Tubulin/microtubules as novel clozapine targets

• Published in: Neuropsychopharmacology reports Vol. 42; no. 1; pp. 32 - 41
• Main Authors: Hino, Mizuki, Kondo, Takeshi, Kunii, Yasuto, Matsumoto, Junya, Wada, Akira, Niwa, Shin‐ichi, Setou, Mitsutoshi, Yabe, Hirooki
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.03.2022; John Wiley and Sons Inc; Wiley
• Subjects: Animals; Antibodies; Antipsychotics; Chromatography; Chromatography, Liquid; Cloning; clozapine; Clozapine - pharmacology; Experiments; HeLa Cells; Humans; Mass spectrometry; Mice; Mice, Inbred ICR; microtubule; Microtubules - metabolism; olanzapine; Original; Peptides; Polymerization; Potassium; Proteins; Psychotropic drugs; Schizophrenia; Scientific imaging; Software; Tandem Mass Spectrometry; tubulin; Tubulin - chemistry; Tubulin - metabolism; Tubulin - pharmacology; United States > US; Japan; microtubule; tubulin; olanzapine; clozapine; schizophrenia
• ISSN: 2574-173X; 2574-173X
• DOI: 10.1002/npr2.12221

Aim Clozapine is currently the only effective drug for treatment‐resistant schizophrenia; nonetheless, its pharmacological mechanism remains unclear, and its administration is limited because of severe adverse effects. By comparing the binding proteins of clozapine and its derivative olanzapine, which is safer but less effective than clozapine, we attempted to clarify the mechanism of action specific to clozapine. Methods First, using the polyproline rod conjugates attached with clozapine or olanzapine, clozapine‐binding proteins in extracts from the cerebra of 7‐week‐old ICR mice were isolated and separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to identify proteins. Second, the effect of clozapine on tubulin polymerization was determined turbidimetrically. Finally, the cellular effects of clozapine were observed in HeLa cells by immunofluorescence microscopy. Results Alpha and β tubulins were the most abundant clozapine‐binding proteins. We also found that clozapine directly binds with α and β tubulin heterodimers to inhibit their polymerization to form microtubules and disturbs the microtubule network, causing mitotic arrest in HeLa cells. Conclusion These results suggest that α and β tubulin heterodimers are targeted by the clozapine and the microtubules are involved in the etiology of schizophrenia. Clozapine‐binding proteins were investigated in mouse brain by using the polyproline rod method. The most abundant clozapine‐binding proteins were α and β tubulins. This figure shows immunofluorescence staining of HeLa cells treated without or with indicated doses of clozapine for 3 hours with anti‐tubulin antibody, indicating that clozapine disrupts the microtubule network in the cell. Scale bar, 20 μm.