A High-Content, Cell-Based Screen Identifies Micropolyin, A New Inhibitor of Microtubule Dynamics Manu De Rycker 1 , Laurent Rigoreau 2 , Sarah Dowding 1 and Peter J. Parker 1,3, * 1 Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, London WC2A 3PX, UK 2 Cancer Research Technology, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK 3 Division of Cancer Studies King's College London, New Hunt's House, Guy's Hospital, St Thomas Street, London SE1 1UL, UK *Corresponding author: Peter J. Parker, peter.parker@cancer.org.uk High-content cell-based screens provide a power- ful tool to identify new chemicals that interfere with complex biological processes. Here, we describe the identification of a new inhibitor of microtubule dynamics (micropolyin) using a high- content screen. Integrated high-resolution imaging allowed for fast selection of hits and progression to target identification. Treatment of cells with micropolyin efficiently causes a pro-metaphase arrest, with abnormal spindle morphology and with the spindle assembly checkpoint activated. The arrest appears to result from interference of micropolyin with microtubule dynamics. We show in vitro that tubulin is indeed the target of micro- polyin and that micropolyin inhibits microtubule polymerization. Our results demonstrate the power of high-content image- and cell-based screening approaches to identify potential new drug candidates. As our approach is unbiased, it should allow for discovery of new targets that may otherwise be overlooked. Key words: cell cycle, chemical genetics, high-content screen, inhibitors, microtubule Received 14 February 2009, revised and accepted for publication 23 March 2009 The study of biological processes using small molecules, chemical genetics, is increasingly used as a parallel approach to traditional genetics to discover new proteins and ⁄ or to investigate their roles [reviewed in (1–3)]. Small molecules have distinct advantages over standard genetic methods, such as temporal control (rapid effect and often rapid reversibility), dose control and straightforward scalability (important for large screens). By analogy with traditional genetics, chemical genetics can be applied both in a forward and reverse way. However, unlike traditional reverse genetics (knock-out, RNA interfer- ence, etc.), general application of small molecules in a reverse chemi- cal genetic way is not yet possible, as compounds that interfere with the function of all proteins are not available. Forward chemical genetic screens provide a way to identify new small molecules to study biological systems and to discover new functions for proteins. These screens involve a phenotypic screen with a small molecule library, typically followed by deconvolution of the target protein(s). The development of automated image acquisition and analysis tech- niques has greatly enhanced the scale at which these types of experi- ments can be performed. Discovery of the targets of the hits is usually the rate-limiting step in this process, as no generally applica- bly methods exist (3,4). In recent years, several successful forward chemical genetic screens have identified new small molecules that have aided in the understanding of protein function and provided new tools for the study of biology, examples of new molecules and their targets are monastrol ⁄ Eg5 (5), melanogenin ⁄ prohibitin (6) and chrolactomycin ⁄ telomerase (7). While the goal of these screens in academia is usually to increase understanding of a biological process, similar screens are performed in the pharmaceutical industry to identify new leads for drug development. Often these two different applications go hand in hand, with new molecules ⁄ targets discovered in academia being developed into drugs (e.g. Eg5) and compounds dis- covered in industry being used as tools in academic labs [e.g. the PI3Kinase inhibitor LY294002(8)]. Cell division and cell migration both play a key role in the success of tumour cells, and both processes are critically dependent on the cyto- skeleton. Microtubules in particular are fundamental for cell division as they make up the mitotic spindle which is responsible for correct separation of the sister chromatids at mitosis (9). During mitosis, microtubule dynamics increase dramatically (four- to 100-fold) com- pared with interphase. This dynamic behaviour is critical for many steps in mitosis, including the 'search and capture' of kinetochores during prophase and the fast oscillations necessary to establish a metaphase plate (10,11). Many small molecules have been identified that inhibit microtubule polymerization (at high concentration) and microtubule dynamics (at 10–100 times lower concentrations). Typical examples are the taxanes, vinca alkaloids and colchicine. Even at low concentrations these compounds efficiently block cells in mitosis by inhibiting microtubule dynamics. At such concentrations, cells arrest in a metaphase-like state with a subset of chromosomes close to the spindle poles and unable to congress (12,13). The arrest is mediated through activation of the spindle-assembly checkpoint (SAC) (14). 599 Chem Biol Drug Des 2009; 73: 599–610 Research Article ª 2009 John Wiley & Sons A/S doi: 10.1111/j.1747-0285.2009.00817.x