Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-03-11
2001-01-16
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Vector, per se
C435S006120, C435S007100, C435S091400, C536S023100
Reexamination Certificate
active
06174722
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to an in-vitro process for analyzing transcription of viral and cellular genes which can be automated and which is suitable for efficient and economical bulk screening with the aim of finding specific chemical lead structures which have a selective effect on gene activity.
BACKGROUND OF THE INVENTION
Screening natural products for bioactive constituents experienced an upswing after it had emerged that rational design of active substances alone does not allow a successful search for active substances. Thus, research focuses not only on libraries of chemical substances and combinatory libraries, but, yet again, on traditional extracts of natural products as sources for substances. This is due mainly to the diversity of the substances which these extracts contain. Model analytical methods prove that extracts of microbial fermentations contain approximately 500 classes of compounds, which differ greatly in their structure. As regards their diversity, they are thus far superior to chemical and combinatory substance libraries.
A factor which limits the pharmacological exploitation of the varied, and as yet largely unresearched, potential of natural products is the number of compatible, meaningful processes with which candidate active substances can be tested. In particular, processes are required which can be employed for identifying highly-specific pharmacologically active substances whose application entails a minimum of side effects.
The process described hereinbelow is based on an approach where substances are tested for their potential of engaging in the very first step of converting genetic information, i.e. the regulation of gene transcription. Such a process is intended to identify substances with direct or indirect, positive or negative effects on transcription.
The transcription strength of a gene is determined by the gene-regulatory elements of this gene, in particular by the promoter, by enhancers or by silencers. The action of the gene-regulatory elements is mediated and converted by transcription factors and cofactors. These transcription factors can have a negative or else positive effect on the transcription rate of a gene and thus contribute to the transcription strength. In the meantime, a large number of transcription factors have been identified as important “molecular switches” in the course of a large number of cellular processes, including signal transduction, cell-cycle control, differentiation and controlled cell death (apoptosis).
Most of the signals, received by the cell, which affect the transcription strength of genes are “registered” by transmembrane proteins, transmitted intracellularly by means of signal transduction chains and converted by transcription factors. Examples of proteins which receive external signals are cAMP-binding proteins, sensors for growth signals (such as the serum response factor, SRF), hormone receptors or transcription factors which participate in cytokin expression, so-called STAT proteins (signal transducers and activators of transcription).
In the meantime, a multiplicity of substances are known which have a direct or indirect effect on the transcription strength of genes. Such substances are employed, inter alia, as pharmacologically active substances in pharmaceuticals, even though the action of these substances is frequently not specific. Taking such pharmaceuticals therefore frequently entails undesired side-effects.
For example, immunological diseases are treated with pharmaceuticals which comprise cyclosporin and steroid derivatives as active substances. Cyclosporin A forms a complex with cyclophilin. The latter inhibits calcineurin, a ubiquitous phosphatase, which dephosphorylates proteins via various metabolic routes. Calcineurin regulates, for example, the transport of a subunit of the transcription factor NFAT from the cytosol into the nucleus (Liu, J. (1993) Immunology Today 14, 290-295). NFAT (nuclear factor of activated T-cells) participates in activation of some immunologically relevant genes. Cyclosporin A (CsA) indirectly regulates expression of these genes via its effect on NFAT (nuclear factor of activated T-cells). However, since cyclosporin A only indirectly regulates NFAT activity, viz. via the ubiquitous calcineurin, cyclosporin A also acts as a vasoconstrictor and as a nephro- and neurotoxin, via other metabolic routes. If a pharmacologically active substance were known with which NFAT could be inhibited specifically, possibly directly, then a medicine containing this active substance would probably cause fewer side-effects.
The pharmacologically active substances which, besides the desired effect, also entail potent side-effects, also include glucocorticoids. Glucocorticoids have been employed for many years in the standard therapy of allergies, rheumatism, inflammations and other diseases caused by an overreactive immune system. They cause, inter alia, inhibition of the activation of the cell-type-specific transcription factor NfkB (Scheinmann, R. I., Cogswell, P. C., Lofquist, A. K. & Baldwin Jr., A. S. (1995) Science 270, 283-286; Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A. & Karin M. (1995) Science 270, 286-290) by stimulating the formation of a cellular NF
K
B inhibitor, viz. I
K
B protein. I
K
B, in turn, prevents the transfer of active NF
K
B dimers into the nucleus and thus the activation of important immunological target genes. Similarly to what has been said for CsA, the effect of glucocorticoids on gene expression is relatively unspecific since glucocorticoids act not only on NF
K
B, but also on other proteins.
These examples make it clear that there exists a great demand for pharmacologically active substances whose profile of action is as specific as possible. To find novel chemical lead structures which have such properties, a great number of substances must be tested for their specific activity.
Despite an identical genetic make-up, individual cells always express specific proteins only, depending on the cell type and/or certain diseases or defects and the respective degree to which these cells are developed and differentiated. The basis of this individuality of cells is considered to be the specific repertoire of gene-regulatory proteins, for example the cell-type-specific and development-specific make-up which provides certain transcription factors and cofactors (accessory proteins) which regulate the coordinated and controlled transcription of distinct genes.
Specific pharmacologically active substances should therefore provide the selective activation or inhibition of the transcription of pathologically relevant genes in cells of a defined type. To identify such active substances, a transcription process is required in which the effect of candidate active substances on the transcription of individual genes, i.e. on the proteins which participate in transcriptional regulation and on the gene-regulatory elements, can be measured directly under defined conditions. Since a multiplicity of candidate active substances must be tested, other prerequisites would be that the process is simple to carry out and that it can be automated.
The first cell-free transcription process was described by Weil et al. (Weil, P. A., Luse, D. S., Segall, J., Roeder, R. G. (1979) Cell 18, 469-484). In this process, concentrated extracts from cell nuclei (so-called S100 extracts) (Weil, P. A., Segall, J., Harris, B. Ng, S. Y., Roeder, R. G. (1979) J. Biol. Chem. 254, 6163-6173), and purified RNA polymerase II were employed for the in-vitro transcription. Without exogenous RNA polymerase II, these concentrated, but not further purified, nuclear extracts were not capable of transcription (Weil, P. A., Luse, D. S., Segall, J., Roeder, R. G. (1979) Cell 18, 469-484; Dignam, J. D., Martin, P. L., Shastry, B. S., Roeder, R. G. (1983) Methods in Enzymology 101, 582-598).
Starting from such nuclear extracts, processes were subsequently developed by means of which transcription factors were isolated using several purification steps. These processes include, inter
Kirschbaum Bernd
Meisterernst Michael
Stahl Wilhelm
Winkler Irvin
Aventis Pharma Deutschland GmbH
Foley & Lardner
Horlick Kenneth R.
Siew Jeffrey
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