Award details

Identification of novel spindle checkpoint inhibitors

Reference	BB/D005736/1
Principal Investigator / Supervisor	Professor Stephen Taylor
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	293,868
Status	Completed
Type	Research Grant
Start date	10/11/2005
End date	09/11/2008
Duration	36 months

Abstract

The aim of this proposal is to identify novel, small molecule spindle checkpoint inhibitors. To do this we will adapt two spindle checkpoint assays into a multi-well format and screen a commercially available compound library. Our approach is to establish relatively simple screens, thereby maximising the chances of success in a timely and cost effective manner. Hits will then be subjected to secondary screens and further analysis to identify inhibitors which have the potential to be useful tools. We will then be in a good position to initiate hypothesis driven experiments. At the same time, we will search compound libraries for analogues and perform structure-activity analyses. This will place us in an ideal position to establish strategic collaborations with chemists to develop the inhibitors into powerful research tools. We will purchase a DIVERSet compound library from ChemBridge, consisting of 10,000 compounds. This collection is assembled using a pharmacophore diversity analysis of ChemBridge's total collection of ~460,000 compounds. Screens of an earlier DIVERSet consisting of ~16,000 compounds yielded numerous research tools including Monastrol and Blebbistatin. The newer collection has been improved, filtering out reactive and coloured compounds, and includes structures that are more stable and more 'lead' like. We will purchase, set up and calibrate equipment to facilitate a semi-automated screening approach, including an automated pipetting system, a plate washer and a plate reader. These three BioTek instruments are cost effective solutions that will meet our requirements and are used in other screening facilities. They are also versatile, providing flexibility to refine the screens if necessary. In addition, all three are compatible with microplate stackers and robotic handlers, which will allow us perform larger and more sophisticated screens in the future. We will conduct two screens. The first will involve plating mitotic arrested HeLa cellsin 384-well plates in the presence of a spindle toxin. Compounds will then be added to the plates for 4-6 hours. If the compound overrides the checkpoint, the cells will exit mitosis and reattach to the plate. Unattached mitotic cells will be then be washed away, the attached cells fixed, stained with Hoechst and the DNA fluorescence measured. Compounds that result in a high fluorescence will be considered hits. In the second screen, asynchronous DLD-1 cells will be plated in 384-well plates and exposed to a spindle toxin plus compounds for 6-8 hours. The cells will then be fixed and stained with a mitotic marker to determine the mitotic index. If a compound overrides the checkpoint, mitotic cells will not accumulate. Therefore, compounds that do not yield a high mitotic index will be considered hits. We will then perform suitable secondary screens followed by 'low resolution' analysis of potential checkpoint inhibitors using flow cytometry, fluorescence microscopy and simple time-lapse studies. Defining a checkpoint profile for each compound will then allow us to focus on a subset of the more interesting compounds. These will be subjected to 'high resolution' experiments including analysis of synchronised populations of cells, 3D microscopy and FPLC based biochemical experiments. The compound library will be a representative subset of a much larger pharmacophore collection. Therefore, a search of commercial compound collections will identify numerous analogues. We will purchase these analogues and perform structure-activity analyses. This information will then facilitate collaborative efforts to synthesise novel analogues in order to further develop the inhibitors. In the meantime, the first generation inhibitors will allow us to undertake our hypothesis driven experiments. Towards the end of the program, we will design experiments to identify the molecular targets of the key compounds.

Summary

THE BIG PICTURE: The fertilisation of a human egg by a sperm generates a single cell, which, following successive rounds of cell division, creates a person comprised of billions of cells. Before each division, the cell must replicate and segregate its genome such that both daughter cells receive all the genetic information required for further growth and development. Because the human genome is divided into 46 chromosomes, the segregation process presents a challenge to the cell: it is critical that each daughter cell receives one copy of each chromosome. If the segregation process is not accurate, a cell may either gain or lose chromosomes. Fortunately, evolution has found a way to address this challenge. A surveillance mechanism, called the spindle checkpoint, monitors the segregation process and delays division until accuracy can be guaranteed. Unfortunately however, chromosome missegregation can still occur and is often associated with age related diseases. For example, Downs syndrome arises when a child inherits an extra copy of chromosome 21. In addition, human cancer cells routinely make errors and consequently have highly irregular numbers of chromosomes. We are interested in understanding how this surveillance mechanism operates at the molecular level and how it goes wrong in diseases associated with aging. THE SPINDLE CHECKPOINT: Chromosome segregation is mediated by a microtubule spindle. Chromosomes attach to the spindle via kinetochores, specialised domains which bind microtubules and move chromosomes to the spindle equator. When all the chromosomes are aligned at the equator, the cell enters anaphase; the chromosomes split longitudinally and segregate to opposite ends of the spindle. The cell then divides down the equator so that each daughter inherits one copy of each chromosome. We now know that the spindle checkpoint operates by delaying anaphase until all the chromosomes are correctly aligned. We know that kinetochores of unaligned chromosomes generate an 'anaphase wait' signal and that once all the kinetochores are attached, this signal is extinguished, giving the all clear for anaphase. Our research focuses on how this wait signal is generated, transmitted and finally extinguished. We have identified protein components of the spindle checkpoint and discovered that they localise to kinetochores, i.e. they are in the right place at the right time to monitor kinetochore-microtubule interactions. In order to understand what these proteins do, we have used traditional genetic approaches, such as gene deletion or gene repression, to deplete checkpoint proteins from cells followed by analysing the subsequent effects. Surprisingly, these experiments suggest that the checkpoint proteins do not just monitor the alignment process: they also appear to be required for chromosome attachment and/or alignment. OUR PLAN: While these observations suggest that there is interplay between the mechanical processes and the regulatory processes, it is possible that our observations are due to the experimental system. Because kinetochores are complex structures, it is possible that depleting a protein may have knock-on structural consequences which inhibit attachment. Therefore, we now wish to use a new approach to study the checkpoint, namely chemical genetics. We plan to use small drug-like molecules to selectively inhibit the checkpoint without depleting proteins from the cell. This way, kinetochore structure should not be affected. We will then be able to rigorously test whether the mechanics and regulation of chromosome segregation are linked. The first step in this process is to identify a panel of checkpoint inhibitors. This we will do by screening a library of 10,000 molecules for ones which inhibit the spindle checkpoint. After identifying inhibitors which will be useful tools, we will then use them in conjunction with microscopy techniques to carefully study chromosome alignment and the spindle checkpoint.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Selective Chemical Intervention In Biological Systems (SCIBS) [2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

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