U.S. patent application number 10/470957 was filed with the patent office on 2004-07-22 for protein kinase signalling.
Invention is credited to Atkin, Julie, Fantino, Emmanuelle, Wilks, Andrew Frederick.
Application Number | 20040142404 10/470957 |
Document ID | / |
Family ID | 3826817 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040142404 |
Kind Code |
A1 |
Wilks, Andrew Frederick ; et
al. |
July 22, 2004 |
Protein kinase signalling
Abstract
The present invention provides a method of selecting or
designing a compound for the ability to regulate JAK activity. The
method comprises assessing the ability of the compound to modulate
the interaction of the pseudo-substrate loop (PSL) with the kinase
like domaain (KLD) of JAK. In addition the present invention
provides compounds which inhibit JAK and methods of treatment of
JAK-associated disease states.
Inventors: |
Wilks, Andrew Frederick;
(Victoria, AU) ; Atkin, Julie; (Victoria, AU)
; Fantino, Emmanuelle; (Victoria, AU) |
Correspondence
Address: |
Kate H Murashige
Morrison & Foerster
Suite 500
3811 Valley Centre
San Diego
CA
92130-2332
US
|
Family ID: |
3826817 |
Appl. No.: |
10/470957 |
Filed: |
January 21, 2004 |
PCT Filed: |
January 30, 2002 |
PCT NO: |
PCT/AU02/00088 |
Current U.S.
Class: |
435/15 ;
435/320.1; 435/325; 435/69.1; 530/388.26; 703/11 |
Current CPC
Class: |
G01N 2500/00 20130101;
C12N 9/1205 20130101; C12Q 1/485 20130101; A61P 11/06 20180101;
A61K 38/00 20130101; A61P 35/00 20180101; C07K 14/71 20130101; G01N
2333/9121 20130101; A61P 37/00 20180101 |
Class at
Publication: |
435/015 ;
435/069.1; 435/320.1; 435/325; 530/388.26; 703/011 |
International
Class: |
G06G 007/48; G06G
007/58; C12Q 001/48; C12P 021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 30, 2001 |
AU |
PR 2791 |
Claims
1. A method of selecting or designing a compound for the ability to
regulate JAK activity, the method comprising assessing the ability
of the compound to modulate the interaction of the pseudo-substrate
loop (PSL) with the kinase like domain (KLD) of JAK.
2. A method of selecting, or designing, a compound which regulates
JAK activity, the method comprising (i) selecting or designing a
compound which has a conformation and polarity such that it
interacts with at least one ligand selected from the group
consisting of residues 667-679, 711-726 and 757-765 of human JAK2
and corresponding regions of JAK family members; and (ii) testing
the compound for the ability to interfere with the binding of the
PSL with the KLD.
3. A method as claimed in claim 1 or claim 2 in which the method
comprises selecting or designing a compound which has a
conformation and polarity such that it interacts with at least one
ligand selected from the group consisting of residues 673, 677,
711-715, 718, 724, 759 and 760 of human JAK2 and corresponding
regions of JAK family members.
4. A method as claimed in claim 1 or claim 2 in which the method
comprises selecting or designing a compound which has a
conformation and polarity such that it interacts with at least two
ligands selected from the group consisting of residues 673, 677,
711-715, 718, 724, 759 and 760 of human JAK2 and corresponding
regions of JAK family members.
5. A method as claimed in claim 1 or claim 2 in which the method
comprises selecting or designing a compound which has a
conformation and polarity such that it interacts with at least
three selected from the group consisting of residues 673, 677,
711-715, 718, 724, 759 and 760 of human JAK2 and corresponding
regions of JAK family members.
6. A method as claimed in claim 1 or claim 2 in which the method
comprises selecting or designing a compound which has a
conformation and polarity such that it interacts with at least four
ligands selected from the group consisting of residues 673, 677,
711-715, 718, 724, 759 and 760 of human JAK2 and corresponding
regions of JAK family members.
7. A compound which interacts with the PSL or the KLD such as to
interfere with the binding of the PSL with the KLD such that the
activity of the JAK is reduced when compared to that of the JAK in
the absence of the compound.
8. A compound as claimed in claim 7 in which the compound binds to
the substrate-binding cleft of the KLD such as to interfere with or
prevent the binding of the PSL to the KLD.
9. A compound as claimed in claim 7 or claim 8 in which the
compound has a conformation and polarity such that it binds to at
least ligand selected from the group consisting of residues
667-679, 711-726 and 757-765 of human JAK2.
10. A compound as claimed in any one of claims 7 to 9 in which the
compound binds to at least one ligand selected from the group
consisting of residues 673, 677, 711-715, 718, 723-724, 759 and 760
of human JAK2.
