title: A negative allosteric modulator modulates GABAB-receptor signalling through GB2 subunits.
abstract: An γ-aminobutyric acid type B (GABAB)-receptor mediates slow and prolonged synaptic inhibition in the central nervous system, which represents an interesting target for the treatment of various diseases and disorders of the central nervous system. To date, only one activator of the GABAB-receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABAB-receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABAB-receptor, although several positive allosteric modulators have been developed, it is only recently that the first negative allosteric modulator (NAM), CLH304a (also named Compound 14), has been reported. In the present study, we provide further information on the mechanism of action of CLH304a, and also show the possibility of designing more NAMs, such as CLH391 and CLH393, based on the structure of CLH304a. First we show that CLH304a inhibits native GABAB-receptor activity in cultured cerebellar granular neurons. We then show that CLH304a has inverse agonist properties and non-competitively inhibits the effect of agonists, indicating that it binds at a different site to GABA. The GABAB-receptor is a mandatory heterodimer made of GB1 subunits, in which agonists bind, and GB2 subunits, which activate G-proteins. By using various combinations made up of wild-type and/or mutated GB1 and GB2 subunits, we show that CLH304a acts on the heptahelical domain of GB2 subunits. These data revealed the possibility of designing innovative NAMs acting in the heptahelical domain of the GB2 subunits, offering novel possibilities for therapeutic intervention based on GABAB-receptor inhibition.
authors.affiliation: Cellular Signalling laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Centre for Brain Science, Huazhong University of Science and Technology, 430074 Wuhan, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Sun
authors.affiliation: National Centre for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Linhai
authors.initials: L
authors.lastname: Chen
authors.affiliation: Cellular Signalling laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Centre for Brain Science, Huazhong University of Science and Technology, 430074 Wuhan, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.affiliation: Cellular Signalling laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Centre for Brain Science, Huazhong University of Science and Technology, 430074 Wuhan, China.
authors.forename: Zhixiong
authors.initials: Z
authors.lastname: Xia
authors.affiliation: Institut de Génomique Fonctionnelle, CNRS, MMR 5203, Université Montpellier 1 et 2, 34094 Montpellier cedex 5, France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
authors.affiliation: National Centre for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China fjnan@simm.ac.cn jfliu@mail.hust.edu.cn.
authors.forename: Fajun
authors.initials: F
authors.lastname: Nan
authors.affiliation: Cellular Signalling laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Centre for Brain Science, Huazhong University of Science and Technology, 430074 Wuhan, China fjnan@simm.ac.cn jfliu@mail.hust.edu.cn.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Liu
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D018755:GABA Agonists / Q000737:chemistry / Q000494:pharmacology*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007651:Keto Acids / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D010636:Phenols / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D021122:Protein Subunits; D018080:Receptors, GABA-B / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: C000609586:CLH304a; D018755:GABA Agonists; D007651:Keto Acids; D010636:Phenols; D021122:Protein Subunits; D018080:Receptors, GABA-B
keywords: GABAB-receptor; cerebellar granular neurons; heptahelical domain; negative allosteric modulator