Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-04
2003-04-22
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S007100, C435S007310, C435S069100, C435S069700, C435S069900, C435S471000, C435S476000, C435S481000, C435S483000, C435S320100, C435S243000, C435S252300, C435S254100, C435S255100, C536S023100, C536S023400
Reexamination Certificate
active
06551786
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to methods for identifying pharmaceutically relevant compounds, and particularly to methods for selecting compounds capable of modulating protein-protein interactions.
BACKGROUND OF THE INVENTION
There has been much interest in protein-protein interactions in the field of proteomics. A number of biochemical approaches have been used to identify interacting proteins. These approaches generally employ the affinities between interacting proteins to isolate proteins in a bound state. Examples of such methods include coimmunoprecipitation and copurification, optionally combined with cross-linking to stabilize the binding. Identities of the isolated protein interacting partners can be characterized by, e.g., mass spectrometry. See e.g., Rout et al.,
J. Cell. Biol.,
148:635-651 (2000); Houry et al.,
Nature,
402:147-154 (1999); Winter et al.,
Curr. Biol.,
7:517-529 (1997). A popular approach useful in large-scale screening is the phage display method, in which filamentous bacteriophage particles are made by recombinant DNA technologies to express a peptide or protein of interest fused to a capsid or coat protein of the bacteriophage. A whole library of peptides or proteins of interest can be expressed and a bait protein can be used to screening the library to identify peptides or proteins capable of binding to the bait protein. See e.g., U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,698; and 5,837,500. Notably, the phage display method only identifies those proteins capable of interacting in an in vitro environment, while the coimmunoprecipitation and copurification methods are not amenable to high throughput screening.
The yeast two-hybrid system is a genetic method that overcomes certain shortcomings of the above approaches. The yeast two-hybrid system has proven to be a powerful method for the discovery of specific protein interactions in vivo. See generally, Bartel and Fields, eds.,
The Yeast Two
-
Hybrid System,
Oxford University Press, New York, N.Y., 1997. The yeast two-hybrid technique is based on the fact that the DNA-binding domain and the transcriptional activation domain of a transcriptional activator contained in different fusion proteins can still activate gene transcription when they are brought into proximity to each other. As shown in
FIG. 1
, in a yeast two-hybrid system, two fusion proteins are expressed in yeast cells. One has a DNA-binding domain of a transcriptional activator fused to a test protein. The other, on the other hand, includes a transcriptional activating domain of the transcriptional activator fused to another test protein. If the two test proteins interact with each other in vivo, the two domains of the transcriptional activator are brought together reconstituting the transcriptional activator and activating a reporter gene controlled by the transcriptional activator. See, e.g., U.S. Pat. No. 5,283,173.
Because of its simplicity, efficiency and reliability, the yeast two-hybrid system has gained tremendous popularity in many areas of research. Numerous protein-protein interactions have been identified using the yeast two-hybrid system. The identified proteins have contributed significantly to the understanding of many signal transduction pathways and other biological processes. For example, the yeast two-hybrid system has been successfully employed in identifying a large number of novel cell cycle regulators that are important in complex cell cycle regulations. Using known proteins that are important in cell cycle regulation as baits, other proteins involved in cell cycle control were identified by virtue of their ability to interact with the baits. See generally, Hannon et al., in
The Yeast Two
-
Hybrid System,
Bartel and Fields, eds., pages 183-196, Oxford University Press, New York, N.Y., 1997. Examples of cell cycle regulators identified by the yeast two-hybrid system include CDK4/CDK6 inhibitors (e.g., p16, p15, p18 and p19), Rb family members (e.g., p130), Rb phosphatase (e.g., PP1-&agr;2), Rb-binding transcription factors (e.g., E2F-4 and E2F-5), General CDK inhibitors (e.g., p21 and p27), CAK cyclin (e.g., cyclin H), and CDK Thr161 phosphatase (e.g., KAP and CDI1). See id. “[T]he two-hybrid approach promises to be a useful tool in our ongoing quest for new pieces of the cell cycle puzzle.” See id at page 193. In another example, the yeast two-hybrid system proved to be a powerful approach in analyzing the yeast pheromone response pathway, a complex multistep signal transduction process in haploid yeast cell mating. See generally, Sprague et al., in
The Yeast Two
-
Hybrid System,
Bartel and Fields, eds., pages 173-182, Oxford University Press, New York, N.Y., 1997. As described in Sprague, various genes were isolated from mutant yeast strains having altered pheromone response patterns. However, it was not clear how the proteins encoded by these genes function in the pheromone response pathway. The yeast two-hybrid system was utilized to test such proteins and mutant forms thereof for their ability to interact with each other. As a result, new insights and better understandings of the complex process were achieved. See id.
Reverse two-hybrid systems have also been developed based the yeast two-hybrid systems and are disclosed in, e.g., U.S. Pat. Nos. 5,525,490; 5,733,726; 5,885,779; Vidal et al.,
Proc. Natl. Acad. Sci. USA,
93:10315-10320 (1996); and Vidal et al.,
Proc. Natl. Acad. Sci. USA,
93:10321-10326 (1996). Such reverse two-hybrid systems can be useful in identifying and selecting compounds capable of modulating a particular protein-protein interaction.
However, such reverse two-hybrid systems in the art typically depend on gene activation in the nucleus of host cells and has generally required that specific protein-protein interactions between fusion proteins occur within the nucleus of host cells. Thus, although certain conventional reverse yeast two-hybrid systems have been used successfully in identifying compounds capable of modulating protein-protein interactions, their usefulness may be limited when it is used in the context of protein-protein interactions that require non-nuclear environment. For example, traditional transcription-based reverse two-hybrid systems are not applicable to interactions between transcriptional factors. In addition, many cell surface proteins and their ligands contain disulfide bonds, which can be disrupted under the intracellular reducing conditions. Additionally, posttranslational protein modifications, particularly glycosylation, typically would preclude the nuclear localization of the modified proteins.
Cytosolic and cell surface protein-protein interactions play major roles in normal cellular functions and biological responses. In particular, many cytosolic and cell surface protein-protein interactions are involved in disease pathways. For example, attacks by pathogens such as viruses and bacteria on mammalian cells typically begin with interactions between viral or bacterial proteins and mammalian cell surface proteins. In addition, many protein-protein interactions between factors in the transcriptional machineries are also valuable drug targets. Therefore, there is a need in the art for improved methods that can be used to identify compounds capable of modulating such protein-protein interactions.
SUMMARY OF THE INVENTION
This invention provides a versatile and sensitive assay system for detecting protein-protein interactions that circumvents the above-described limitations inherent in prior art methods. Particularly, the present invention utilizes the so-called inteins, which are peptide sequences capable of directing protein trans-splicing both in vivo and in vitro. An intein is an intervening protein sequence in a protein precursor that is excised from the protein precursor during protein splicing. Protein splicing results in the concomitant ligation of the flanking protein fragments, i.e., the exteins, with a native peptide bond, thus forming a mature extein protein and the free intein. It is now known t
Guzo David
Lambertson David A.
Myriad Genetics Inc.
Myriad IP Dept.
Zhang Jay Z.
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