Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-10-18
2002-06-25
Nguyen, Dave T. (Department: 1633)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S350000, C530S329000, C530S325000, C530S326000, C530S327000, C530S328000, C514S002600
Reexamination Certificate
active
06410693
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to signal transduction inhibitors.
BACKGROUND OF THE INVENTION
The c-Jun NH2-terminal kinase (JNK) is a member of the stress-activated group of Mitogen Activated Protein kinases (MAP kinases) implicated in the control of cell growth. The JNK signal transduction pathway is activated in response to environmental stress and by the engagement of several classes of cell surface receptors, including cytokine receptors, serpentine receptors, and receptor tyrosine kinases. Whitmarsh et al.,
J. Mol. Med.,
74:589 (1996). In addition, genetic studies of Drosophila have demonstrated that JNK is required for early embryonic development. Sluss et al.,
Genes
&
Dev.,
10:2745 (1996); Riesgo-Escovar et al.,
Genes
&
Dev.
10:2759 (1996). In mammalian cells, JNK has been implicated in the immune response, oncogenic transformation, and apoptosis. JNK mediates these-effects, at least in part, by increasing the expression of target genes. Targets of the JNK signal transduction pathway include the transcription factors c-Jun, ATF2, and Elk-1. Whitmarsh et al., supra.
JNK is activated in the liver by metabolic oxidative stress. Mendelson et al.,
Proc. Natl. Acad. Sci. USA
93:12908-12913 (1996). Activation of JNK also occurs in the kidney during stress, for example, during Bchemic renal failure. Demari et al.,
Am. J. Physiol.
272:F292-F298 (1997). JNK is also activated during cardiovascular disease such as ischemia Ireperfusion and during organ transplantation. Pombo et al.,
J. Biol. Chem.
269:26546-26551 (1994); Force et al.,
Circ. Res.
78:947-53 (1996).
While JNK is located in both the cytoplasmic and the nuclear compartments of quiescent cells, activation of the JNK signal transduction pathway is associated with accumulation of JNK in the nucleus. Mechanisms governing this sub-cellular distribution have not been previously elucidated.
Anchor or tethering proteins play an important role in the regulation of multiple signal transduction pathways. These anchor proteins, which include the nuclear factor kappa B (NFkB) inhibitor IkB, the A kinase anchor protein (AKAP) group of proteins that bind the type II cyclic adenosine monophosphate (AMP) dependent protein kinase, and the p190 protein that binds Ca
2+
-calmodulin-dependent protein kinase II, localize their tethered partners to specific sub-cellular compartments. Verma et al.,
Genes
&
Dev.,
9:2723 (1995); McNeill et al.,
J. Biol. Chem.,
270:10043 (1995); Faux et al.,
Trends Biochem. Sci.,
21:312 (1996)). Anchor proteins also target enzymes to specific substrates, and create multi-enzyme signaling complexes, such as the Ste5 MAP kinase scaffold complex and the AKAP79 kinase/phosphatase scaffold complex. Choi et al.,
Cell,
78:499 (1994); Klauck et al.,
Science,
271:1589 (1996); Faux et al.,
Cell,
85:9 (1996)).
SUMMARY OF THE INVENTION
The invention, which is based on the discovery of a cytoplasmic anchor protein, JNK-interacting protein 1 (JIP-1; SEQ ID NO:1), features JIP-1 polypeptides and nucleic acids, therapeutic compositions containing these polypeptides and nucleic acids, and methods of administering these compositions. JIP-1 specifically binds to and inhibits the biological effects of JNK, including the initiation of apoptosis and oncogenic transformation. JIP-1 is therefore useful as a therapeutic agent for treating pathological conditions characterized by apoptosis or transformation. For example, JIP-1 compositions can be used to treat neurodegenerative diseases characterized by apoptosis, including Parkinson's disease and Alzheimer's disease; and blood clots, which left untreated could result in stroke and associated memory loss. Other conditions that can be treated using the compositions and methods of the invention are autoimmune diseases such as arthritis; other conditions characterized by inflammation; and malignancies, such as leukemias, e.g., chronic myelogenous leukemia (CML). other conditions that can be treated with JIP-1 compositions include oxidative damage to organs such as the liver and kidney, and heart disease, particularly damage due to ischemia/reperfusion and cardiomyopathy. JIP-1 compositions can also be used to treat donor organs for transplantation. These organs are exposed to substantial environmental stress, the effects of which are blocked by JNK inhibitors such as JIP-1.
