Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-23
2001-02-13
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007900, C435S007920
Reexamination Certificate
active
06187552
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an in vitro method for identifying compounds that inhibit the binding of human JAK2 protein to cytokine receptors.
BACKGROUND OF THE INVENTION
Asthma is characterized by reversible airway obstruction, airway inflammation, and increased airway responsiveness to various stimuli. [1] Increases in the numbers and activation state of eosinophils and mast cells in asthmatic bronchial epithelium are well documented. [2] Additionally, the activation state of airway T lymphocytes is increased. [2] Inhaled &bgr;2-adrenergic receptor agonists control acute airway hyperresponsiveness in some individuals but do not affect the underlying chronic inflammation. When used chronically, glucocorticoids decrease eosinophil accumulation in the lung and reduce symptoms in most, but not all, patients with asthma. However, glucocorticoids inhibit the activation of all inflammatory cells, which results in potential side effects due to immunosuppression. [3]
In patients with asthma, but not in normal individuals, degranulated eosinophils are found below the basement membrane among lung epithelial cells. [4] Also in asthmatics, the number of eosinophils in bronchoalveolar lavage fluid and peripheral blood are elevated, and these levels are correlated with the severity of the disease. [4] When activated, eosinophils release cytotoxic mediators including eosinophil cationic protein, major basic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin. [5] The resulting damage to lung epithelium leads to chronic inflammation and the symptoms of asthma.
The number of cells expressing IL-3, IL-4, IL-5, and GM-CSF mRNA is increased in asthmatic airways. [6] As a result, levels of IL-3, IL-5, and GM-CSF protein are increased in abundance in asthmatic airways. [7,8]The functions of these three cytokines overlap in many ways. IL-3 and GM-CSF are important in the development and maturation of eosinophils in the bone marrow. [9] IL-5 and GM-CSF induce release of eosinophils from the bone marrow and prolong the survival of eosinophils in vitro by preventing apoptosis. [10,11] GM-CSF and IL-5 prime eosinophils to respond more strongly to activators such as platelet activating factor and formyl-Met-Leu-Phe [12,13] and eotaxin [14], which was discovered in the airway of animal models of lung inflammation. [15] Antibodies against IL-5 have been tested for anti-inflammatory effects in animal models of acute lung inflammation. In guinea pigs, anti-IL-5 antibody completely inhibited eosinophil influx into the lungs following antigen challenge [16], but the antibody only partially decreased eosinophilia in similar experiments using mice and monkeys. [17,18] The effect of anti-IL-5 antibodies or IL-5 receptor antagonists [19] in chronic asthma in humans is unknown.
IL-3, IL-5, and GM-CSF receptors are heterodimers consisting of an &agr; subunit unique to each cytokine receptor and a common &bgr; subunit (&bgr;
c
) [20] Both subunits are required for high-affinity ligand binding. [20] The &agr; subunit of these receptors is almost entirely extracellular, leaving the membrane-spanning &bgr;
c
subunit responsible for transducing cytoplasmic signals following cytokine binding. Activation of &bgr;
c
signaling by IL-3, IL-5, or GM-CSF leads to autophosphorylation of the tyrosine kinase JAK2 and activation of the mitogen-activated protein (MAP) kinase pathway. [21-25]
The &bgr;
c
subunit does not contain a consensus protein kinase domain [26]; however, &bgr;
c
binds directly to JAK2 [21,27], suggesting that this is the most proximal kinase involved in signaling. Binding of IL-3, IL-5, or GM-CSF to their receptor complexes likely leads to aggregation of the receptors and activation of JAK2. [28] This is a reasonable hypothesis given that overexpression of JAK2 using baculovirus vectors in insect cells leads to autophosphorylation of JAK2 once the amount of protein accumulates to a critical level. [29]
After JAK2 is activated by autophosphorylation, it phosphorylates the &bgr;
c
subunit of the receptor. This leads to recruitment of signal transducers and activators of transcription (STATs) via binding of their SH2 domains to tyrosine phosphates on the receptor complex (FIG.
1
). The STATs are in turn phosphorylated, probably by JAK2, form dimers with other phosphorylated STATs, and are translocated to the nucleus where they bind directly to DNA. [30,31] Using this mechanism, IL-3, IL-5, and GM-CSF can activate STAT1, STAT3, and two forms of STAT5. [21,32,33]
Truncation and deletion mutagenesis has shown that separate regions of the &bgr;
c
cytoplasmic domain are responsible for activating the JAK2 and MAP kinase pathways. [24,27,34] For example, 62 membrane-proximal cytoplasmic amino acids [SEQ ID NO:3] are necessary and sufficient for activating JAK2 and upregulating transcription of c-myc and pim-1. [24] Pim-1 is a serine/threonine kinase that has anti-apoptotic activity. [35] The more C-terminal region of &bgr;
c
including amino acids 626-763 is necessary for activation of the MAP kinase pathway and increased transcription of c-fos and c-jun. [24] Only the 62 membrane-proximal residues of &bgr;
c
as shown in [SEQ ID NO:3] are required for cell survival and proliferation when other growth factors activate the MAP kinase pathway. [36] This membrane proximal region contains two motifs known as Box 1 and Box 2, which are loosely conserved among many cytokine receptors. Box 1 may be necessary for JAK binding, as deletion of Box 1 or mutation of specific prolines in the motif can decrease JAK binding. [37]
JAK2 is also activated by other cytokine receptors, including those for IL-6 and IL-10 [28], as well as erythropoietin and growth hormone. [38,39] The N-terminal region of JAK2 is necessary for binding to the growth hormone receptor [39] and probably other receptors, as well. JAK2 deletion mutagenesis demonstrated that the N-terminal 239 amino acids are required for binding &bgr;
c
and that the N-terminal 294 residues are sufficient to bind &bgr;
c
. [40] Deletion of the N-terminus does not affect the kinase activity of JAK2 expressed in insect cells. [29] The tyrosine kinase domain of JAK2 is present near the C-terminus of the protein [41], and this domain must be functional to support IL-3- and erythropoietin-induced proliferation and survival. [38,42]
In view of the continuing need for the identification of potential therapeutics for the treatment of asthma, a binding assay which identifies compounds that effect the binding of &bgr;
c
with JAK2 would be a valuable tool.
SUMMARY OF THE INVENTION
A method of screening for compounds potentially useful for treating or preventing asthma, the method comprising contacting a first molecule comprising at least the N-terminal 294 residues of JAK2 protein as shown in [SEQ ID NO:5] with a second molecule comprising at least 62 membrane-proximal cytoplasmic amino acids of &bgr;
c
subunit of the IL-3, IL-5 and GM-CSF receptors as shown in [SEQ ID NO:3] in the presence of a candidate compound. Following this contacting step, a detection step is conducted to determine whether a complex forms between the first and second molecules to determine whether the candidate compound inhibits the formation of the complex, the inhibition being an indication that the candidate compound is potentially useful for treating asthma.
A second method of screening for compounds potentially useful for treating or preventing asthma, the method comprising contacting a first molecule comprising at least the N-terminal 294 residues of JAK2 protein as shown in [SEQ ID NO:5] with a second molecule comprising at leas
Kaytes Paul S.
Roberds Steven L.
Darnley , Jr. James D.
Kemmerer Elizabeth
O'Hara Eileen B.
Pharmacia & Upjohn Company
Rehberg Edward F.
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