Tubulin/microtubules as novel clozapine targets

Abstract

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.

Keywords: clozapine; microtubule; olanzapine; schizophrenia; tubulin.

FIGURE 1

Clozapine causes mitotic arrest in HeLa cells. A, Immunofluorescence images of HeLa cells stained with 4ʹ,6‐diamidino‐2‐phenylindole (DAPI) (cyan) and anti‐mitotic protein antibody (MPM‐2) (green). (1) A mitotic cell (metaphase) in the center (arrow) and interphase cells. Cells in prophase (2), prometaphase (3), metaphase (4) anaphase (5), telophase (6), and late telophase/cytokinesis (7) are also shown. Scale bar, 20 μm (b) Percentage of cells in mitotic phase treated with 50 μM clozapine for 5 h. The p‐value was calculated using Student’s t test. Error Bars: Standard Error of Mean

FIGURE 2

Isolation and identification of clozapine and olanzapine‐binding proteins in the mouse brain. A, Schematics of clozapine and olanzapine conjugates. Amino acids in polypeptides are represented by single letter codes. The HRV‐3C protease cleavage site is indicated. B, Silver stained sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) image of clozapine/olanzapine‐binding proteins in mouse brains isolated using clozapine and olanzapine conjugates. The clozapine/olanzapine‐binding proteins were isolated from phosphate‐buffered saline (PBS)‐soluble and Nonidet P‐40 (NP‐40)‐soluble fractions of mouse brain using clozapine and olanzapine conjugates and separated by SDS‐PAGE and stained by silver staining. M: The positions of molecular weight markers in kDa. Arrowheads indicate the position of α and β tubulins. C, Silver stained SDS‐PAGE images of the results of three independent trials and sequenced bands. The numbers labeled on each band correspond to those in Table 1

FIGURE 3

Clozapine inhibits tubulin polymerization. Purified tubulin heterodimer polymerized in 20% dimethyl sulfoxide (DMSO) without or with clozapine (5, 10, and 20 μM) and monitored turbidimetrically by absorbance at 350 nm. DMSO‐induced polymerization of tubulin to microtubules was inhibited by clozapine in dose‐dependent manner

FIGURE 4

Clozapine disrupts the microtubule network in HeLa cells. Immunofluorescence images of microtubules in HeLa cells treated without or with clozapine (50 and 100 μM) for 3 h. Arrow indicates the indentation of the nucleus. Arrowheads indicate membrane blebs. Scale bar, 20 μm.