Selective Inhibition of Spindle Microtubules by a Tubulin-Binding Quinazoline Derivative

Abstract

In the research field of tubulin-binding agents for the development of anticancer agents, hidden targets are emerging as a problem in understanding the exact mechanisms of actions. The quinazoline derivative 1-(4-methoxyphenyl)-1-(quinazolin-4yl)ethan-1-ol (PVHD121) has anti-cell proliferative activity and inhibits tubulin polymerization by binding to the colchicine site of tubulin. However, the molecular mechanism of action of PVHD121 in cells remains unclear. Here, we demonstrate that PVHD121 delays mitotic entry and efficiently causes mitotic arrest with spindle checkpoint activation, leading to subsequent cell death. The dominant phenotype induced by PVHD121 was aberrant spindles with robust microtubules and unseparated centrosomes. The microtubules were radially distributed, and their ends appeared to adhere to kinetochores, and not to centrosomes. Extensive inhibition by high concentrations of PVHD121 eliminated all microtubules from cells. PVHD277 [1-(4-methoxyphenyl)-1-(2-morpholinoquinazolin-4-yl)ethan-1-ol], a PVHD121 derivative with fluorescence, tended to localize close to the centrosomes when cells prepared to enter mitosis. Our results show that PVHD121 is an antimitotic agent that selectively disturbs microtubule formation at centrosomes during mitosis. This antimitotic activity can be attributed to the targeting of centrosome maturation in addition to the interference with microtubule dynamics. Due to its unique bioactivity, PVHD121 is a potential tool for studying the molecular biology of mitosis and a potential lead compound for the development of anticancer agents. SIGNIFICANCE STATEMENT Many tubulin-binding agents have been developed as potential anticancer agents. The aim of this study was to understand the subcellular molecular actions of a quinazoline derivative tubulinbinding agent, 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1ol (PVHD121). As expected from its binding activity to tubulin, PVHD121 caused aberrant spindles and inhibited mitotic progression. However, in addition to tubulin, PVHD121 also targeted an unexpected biomolecule involved in centrosome maturation. Due to targeting the biomolecule just before entering mitosis, PVHD121 preferentially inhibited centrosome-derived microtubules rather than chromosome-derived microtubules during spindle formation. This study not only revealed the molecular action of PVHD121 in cells but also emphasized the importance of considering possible tubulin-independent effects of tubulinbinding agents via hidden targeted biomolecules for future use.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules

“…16,17 The cellular effects of these compounds, however, are thought to be distinct from colchicine, which has raised the possibility that there is an alternative cellular target. 17,18 Cells treated with relatively high concentrations of PVHD121 or PVHD303 have abnormal spindles and no microtubules, consistent with inhibition of tubulin polymerization. However, at concentrations close to the anti-proliferative IC 50 of the respective compounds, cells exhibit a more specific defect in centrosome-specific micronucleation.…”

Discovery of a Potent Anticancer Agent PVHD303 with in Vivo Activity

“…It should be noted that 2-aminoquinazolines were found to be fluorescent during our previous structure−activity relationship (SAR) study, and one of them, 2-morpholino derivative 2, has been used as a molecular probe in the previously mentioned study. 7,12 The previous SAR study based on the structure of 1 led to the discovery of quinazoline 3a with 10 times higher activity, that is, an IC 50 value of 0.027 μM against the human lung cancer cell line A549. 6 In addition, other 2-substituted quinazolines, such as 2-trichloromethyl and 2-methoxy, and 2-methythio analogs also exhibited potent activities comparable to 3a.…”

“…In our subsequent study, it was disclosed that 1 selectively disturbed the microtubule formation at centrosomes during mitosis, causing aberrant spindle formation and subsequent cell death . This observation indicates that 1 interacts with biomolecules other than tubulin at the colchicine site and that investigating the molecular action of 1 and its derivatives will lead to the detection of a new drug target molecule.…”

“…In mitotic cells treated with 12 nM, we observed randomly arranged microtubule bundles around the condensed chromosomes and unseparated centrosomes in more than half of the mitotic cells. The appearance of unseparated centrosomes is one of the characteristics of 1 -arrested mitotic cells, suggesting that 9e and 1 share the same mechanism of antimitotic activity other than binding to the tubulin dimer. Even at 16 nM (higher concentration than the IC 50 ), fewer than half of 9e -arrested mitotic cells exhibited diffuse α-tubulin distribution without microtubules.…”

“…10,11 In our subsequent study, it was disclosed that 1 selectively disturbed the microtubule formation at centrosomes during mitosis, causing aberrant spindle formation and subsequent cell death. 12 This observation indicates that 1 interacts with biomolecules other than tubulin at the colchicine site and that investigating the molecular action of 1 and its derivatives will lead to the detection of a new drug target molecule. It should be noted that 2-aminoquinazolines were found to be fluorescent during our previous structure−activity relationship (SAR) study, and one of them, 2-morpholino derivative 2, has been used as a molecular probe in the previously mentioned study.…”