title: Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis.
abstract: Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 µm range, the most potent being trifluoperazine (IC50 2.6 ± 0.7 µm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia.|Present address: Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany.
authors.forename: James A
authors.initials: JA
authors.lastname: Daniel
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia.
authors.forename: Ngoc
authors.initials: N
authors.lastname: Chau
authors.affiliation: Centre for Chemical Biology, Chemistry, The University of Newcastle, Callaghan, NSW, 2308, Australia.
authors.forename: Mohammed K
authors.initials: MK
authors.lastname: Abdel-Hamid
authors.affiliation: Epithelial Cancer Group, The University of Queensland Diamantina Institute, Translational Research Institute, University of Queensland, Woolloongabba, QLD, 4102, Australia.
authors.forename: Lingbo
authors.initials: L
authors.lastname: Hu
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, 13125, Berlin, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: von Kleist
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia.
authors.forename: Ainslie
authors.initials: A
authors.lastname: Whiting
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia.
authors.forename: Sai
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Maamary
authors.affiliation: Epithelial Cancer Group, The University of Queensland Diamantina Institute, Translational Research Institute, University of Queensland, Woolloongabba, QLD, 4102, Australia.
authors.forename: Shannon R
authors.initials: SR
authors.lastname: Joseph
authors.affiliation: Epithelial Cancer Group, The University of Queensland Diamantina Institute, Translational Research Institute, University of Queensland, Woolloongabba, QLD, 4102, Australia.
authors.forename: Fiona
authors.initials: F
authors.lastname: Simpson
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, 13125, Berlin, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
authors.affiliation: Centre for Chemical Biology, Chemistry, The University of Newcastle, Callaghan, NSW, 2308, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: McCluskey
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
mesh_terms: D014150:Antipsychotic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D010640:Phenothiazines / Q000494:pharmacology*; D014168:Transferrin / Q000378:metabolism
chemical_list: D014150:Antipsychotic Agents; D002966:Clathrin; D010640:Phenothiazines; D014168:Transferrin; D034281:Dynamins; C031637:phenothiazine
keywords: antipsychotic drugs; calmidazolium; chlorpromazine; drug discovery; dynamin; flunarizine; fluphenazine; phenothiazines; small molecule inhibitors; stemetil; trifluoperazine