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In vitro screening strategies for nicotinic receptor ligands
- Source :
- Biochemical Pharmacology. 74:1172-1181
- Publication Year :
- 2007
- Publisher :
- Elsevier BV, 2007.
-
Abstract
- A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat alpha7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC(50) values of 5.5 and 1.6 microM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known alpha7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration-response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC(50) values for acetylcholine-evoked currents in GH4C1/alpha7 cells were 55 and 576 microM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of alpha7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC(50) values are highly comparable to those obtained using conventional manual patch clamp.
- Subjects :
- Agonist
Nicotine
alpha7 Nicotinic Acetylcholine Receptor
medicine.drug_class
Voltage clamp
Receptors, Nicotinic
Biology
Pharmacology
Ligands
Biochemistry
Membrane Potentials
Allosteric Regulation
Calcium flux
medicine
Animals
Fluorometry
Nicotinic Agonists
Patch clamp
Receptor
Acetylcholine receptor
Rats
Nicotinic agonist
Calcium
medicine.drug
Subjects
Details
- ISSN :
- 00062952
- Volume :
- 74
- Database :
- OpenAIRE
- Journal :
- Biochemical Pharmacology
- Accession number :
- edsair.doi.dedup.....3c77463e4c033ae0a5842ee26a1b8577