11. A compound as claimed in any one of claims 7, to 9 in which the
compound binds to at least two ligands selected from the group
consisting of residues 673, 677, 711-715, 718, 723-724, 759 and 760
of human JAK2.
12. A compound as claimed in any one of claims 7 to 9 in which the
compound binds to at least three ligands selected from the group
consisting of residues 673, 677, 711-715, 718, 723-724, 759 and 760
of human JAK2.
13. A compound as claimed in any one of claims 7 to 9 in which the
compound binds to at least four ligands selected from the group
consisting of residues 673, 677, 711-715, 718, 723-724, 759 and 760
of human JAK2.
14. A compound as claimed in any one claims 7 to 13 in which the
compound is comprised at least in part of amino acids.
15. A compound as claimed in claim 14 in which the amino acids are
derived from the sequence of the PSL.
16. A compound as claimed in claim 14 or claim 15 in which the
compound is a peptide.
17. A compound as claimed in claim 16 in which the peptide is
cyclic.
18. A therapeutic composition comprising an compound as claimed in
any one of claims 7 to 17.
19. A compound which regulates JAK, the compound having the same or
similar physico-chemical properties to a peptide comprising
following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 in which: X1
is any amino acid, preferably proline or glycine; X2 is any amino
acid, preferably alanine, valine, leucine or isoleucine; X3 is any
amino acid, preferably glutamate or aspartate; X4 is phenylalanine
or tyrosine, preferably phenylalanine; X5 is leucine or methionine
or isoleucine; X6 is arginine or lysine; X7 is methionine or
leucine or isoleucine, preferably methionine; X8 is isoleucine or
leucine or methionine, preferably isoleucine.
20. A compound as claimed in claim 19 in which the compound has the
same or similar physico-chemical properties to the peptide
PAEFMRMI.
21. A compound as claimed in claim 19 or 20 in which the compound
is a peptide comprising the following amino acid sequence:
X1-X2-X3-X4-X5-X6-X7-X8 in which X1 is any amino acid, preferably
proline or glycine; X2 is any amino acid, preferably alanine,
valine, leucine or isoleucine; X3 is any amino acid, preferably
glutamate or aspartate; X4 is phenylalanine or tyrosine, preferably
phenylalanine; X5 is leucine or methionine or isoleucine; X6 is
arginine or lysine; X7 is methionine or leucine or isoleucine,
preferably methionine; X8 is isoleucine or leucine or methionine,
preferably isoleucine.
22. A compound as claimed in any one of claims 19 to 21 in which
the compound is the peptide PAEFMRMI.
23. A compound obtained by the method of any one of claims 1 to
6.
24. A method of treating a subject suffering from a JAK-associated
disease state, the method comprising administering to the subject a
compound as claimed in any one of claims 7 to 23.
25. A method as claimed in claim 24 in which the JAK-associated
disease stateis selected from the group consisting of Asthma,
Eczema, Food Allergy, Inflammatory Bowel Disease, Crohn's Disease,
Leukaemia, Lymphoma, Cutaneous Inflammation, Immune Suppression By
Solid Tumour and Prostate Cancer.
26. The use of a compound as claimed in any of claims 7 to 23 in
the preparation of a medicament for the treatment of a
JAK-associated disease state.
27. A method of designing or selecting a compound which modulates
JAK activity, the method comprising subjecting a compound obtained
by a method as claimed in any one of claims 1 to 6 to biological
screens and assessing the ability of the compound to modulate JAK
activity.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of regulators of
the JAK family of protein tyrosine kinases. More particularly, the
present invention relates to assays and screens for chemical
entities which regulate the activity of the JAK family of protein
tyrosine kinases. The invention further relates to the use of these
chemical entities in therapeutic situations where the regulation of
a protein tyrosine kinase, in particular a member of the JAK family
of protein tyrosine kinases is indicated.
BACKGROUND OF THE INVENTION
[0002] Since the immune system is central to the protection of an
individual from an external biological threat, diseases of the
immune system are therefore a consequence of one or a combination
of three problems with the immune system.
[0003] Underproduction or suppression of the immune system (e.g.
AIDS or SIDS);
[0004] Overproduction of cells of the immune system (e.g Leukemia
or Lymphoma);
[0005] Overproduction of the effects of the immune system (e.g.
[0006] Inflammation);
[0007] Inappropriate activation of the effects of the immune system
(e.g. allergy).
