Identification of novel microtubule inhibitors effective in fission yeast and human cells and their effects on breast cancer cell lines.

Jun Morishita,Paul Nurse

doi:10.1098/rsob.210161

Abstract

Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by βIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.

Highlights

Microtubules are dynamic protein polymers composed of α- and β-tubulins
The observed defects could be grouped into three classes: (i) microtubules are undetectable, and α-tubulin accumulates in the nucleus (as seen with compound L1 in figure 1c(ii)); (ii) mitotic cells exhibit only short bipolar spindles (as seen with compound P2 in figure 1c(iii)); and (iii) tubulin is found as a single dot at the SPB without the formation of short or long microtubules (as seen with compound B8 in figure 1c(iv) and figure 1d)
Unlike most previous small molecule screens, which identified tubulin inhibitors through their cytotoxic effects on human tissue culture cells, we first employed fission yeast for rapid and cost-effective primary screening followed by screening in human tissue culture cells

Summary

Introduction

Microtubules are dynamic protein polymers composed of α- and β-tubulins. They are found in eukaryotic cells where they form cytoskeletal architectures supporting a wide range of cellular functions, such as chromosome segregation, cell shape, intracellular transport and cellular movement [1,2]. When proliferating cells are exposed to microtubule inhibitors, bipolar spindle formation and microtubule attachment to kinetochores are inhibited [3], activating the spindle assembly checkpoint (SAC) This causes cell cycle arrest prior to the metaphase–anaphase transition [4], eventually leading to apoptosis in tissue culture cells [5]. Anti-microtubule drugs such as colchicine, nocodazole (NOC), benomyl and paclitaxel are commonly used to study the diverse functions of microtubules and to synchronize cells at mitosis. Drugs that both stabilize microtubules (e.g. taxanes such as paclitaxel) and destabilize microtubules (e.g. vinca alkaloids, such as vinblastine) are used as anti-cancer agents [6,7]. Most anti-microtubule drugs bind one of four main sites within microtubules impacting tubulin stability: the laulimalide site (stabilizing), taxane/

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