhibited Ca-sensitive chloride currents (ICI(Ca)) evoked by caffeine (10 mM) and by noradrenaline (10μM) by the extend of 58% and 96%, respectively. In addition, at a holding potential of -2 mV in potassium -containing solutions, NPPB (10 μM) inhibited charybdotoxin-sensitive potassium-currents (IBK(Ca)) induced by noradrenaline (10 μM) and acetylcholine (10 μM) by approximately 90%. NPPB's inhibitory effects of volume-activated taurine, glucose, and uridine influxes was studied. The IC50 for the inhibition of the volume- activated fluxes by NPPB was around 12 μM. [1-3]
参考文献:
[1] Wangemann, Ph, et al. "Cl?-channel blockers in the thick ascending limb of the loop of Henle Structure activity relationship." Pflügers Archiv 407.2 (1986): S128-S141.
[2] Kirkup, A. J., G. Edwards, and A. H. Weston. "Investigation of the effects of 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB) on membrane currents in rat portal vein." British journal of pharmacology 117.1 (1996): 175-183.
[3] Kirk, Kiaran, J. C. Ellory, and J. D. Young. "Transport of organic substrates via a volume-activated channel." Journal of Biological Chemistry 267.33 (1992): 23475-23478.
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Cell experiment: |
Cells are seeded in the 96-well microtiter plate at a density of 5×103 cells per well and incubated at 37°C for 24 h in a humidified 5% CO2 atmosphere. After removing the culture medium, fresh media containing various concentrations of NPPB is added, and incubated for 24 h. Next, 100 μL of Thiazolyl blue tetrazolium bromide at 0.5 mg/mL is added to each well and incubated at 37°C for 1 h. Cells are then dissolved in 100 μL of DMSO, and the absorbance is measured at 570 nm with a Microplate Reader. Concentration-response curves of NPPB are fitted to a Hill equation to obtain GI50 and GI80 (50% and 80% growth inhibition concentrations, respectively) values[2]. |
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参考文献: [1]. Li J, et al. Enhancement of an outwardly rectifying chloride channel in hippocampal pyramidal neurons after cerebral ischemia. Brain Res. 2016 Aug 1;1644:107-17. |
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