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 (IC 50 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.

Distinct functions of dynamin isoforms in tumorigenesis and their potential as therapeutic targets in cancer

“…The mechanism involved could possibly be that this group of inhibitors block dynamin activity and inhibit endocytosis in tumor cells, as observed in HeLa and human neuroblastoma SH-Sy5Y cells [92]. Chlorpromazine marketed under the trade names Thorazine and Largactil among others, is a cationic amphiphilic phenothiazine-derived anti-psychotic drug which inhibits endocytosis by blocking dynamin 2 function [93]. Apart from above-noted dynamin inhibitors, chlorpromazine has been shown to have a relative high specificity for brain cancer [94, 95].…”

“…Moreover, it has been proven to inhibit glioblastoma growth in rat brains when used in combination with Nitrosureas. Phenothiazine-derived antipsychotic drugs (APDs), which are the cationic amphi-pathic drugs, also inhibit clathrin-mediated endocytosis [93]. It is proposed that potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs [93].…”

A Single Atom Change Facilitates the Membrane Transport of Green Fluorescent Proteins in Mammalian Cells

“…The uptake is blocked under these conditions (Figure B, Figure S14), suggesting that the proteins are taken up by the cells via an energy‐dependent pathway, involving either the clathrin‐mediated or caveolar endocytosis. As several proteins are transported via the clathrin‐mediated endocytosis (CME), we studied the uptake in the presence chlorpromazine (CPZ), a cationic amphipathic compound that inhibits the CME by depleting clathrin from the plasma membrane . As observed for SY, no inhibition of cellular uptake was observed in the presence of CPZ (Figure A,B, Figure S14), suggesting that a clathrin‐independent endocytosis (CIE) is probably responsible for the cellular uptake.…”

Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells

“…[32][33][34] Similarly, clathrin also mediated the internalization of CONPs. chlorpromazine, 35 the inhibitor of clathrin-mediated endocytosis, and wortmannin, 36 an indirect inhibitor of clathrin-mediated endocytosis, could improve cell viability of A375 cells in a dose-dependent manner. In line with the viability improvement, GF-AAS results indicated that chlorpromazine significantly reduced the content of intracellular Cu in A375 cells exposed to CONPs compared with control cells, which further validated clathrinmediated endocytosis is the major pathway of internalization for CONPs into melanoma cells.…”