High-throughput screening assay for identifying substances capable of modulating cell survival and/or proliferation
OwnerBarbara Ann Spruce, Jeremy John Lambert +2inventors
InventorsBarbara Ann Spruce, Jeremy John Lambert, Peter Downes, Nicholas Reid Leslie
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Abstract
The present invention relates to an assay particularly suited as a high-throughput screening assay for identifying novel compounds for use, for example, as anti-tumour, angiogenesis-modulating and/or anti-inflammatory agents, the assay being based on determining calcium levels within normal and abnormal cells.
Claims19
1) A method for identifying a substance capable of modulating cell survival and/or proliferation, which method comprises:
- a) providing a test substance to a cell or cell population that displays abnormal properties of survival and/or proliferation regulation and to a cell or cell population that displays normal properties of cell survival and/or proliferation regulation;
- b) observing an effect said test substance has on a level of cytosolic calcium within the abnormal and normal cells;
- and
- c) comprising any effects said test substance has on the level of cytosolic calcium within the abnormal and normal cells, so as to identify agents which affect the level of cytosolic calcium within the abnormal cells whilst minimally or substantially not affecting the level of cytosolic calcium within the normal cells.
6) The method according to any preceding claim for identifying a novel sigma (antagonistic) ligand.
7) The method according to any preceding claim wherein the abnormal cell or cell population is obtained from a tumour cell or cell line, a lens epithelial, or microvascular endothelial cell of cell line, a cell or cell line in which NFKappaB is dysregulated, a PTEN null cell or cell line or a persistent inflammatory cell or cell line.
8) The method according to any preceding claim wherein the normal cell or cell population is/are obtained from adult fibroblasts cerebellar granule neurones, mammary epithelial cells, or prostate epithelial cells.
9) The method according to any preceding claim wherein any alternation in the level of cytosolic calcium is observed within a few seconds to 10 minutes.
10) The method according to any preceding claim wherein the assay is conducted for 10-60 minutes in order to observe if changes in calcium level over the time course of the assay.
11) The method according to any claim wherein the cytosolic calcium levels are detected by use of fluorescent probes, which shows a spectral response upon binding calcium and which are detected using fluorescence microscopy, flow cytometry fluorescence spectroscopy, a CCD camera, a luminometer, a FLIPR-flumetric imaging based plate reader or a VIPR voltage ion probe reader.
12) The method according to any preceding claim further comprising conducting an assay to determine a degree of PLC activation in said abnormal and/or normal cells.
17) The method according to any preceding claim further comprising the step of detecting the inhibition of PKB activity.
18) The method according to any preceding claim further comprising the step of conducting a sigma radio ligand binding assay.
19) The method according to any preceding claim further comprising the step or step of conducting one or more additional assays/tests as shown in FIG. 6.
Dependencies are inferred from the claim text.