High throughput screening method for compounds

Toxicology studies are required to determine the adverse effects of drug candidates and the effect of dosage on toxicity. To assess toxicity, Medicilon offers a wide range of GLP and non-GLP toxicology services. Email:marketing@medicilon.com.cn web:www.medicilon.com

The present ivention relates to a high through put screening method for assaying the non-, pro-, or anti-apoptonic or proliferative or necrotic activity of test compounds in cells, using vectors coding for a specific marker protein, cell lines and spheroids transfected with said vectors.

Thus, it is the major aspect of the present invention to provide an improved screening method, that allows for a high through put screening of compounds to be tested for their non-, pro-, or anti-apoptotic, or proliferative or necrotic effects on tumour cell lines or more particularly on primary cells, cell lines or spheroids of various origins representing healthy tissue- and/or organ-models or disease models. Said improved screening method has to be industrially applicable on a cost effective level.

More importantly, the main embodiment of the present invention allows for a clear and unambigous assessment of non-, pro-, or anti-apoptotic, or proliferative or necrotic activity of test compounds on test cells, which is achieved by a two-step screening assay:

A primary screening step is carried out in order to clearly discriminate between two main groups of different activities. The first group represents the pro-apoptotic and/or necrotic activity of test compounds, whereas the other group represents the non- and/or anti-apoptotic and/or proliferative activity of test compounds.

The secondary screening then allows the clear discrimination between pro-apoptotic and necrotic activity, and between non-, anti-apoptotic and proliferative activity.

Another major embodiment of the present invention a single cell imaging scanning system with an appropriate throughput capacity is applied as fluorescence detecting device in the primary screening, which enables a clear discrimination already at this level between non-, pro, or anti-apoptotic, or proliferative or necrotic activity of test compounds.

In yet a further embodiment the method of the present invention is applied for drug screening.

A further important aspect of the invention is the applicability of the herein described method for toxicological studies by assaying the necrotic/toxic activity of test compounds.

The screening method according to the present invention comprises stably transfecting a group of cells, either cells of a tumor cell line, or cells applicable as healthy tissue-and/or organ-model or disease model such as cell lines and primary cells of human or animal origin isolated from healthy individuals/animals or patients/animals suffering of diseases such as cancers, autoimmune diseases, organ transplantation derived pathogenesis, cardiovascular diseases and degenerative diseases of various origin or the like or spheroids with a vector coding for and expressing a marker protein in the respective cells or cell line. The transfected cells are transferred into 96 or 384 or 1536 well plates in a suitable culture medium. Afterwards the respective test compound is added in series of dilutions. The presence and/or activity, i.e. the decrease or increase, respectively, of the expressed marker protein in said group of cells is monitored by conventional methods, and compared with the results observed with a parallel group of the same test cells, which was not exposed to the test compound. With the screening method of the present invention an average of several hundreds to thousands test compounds can be evaluated per day.

These and other features, aspects, and advantages of the invention will become better understood with reference to the accompanying Figures, the description and appended claims.

The present invention relates to the assessment of the non-, pro-, or anti-apoptotic or proliferative or necrotic activity of test compounds on test cell systems of various origins in an industrially applicable assay for purposes such as drug screening and toxicology studies. Test compounds can have different activities on the test cells, i.e. non-, pro-, or anti-apoptotic, or proliferative and/or necrotic activity.

In a primary screening, the overall fluorescence activity of the test cells within a single well is measured with an appropriate fluorescence-detecting device, e.g. a fluorescence plate-reader. An appropriate cut-off value is set in order to clearly discriminate between two main groups of different activities. The first group represents the pro-apoptotic and/or necrotic activity of test compounds, whereas the other group represents the non- and/or anti-apoptotic and/or proliferative activity of test compounds.

In a secondary screening, the single-cell fluorescence activity of each test cell within a population of test cells is measured with an appropriate fluorescence detecting device, e.g. flow cytometry, microfluidic devices (chip technology) and single cell imaging scanning systems. This measurement allows the clear discrimination between pro-apoptotic and necrotic activity, and between non-, anti-apoptotic and proliferative activity.

The combination of primary and secondary screening allows the clear assessment of non-, pro-, or anti-apoptotic, or proliferative or necrotic activity of test compounds on test cells.

A single cell imaging, scanning systems with an appropriate throughput capacity can also be used as fluorescence detecting device in the primary screening, in order to clearly discriminate at that level between non-, pro, or anti-apoptotic, or proliferative or necrotic activity of test compounds.

Test compounds comprise synthetic or natural compounds, chemical or peptide structures or a combination thereof, proteins or recombinant proteins, pure compounds or a combination of pure compounds or extracts, such as plant extracts, extracts of marine micro- and macro-organisms and extracts of microbial fermentations. Test compounds are incubated with test cells either alone or in combination with known pro- or anti-apoptotic compounds or stimuli of various origins.