The invention features a substantially pure JIP-1 polypeptide. A “JIP-1 polypeptide” is a protein having an amino acid sequence that specifically binds JNK to the same extent, or at least 10% of the binding activity of wildtype JIP-1. Such polypeptides can be from 5 to 200 amino acids in length, e.g., from 10 to 100 amino acids in length, or from 20 to 50 amino acids in length. Such polypeptides include the JNK Binding Domain (JBD), or portions thereof, of JIP-1 (e.g., amino acids 148 to 174, forming the “core” of the JBD of wildtype JIP-1, shown in
FIG. 2
, and having the sequence SGDTYRPKRPTTLNLFPQVPRSQDTLN; SEQ ID NO:3). JIP-1 polypeptides are preferably derived from a mammal, such as a mouse or a human.
In various embodiments, the polypeptide is soluble, the polypeptide includes the JNK-binding domain of JIP-1 or a portion thereof, the polypeptide is at least 80%, 90%, or 100% identical to the amino acid sequence from amino acid 148 to amino acid 174 of JIP-1 (the core JNK-binding domain; SEQ ID NO:3), or the polypeptide has an amino acid sequence identical to the amino acid sequence from amino acid 148 to amino acid 174 of JIP-1 (SEQ ID NO:3), or the polypeptide is at least 80%, 90%, or 100% identical to the amino acid sequence from amino acid 127 to amino acid 281 of JIP-1 (the JNK-binding domain; SEQ ID NO:4).
The polypeptides of the invention can be modified to enhance their uptake by cells. Such modifications increase the hydrophobicity of molecules to facilitate passage through the lipid bilayer of the cell membrane. For example, polypeptides can be complexed with myristic acid or packaged in liposomes. Alternatively, JIP-1 polypeptides can be complexed with hydrophobic moieties (e.g., lipids) or peptides that increase the delivery of proteins into cells.
The invention also includes peptide mimetics of JIP-1 polypeptides. A “peptide mimetic” of a known polypeptide is a compound that mimics the activity of the peptide or polypeptide, but which is composed of molecules other than, or in addition to, amino acids.
By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), and thus includes peptides, proteins, and fusion proteins.
A “substantially identical” polypeptide sequence differs from a given sequence only by conservative amino acid substitutions or by one or more nonconservative substitutions, deletions, or insertions located at positions which do not destroy the function of the polypeptide compared to wildtype JIP-1. Polypeptides of the invention can be 70%, 80%, 85%, 90%, or 95% identical to wildtype JIP-1.
A “substantially pure” preparation is at least 60% by weight of the compound of interest, e.g., a JIP-1 polypeptide or fragment of a JIP-1 polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and more preferably at least 95% by weight of the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or High Pressure Liquid Chromatography (HPLC) analysis.
The polypeptides of the invention include, but are not limited to, recombinant polypeptides, natural polypeptides, and synthetic polypeptides, as well as preproteins or proproteins and biologically active fragments. A “biologically active fragment” of JIP-1 is a fragment having at least 50%, 70%, 80%, 90%, 95%, or 100% or greater, of the activity of naturally occurring or synthetic, full length JIP-1.
The polypeptides of the invention can be physically linked to another polypeptide, e.g., a marker polypeptide. For example, the polypeptide can be fused to a hexa-histidine tag to facil
Davis Roger J.
Dickens Martin
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