The actin cytoskeleton is a highly organised and dynamic system that processes both external and internal signalling cues into changes in the structure of cellular architecture.  The local organisation of the actin cytoskeleton is responsible for the regulation of many cellular processes such as cell motility, adhesion, endocytosis and invasion.  Dysregulation of actin organisation is seen in many tumour types and disease processes, often through the involvement of actin binding proteins.  Due to the high sequence similarity between the 6 actin isoforms expressed in humans, actin is not a drug target due to the cardiotoxic side-effects of current actin-targeting drugs.  Actin binding proteins are a diverse group of proteins that have distinct functions and tissue and cellular expression profiles, and as such present as more attractive therapeutic targets.  This project aims to identify compounds that impact on the organisation of the actin cytoskeleton either through signalling pathways or direct actin binding proteins.  We have screened a chemical library to identify compounds that reorganise the actin cytoskeleton in ways different from known anti-actin drugs. Using a high content 95 parameter image-based approach, we have screened a 114,400 compound high diversity chemical library.  Changes in filamentous actin structures within the cells were easily visualised by staining with fluorescently labelled phalloidin.  Through the use of custom designed image analysis algorithms, unsupervised statistical clustering and systems-based analyses, we have identified 1306 compounds that impact actin filament organisation and fall into distinct phenotypic groupings.  This suggests that there are a discrete number of ways that polymeric actin can be reorganised in a mammalian cell.  The newly identified compounds produce nodal perturbations in the actin cytoskeleton which can be used to establish functional relationships between pathways that organise the actin cytoskeleton when the drug target is unknown.