title: Development of Automated Patch Clamp Assay for Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists in Automated QPatch-16.
abstract: The α7 nicotinic acetylcholine receptor (α7 nAChR) is an important and challenging target for drug discovery in the area of neuropsychiatric disorders. The current screening for chemicals targeting α7 nAChRs is primarily achieved by the use of low-throughput assay two-electrode voltage clamp (TEVC) in nonmammalian Xenopus oocytes. Automated patch clamp system has emerged as an attractive approach compared to conventional electrophysiology. To develop a mammalian cell-based functional assay in an automated electrophysiology system, we in this study generated a stable expression of α7 nAChRs in GH3 cells that originated from a rat pituitary tumor cell line and utilized automated QPatch-16 to test a set of tool compounds and chemicals identified as α7 agonists by TEVC. For the improvement of evaluating weak or partial α7 nAChRs agonists, we achieved enhancement of the signal-to-noise ratio by the addition of a positive allosteric modulator PNU-120596, which only activates α7 current in the presence of agonist. This improved assay was further validated by using known α7 partial agonists, such as RG3487, EVP-6124, and A-P90. Using this validated assay, we were able to identify a novel agonist 140507C that partially activates α7 nAChRs. Taken together, our results validate the use of QPatch-16 for evaluation α7 partial agonists, demonstrating its utility as an effective tool for α7 ion channel drug discovery.
authors.affiliation: 1 Department of Molecular and Cellular Pharmacology, State Key Laboratory of Natural and Biomimetic Drugs, Peking University School of Pharmaceutical Sciences , Beijing, China .
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Hao
authors.forename: Jingshu
authors.initials: J
authors.lastname: Tang
authors.forename: KeWei
authors.initials: K
authors.lastname: Wang
mesh_terms: D000818:Animals; D001331:Automation; D001643:Bridged Bicyclo Compounds / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007191:Indazoles / Q000737:chemistry / Q000494:pharmacology; D007555:Isoxazoles / Q000737:chemistry / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000737:chemistry / Q000494:pharmacology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D010671:Phenylurea Compounds / Q000737:chemistry / Q000494:pharmacology; D011812:Quinuclidines / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D013876:Thiophenes / Q000737:chemistry / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000819:agonists*
chemical_list: C508388:1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; C573817:7-chloro-N-quinuclidin-3-yl-benzo(b)thiophene-2-carboxamide; D001643:Bridged Bicyclo Compounds; D007191:Indazoles; D007555:Isoxazoles; D018722:Nicotinic Agonists; D010671:Phenylurea Compounds; D011812:Quinuclidines; D013876:Thiophenes; D064569:alpha7 Nicotinic Acetylcholine Receptor; C554954:N-((3S)-1-azabicyclo(2.2.2)oct-3-yl)-1H-indazole-3-carboxamide hydrochloride