[0008] Treatments of diseases of the immune system are therefore
aimed at either the augmentation of immune response or the
suppression of inappropriate responses. Since cytokines play a
pivotal role in the regulation of the immune system, they are
appropriately considered to be key targets for therapeutic
intervention in immune pathologies. Similarly, the intracellular
signal transduction pathways that are regulated by cytokines are
potential points of therapeutic intervention in diseases that
involve overproduction of cytokine signalling. The JAK family of
protein tyrosine kinases (PTKs) play a central role in the cytokine
dependent regulation of the proliferation and end function of
several important cell types of the immune system. As such they
represent excellent, well-validated targets for the purpose of drug
discovery; the notion being that potent and specific inhibitors of
each of the four JAK family members will provide a means of
inhibiting the action of those cytokines that drive immune
pathologies, such as asthma (e.g. IL-13; JAK1, JAK2), and
leukemia/lymphoma (e.g. IL-2: JAK1 and JAK3). Furthermore, certain
types of cancer such as prostate cancer develop autocrine
production of certain cytokines as a selectable mechanism of
developing growth and/or metastatic potential. An example of this
is cancer of the prostate, where IL-6 is produced by and stimulates
the growth of prostate cancer cell lines such as TSU and TC3
(Spiotto M T, and Chung T D, 2000). Interestingly, levels of IL-6
are elevated in sera of patients with metastatic prostate
cancer.
[0009] A great deal of literature covers the area of cytokine
signalling. The present inventors have focussed on the JAK/STAT
pathway that is involved in the direct connection of cytokine
receptor to target genes (such as cell cycle regulators (e.g. p21)
and anti-apoptosis genes (such as Bcl-X.sub.L)).
[0010] The JAK/STAT Pathway
[0011] The delineation of a particularly elegant signal
transduction pathway downstream of the non-protein tyrosine kinase
cytokine receptors has recently been achieved. In this pathway the
key components are: (i) A cytokine receptor chain (or chains) such
as the Interleukin-4 receptor or the Interferon .gamma. receptor;
(ii) a member (or members) of the JAK family of PTKs; (iii) a
member(s) of the STAT family of transcription factors, and (iv) a
sequence specific DNA element to which the activated STAT will
bind.
[0012] The general principles of the JAK/STAT pathway are shown
below, for the IFN.gamma. receptor, an example of the class II
cytokine receptors. Although the same basic mechanism is initiated
by each family of cytokine receptors, there remain discrepancies in
detail which are at present unresolved, although they presumably
define the specificity of the cellular response to particular
cytokines.
[0013] A review of the JAK/STAT literature offers strong support to
the notion that this pathway is important for the recruitment and
marshalling of the host immune response to environmental insults,
such as viral and bacterial infection. This is well exemplified in
Table 1 and Table 2. Information accumulated from gene knock-out
experiments have underlined the importance of members of the JAK
family to the intracellular signalling triggered by a number of
important immune regulatory cytokines (Table 7). The therapeutic
possibilities stemming from inhibiting (or enhancing) the JAK/STAT
pathway are thus largely in the sphere of immune modulation, and as
such are likely to be promising drugs for the treatment of a range
of pathologies in this area. In addition to the diseases listed in
Tables 1 and 2, inhibitors of JAKs could be used as
immunosuppresive agents for organ transplants and autoimmune
diseases such as lupus, multiple sclerosis, rheumatoid arthritis,
Type I diabetes, autoimmune thyroid disorders, Alzheimer's disease
and other autoimmune diseases. Additionally, treatment of cancers
such as prostate cancer by JAK inhibitors is indicated.
1TABLE 1 Cell Types Disease Type Involved Characteristics Atopy
Allergic Asthma (Mast Cells T-cell activation of B-cells Atopic
Dermatitis (Eczema) (Eosinophils followed by IgE mediated Allergic
Rhinitis (T-Cells activation of resident Mast (B-Cells cells and
Eosinophils Cell Mediated Hypersensitivity Allergic Contact
(T-cells T-cell hypersensitivity Dermatitis Hypersensitivity
(B-cells Pneumonitis Rheumatic Diseases Systemic Lupus
Erythematosus (SLE) Rheumatoid Arthritis (Monocytes Cytokine
Production Juvenile Arthritis (Macrophages (e.g. TNF, IL-1, CSF-1,
Sjogren's Syndrome (Neutrophils GM-CSF) Scleroderma (Mast Cells
T-cell Activation Polymyositis (Eosinophils JAK/STAT activation
Ankylosing Spondylitis (T-Cells Psoriatic Arthritis (B-Cells Viral
Diseases Epstein Barr Virus (EBV) Lymphocytes JAK/STAT Activation
Hepatitis B Hepatocytes JAK/STAT Activation Hepatitis C Hepatocytes
JAK/STAT Inhibition HIV Lymphocytes JAK/STAT Activation HTLV 1
Lymphocytes JAK/STAT Activation Varicella-Zoster Fibroblasts
JAK/STAT Inhibition Virus (VZV) Human Papilloma Epithelial cells
JAK/STAT Inhibition Virus (HPV) Cancer Leukemia Leucocytes
(Cytokine production Lymphoma Lymphocytes (JAK/STAT Activation
[0014] There are many different types of protein kinase. Each type
has the ability to add a phosphate group to an amino acid in a
target protein. The phosphate is provided by hydrolyzing ATP to
ADP. Typically, a protein kinase has an ATP-binding site and a
catalytic domain that can bind a portion of the substrate protein.
