Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1997-11-26
2001-11-06
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S242000, C435S252300, C435S320100, C536S023100, C530S350000
Reexamination Certificate
active
06312941
ABSTRACT:
FIELD OF THE INVENTION
The invention generally relates to compositions and methods for identifying cytokine, hormone and growth factor signaling pathway agonists and antagonists.
BACKGROUND OF INVENTION
Growth hormone (GH) is well recognized as an important regulator of body growth and metabolism. It is currently used to treat children of short stature, growth hormone deficient adults, surgical patients (to prevent muscle wasting), and burn patients (increase growth rate of skin), to name a few of its therapeutic uses. Other cytokines, hormones and growth factors that act by a similar mechanism regulate a whole host of important body functions, including milk production, the immune system, nerve growth and regeneration, appetite and body fat composition.
As the therapeutic potential of GH (and GH suppression) becomes apparent, a more mechanistic understanding of GH action at the cellular and molecular levels is needed. The initial steps in the pathway following the binding of GH to its receptor have recently been established. It is now known that growth hormone receptor (GHR) forms a complex with a tyrosine kinase. See Argetsinger et al., “Identification of JAK2 as a Growth Hormone Receptor-Associated Tyrosine Kinase,”
Cell
74:237 (1993). The kinase, termed JAK2, is a member of the Janus family of tyrosine kinases. In addition to having a kinase domain, these proteins are characterized by the presence of a second kinase-like domain and the absence of Src homology 2 (SH2), SH3, and membrane-spanning domains. Ligand binding to the cytokine receptor appears to activate the kinase, causing tyrosyl phosphorylation of both JAK2 and the receptor involved, such as GHR for GH mediated signaling. The steps beyond this point are largely unclear, although it has been presumed that other intracellular proteins are recruited to JAK2-receptor complexes.
It is important that the other members of the pathway be identified, given that direct use of some growth factors, such as GH is associated with undesirable side-effects. A better understanding of the signaling pathway and elucidation of the cellular mechanisms by which these agents act is required, in order to define the origin of diseases caused by their malfunction as well as design specific therapeutics.
SUMMARY OF THE INVENTION
The invention generally relates to compositions and methods for identifying cytokine, hormone and growth factor signaling pathway agonists and antagonists, and more particularly, methods and compositions for screening compounds and identifying compounds that will modulate the interactions of cytokine receptors with their intracellular ligands, as well as between their intracellular ligands and other members of the signaling pathway. It is not intended that the present invention be limited to particular cytokines or particular signaling pathways. The present invention contemplates that the methods and compositions described herein will be useful with a variety of cytokines, hormones and growth factors, including but not limited to GH, interferon-gamma (IFN-&ggr;), platelet-derived growth factor (PDGF) epidermal growth factor (EGF), and nerve growth factor (NGF) to identify compounds that will modulate the interactions of receptors that bind these extracellular ligands, as well as between their intracellular ligands and other members of the signaling pathway.
In one embodiment, the present invention contemplates screening compounds and identifying compounds that modulate the interactions of receptors with tyrosine kinases, and in particular JAK2. Furthermore, the present invention contemplates identifying JAK2 substrates and JAK2-binding ligands, and compounds that will modulate the interaction of JAK2 kinase with JAK2 substrates and JAK2 binding ligands.
In one embodiment, the present invention contemplates identifying compounds that modulate the interaction of SH2-B&bgr;, which binds to activated, Tyr phosphorylated JAK2. In other embodiments, the present invention contemplates identifying compounds that modulate the interaction of SH2-B&bgr;, which binds to unphosphorylated JAK2.
In preferred embodiments, SH2-B&bgr; (and in particular, fragments of SH2-B&bgr;) are useful in drug screening assays designed to identify drugs that interfere with the specific binding of JAK2 kinases with their substrate as well as JAK2 kinase activity, and thereby block the activation of downstream signaling molecules. In other embodiments, the present invention contemplates identifying compounds that modulate the interaction of SH2-B&bgr;, which may bind to kinases other than JAK2.
Moreover, the present invention contemplates identifying ERK½ substrates and ERK½-binding ligands, and compounds that will modulate the interaction of MAP kinase with ERK½ substrates and ERK½ binding ligands.
In other embodiments, the invention provides an isolated SH2-B&bgr; polypeptide, or a fragment thereof, having JAK2 kinase-specific binding affinity. The invention provides nucleic acids encoding the SH2-B&bgr; polypeptide and SH2-B&bgr; fragments as part of expression vectors for introduction into cells. The invention provides methods of identifying intracellular molecules which interact with SH2-B&bgr; or SH2-B&bgr; fragments, as well as exogenous agents (i.e. drugs) which disrupt the binding of SH2-B&bgr; and/or fragments thereof to such intracellular targets.
The claimed polypeptide SH2-B&bgr; and SH2-B&bgr; fragments find particular use in screening assays for agents or lead compounds for agents useful in the diagnosis, prognosis or treatment of disease, particularly disease associated with undesirable cell growth, cell movement, apoptosis, differentiation and/or cytokine/growth factor signal responsiveness. One such assay involves forming mixtures of 1) SH2-B&bgr; (or fragments thereof) and 2) an intracellular SH2-B&bgr;-binding ligand, in the presence or absence of 3) a prospective drug candidate. The mixtures are made under conditions that permit the binding of the intracellular SH2-B&bgr;-binding ligand to SH2-B&bgr; (or fragments thereof) and the mixtures are then analyzed for the presence of such binding. A difference in such binding in the presence of such a drug candidate indicates that the agent is capable of modulating the binding of SH2-B&bgr; (or fragments thereof) to an intracellular SH2-B&bgr;-binding ligand.
It is not intended that the present invention be limited by the species (human, murine, rat, etc.) of the binding ligands described above. The polypeptide SH2-B&bgr; and SH2-B&bgr; fragments are shown below to bind across species. Moreover, the nucleic acid sequences described herein allow for the identification of homologues in other species by various methods, including but not limited to amplification (e.g. PCR) using primers designed from the nucleic acid sequence of one species (e.g. rat) on the nucleic acid template of another species (e.g. human).
In one embodiment, the present invention contemplates an isolated nucleic acid encoding at least a fragment of a protein having the amino acid sequence set forth in SEQ ID NO:2. It is not intended that the present invention be limited by the size or nature of the fragment (although it is preferred that such fragments are capable of binding kinases). In one embodiment, said nucleic acid encodes full-length SH2-B&bgr; as set forth in (SEQ ID NO:2) and said nucleic acid comprises SEQ ID NO:1. In another embodiment, said nucleic acid encodes a fragment comprising either residues 527-670, residues 1-555, or residues 1-631 of the amino acid sequence set forth in SEQ ID NO:2. In yet another embodiment, said nucleic acid encodes a fusion protein.
It is not intended that the present invention be limited as to the specific nature of the nucleic acid encoding the peptides described above. In one embodiment, said nucleic acid is contained in a vector. In another embodiment, said vector is in a host cell.
The present invention also contemplates complexes of ligands. In one embodiment, the present invention contemplates a composition, comprising a SH2-B&bgr;—ki
Carter-Su Christin
Karow David S.
Rui Liang-you
Celsa Bennett
Medlen & Carroll LLP
The Regents of the University of Michigan
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