title: Mapping the binding pocket of a novel, high-affinity, slow dissociating tachykinin NK3 receptor antagonist: biochemical and electrophysiological characterization.
abstract: The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3.
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland. Electronic address: pari.malherbe@bluewin.ch.
authors.forename: Pari
authors.initials: P
authors.lastname: Malherbe
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Knoflach
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Anne
authors.initials: A
authors.lastname: Marcuz
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Bohnert
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Weber
authors.affiliation: Discovery Chemistry, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Henner
authors.initials: H
authors.lastname: Knust
authors.affiliation: Discovery Chemistry, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Hasane
authors.initials: H
authors.lastname: Ratni
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Will
authors.initials: W
authors.lastname: Spooren
authors.affiliation: Discovery Neuroscience, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Ballard
authors.affiliation: Discovery Chemistry, F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, Grenzacherstrasse 124, CH4070, Basel, Switzerland.
authors.forename: Caterina
authors.initials: C
authors.lastname: Bissantz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D014150:Antipsychotic Agents / Q000494:pharmacology; D001665:Binding Sites; D002219:Carbamates / Q000493:pharmacokinetics / Q000494:pharmacology*; D002491:Central Nervous System Agents / Q000494:pharmacology; D059290:Dopaminergic Neurons / Q000187:drug effects / Q000502:physiology; D005260:Female; D005849:Gerbillinae; D006168:Guinea Pigs; D057809:HEK293 Cells; D006801:Humans; D008124:Locomotion / Q000187:drug effects / Q000502:physiology; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018377:Neurotransmitter Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D065842:Pars Compacta / Q000187:drug effects / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology; D010880:Piperidines / Q000493:pharmacokinetics / Q000494:pharmacology*; D018041:Receptors, Neurokinin-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D018042:Receptors, Neurokinin-3 / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives / Q000494:pharmacology; D046509:Tissue Culture Techniques
chemical_list: C000599354:4-(3,4-dichlorophenyl)-1-(1-(1-methylcyclopropanecarbonyl)piperidine-4-5 carbonyl)pyrrolidin-3-yl)methylcarbamic acid 4-fluoro-phenyl ester; D014150:Antipsychotic Agents; D002219:Carbamates; D002491:Central Nervous System Agents; D018377:Neurotransmitter Agents; D010446:Peptide Fragments; D010880:Piperidines; D018041:Receptors, Neurokinin-2; D018042:Receptors, Neurokinin-3; C054724:senktide; D013373:Substance P; C091554:SR 142801
keywords: Dual NK2/NK3 antagonist; Firing frequency; Inositol phosphate accumulation; Locomotor activity; NK(3) modeling; RO5328673