Screening system

Details Screening system Screening system

2001-03-09

2004-11-23

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

Reexamination Certificate

active

06821728

ABSTRACT:

CROSS REFERENCES TO RELATED APPLICATIONS
This application claims the priority of European patent application no. 98111428.3, filed Jun. 22, 1998, the disclosure of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The present invention concerns a screening system, to identify lead compounds. Said screening system is especially suitable for the screening of small molecules, whereby peptides are the much preferred small molecules.
BACKGROUND ART
Screening compounds in order to find molecules for further drug development, is one of the crucial steps in such drug development. Up to now, product screening is performed in vitro, e.g. by so called high-throughput screening systems or by phage display. Phage display systems can identify proteins or peptides which are expressed on the surface of a bacteriophage particle (see R. Cortese et al.,
Selection of biologically active peptides by phage display of random peptide libraries
, Current Opinion in Biotechnology 1996, 7, 616-621, 1996). Only in the case of a successful interaction between the two proteins, the bacteriophage can infect the bacterial
E. coli
host. This event becomes visible since this successful bacteriophage will spread and form plaques which consist of lysed
E. coli
cells. High throughput screening systems suitable for the screening of small molecules are e.g. described in WO 9710253 by Merck & Co. Inc. Such systems are generally applied for the screening of e.g. plant extracts, etc. in vitro.
These screening systems of the state of the art are very laborious and time-consuming.
The disadvantage of these screening systems of the state of the art is that they are far away from natural conditions and very slow. In particular, no post-translational modifications of proteins occur in the phage display system. Additionally, the interactions of the phage system occur in vitro, whereas the interesting protein—protein interactions in mammals, including man, actually occur in the cell, many of them in the nucleus.
It is also known to study protein—protein interactions in vivo by using two hybrid proteins. The protein of interest is fused to a DNA binding domain, e.g. a GAL4 DNA binding domain, whereas a library of potential interaction candidates is fused to a transcription activation domain, e.g. a GAL4 transcription activation domain. Only in the case of successful interaction between the protein of interest and a real interacting candidate, the transcriptional activation domain is brought to the reporter gene (lacZ), which leads to a blue colouring of this cell (see Fields and Song,
A novel genetic system to detect protein—protein interactions
, Nature 340, 245, 1989). Such systems are additionally described in reviews and patent literature (see e.g. S. Fields and R. Sternglanz, The Two-hybrid System: An Essay for Protein-protein Interactions, Reviews, TIG August 1994, Vol. 10, No. 8, p. 286-292, and
The Matchmaker Two
-
hybrid System
, published by Clontech, as well as U.S. Pat. Nos. 5,283,173 and 5,468,614 both assigned to the Research Foundation of the State University of New York). A further two-hybrid system, which is a modification of the classical one, and which—due to the modification—is dependent on post-translational modifications of one of the proteins, is described in U.S. Pat. No. 5,637,463. Such two-hybrid systems are disclosed to have three major applications: Testing known proteins for interaction, defining domains or amino acids critical for an interaction, and screening libraries for proteins that bind some target protein. It is also known to modify the existing classical two-hybrid screening. Instead of fusing a library of cellular proteins to an activation domain, peptide libraries are fused to the GAL4 activation domain (see WO97/41255, WO95/34646, WO92/05286, FR 2720068). It is also known to fuse a peptide library to a DNA binding domain (see U.S. Pat. No. 5,498,530). The selection principle is identical to the classical two-hybrid (see Young, Wo and Fields,
Protein—peptide interactions analysed with the yeast two-hybrid system
, Nucl. Acids Res. 23, 1152, 1995).
Another DNA-binding domain and activation domains e.g. disclosed in connection with screening for peptides which interact with the cyclin dependent kinase II. Here, the Cdk2 was fused to a LexA DNA binding domain, and the peptide library was cloned into the active loop of the
E. coli
thioredoxin, which was also fused to an activation domain (see Colas, Cohen, Jessen, Grishina, McCoy & Brent,
Genetic selection of peptide aptamers that recognise and inhibit cyclin
-
dependent kinase II
, Nature, 30, 554, 1996 and WO 94/10300). Information about other reporter genes can be found in Vidal, Brachmann, Fatai, Harlow and Böcke, Reversed two-hybrid to detect dissociation of protein—protein and DNA protein interactions, Proc. Natl. Acad. Sci. USA 93, 10315, 1996, and Vidal, Brown, Chen, Böcke and Harlow,
Genetic characterisation of a mammalian protein—protein interaction domain by using a yeast reverse two
-
hybrid system
, Proc. Natl. Acad. Sci. USA 93, 10321, 1996.
Other two-hybrid systems were described by A. Aronheim et al.,
Isolation of an AP
-1
repressor by a novel method for detecting protein—protein interactions
, Mol. Cell. Biol. 17, 3094-3102, 1997; and by I. Stagljar et al.,
A genetic system based on split
-
ubiquitin for the analysis of interactions between membrane proteins in vivo
, Proc. Natl. Acad. Sci. USA 95, 5187-5192, 1998. In the Aronheim report a two-hybrid system based on the mammalian GDP-GTP exchange factor (GEF)hSoS is described.
Also known are so-called three-hybrid systems to detect RNA-protein interactions (see e.g. D. J. SenGupta et al.,
A three
-
hybrid system to detect RNA
-
protein interactions in vivo
, Proc. Acad. Natl. Sci. USA 93, 8496-8501, 1996), or mediating proteins (WO97/24457).
The two-hybrid method, as it is described in the above mentioned literature, thus allows to study molecular interactions that might be the causes of diseases. However, it does not show how to use such systems in the search for active substances and the development of novel drugs.
It is also already known to use the two-hybrid technology to find exogenous small molecules, which inhibit protein—protein interaction (see WO97/41255, U.S. Pat. No. 5,569,588, WO92/05286). Hybrid proteins, of which the interaction should be disrupted, can also be expressed as fusions to a DNA binding domain (LexA) and an activation domain (B42). The small molecule can be covalently linked to a polymer bead, which is photoreleasable from the small molecule (see Huang & Schreiber,
A yeast genetic system for selecting small molecule inhibitors of protein—protein interaction in nano droplets
, Proc. Natl. Acad. Sci. USA 94, 1336, 1997).
This technology is not suitable for a screening of interacting peptides, since it is laborious in view of the production of peptides, and the analysis of the possibly interacting peptides.
Attempts to screen HIV-1 Rev protein inhibitors have already been made (U.S. Pat. No. 5,691,137 to Brandeis University, Waltham, Mass.). In said attempts the re-porter gene used was CUP 1 modified by insertion of an intron sequence, and a HIV-1 Rev response element 3′ of the CUP 1 open reading frame. Upon interaction of Rev with its response element, the splicing of the pre-mRNA is inhibited leading to a inactive protein.
DISCLOSURE OF THE INVENTION
Hence, it is a general object of the invention to provide a much faster screening system allowing the trial of many different effector peptides with agonist or antagonist effect in parallel, whereby the time and costs of the first step in drug development can be dramatically reduced.
This object is achieved by providing a screening system for agonists or antagonists comprising a eukaryotic host cell stably transformed with a selection system enabling the survival of the cells in the case of desired interaction between at least one target protein and an effector peptide, and a peptide expressing system,
whereby said selection system comprises
at least one

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