The JAK family of protein tyrosine kinases (PTKs) play a central
role in the cytokine dependent regulation of the proliferation and
end function of several important cell types of the immune
system.
[0015] The JAK family of Protein Tyrosine Kinases (PFKs) represent
excellent drug discovery targets for the following reasons:
[0016] They are proven key players in the cellular response to a
number of important cytokines (from gene Knock-out and biochemical
studies);
[0017] Whilst each of the JAK family members are relatively widely
expressed, their PTK activity is activated only at sites where
cytokine levels are relatively high, i.e. at a local site of
inflammation;
[0018] They are enzymes permitting effective inhibition of signal
amplification and facilitating drug design;
[0019] Therapeutic applications in which inhibitors of particular
JAK kinases may be useful are outlined in Table 2 below:
2TABLE 2 Diseases Potentially Treatable By JAK-Based Drug Therapies
JAK family Strength Target Disease Cytokine member of Association
Asthma IL-4 & IL-9 JAK1 & JAK3 +++ IL-13 JAK1 & JAK2
+++ IL-5 JAK2 +++ Eczema IL-4 JAK1 & JAK3 +++ IFN-.alpha. JAK1
& JAK2 +++ Food Allergy IL-4 JAK1 & JAK3 +++ Inflammatory
Bowel IL-4 JAK1 & JAK3 +++ Disease & Crohn's Disease
Leukaemia And (IL-2) JAK3, JAK1 & +++ Lymphoma JAK2 Cutaneous
Inflammation GM-CSF & JAK1 & JAK2 +++ IL-6 Immune
Suppression By IL-10 JAK1 & TYK2 +++ Solid Tumour Multiple
Myeloma IL-6 JAK1, JAK2 & +++ Tyk2 Prostate Cancer IL-6 JAK1,
JAK2 & +++ Tyk2
[0020]
3TABLE 3 A list of Cytokines that use the JAK/STAT pathway for
Signalling CYTOKINE JAK1 JAK2 JAK3 TYK2 IL-2, IL-4, IL-7, IL-9,
IL15 (IL-13) + (+) + (+) IL-13 + + (+) IL-3, IL-5, GM-CSF + IL-6,
IL-11, OSM, CNTF, LIF + + + IL-12 + + Leptin + GH, PRL, Epo, Tpo +
IFN.alpha., IFN.beta., IL-10 + + IFN.gamma. + +
[0021] A direct comparison of the four mammalian JAK family members
revealed the presence of seven highly conserved domains (Harpur et
al, 1992). In seeking a nomenclature for the highly conserved
domains characteristic of this family of PTKs, the classification
used herein was guided by the approach of Pawson and co-workers
(Sadovski et al, 1986) in their treatment of the SRC homology (SH)
domains. The domains have been enumerated accordingly with most
C-terminal homology domain designated JAK Homology domain 1 (JH1).
The next domain N-terminal to JH1 is the kinase-related domain,
designated here as the JH2 domain. Because of its overall
similarity to other kinase domains it is also known as the
Kinase-Like Domain or KLD. Each domain is then enumerated up to the
JH7 located at the N-terminus (FIG. 1 shows a schematic
representation of this nomenclature). The high degree of
conservation of these JAK homology (JH) domains suggests that they
are each likely to play an important role in the cellular processes
in which these proteins operate. However, the boundaries of the JAK
homology domains are arbitrary, and may or may not define
functional domains. Nonetheless, their delineation is a useful
device to aid the consideration of the overall structural
similarity of this class of proteins.
The PTK Domain
[0022] The feature most characteristic of the JAK family of PTKs is
the possession of two kinase-related domains (JH1 and JH2/KLD)
(Wilks et al, 1991). The putative PTK domain of JAK1 (JH1) contains
highly conserved motifs typical of PTK domains, including the
presence of a tyrosine residue at position 1022 located 11 residues
C-terminal to sub-domain VII that is considered diagnostic of
membership of the tyrosine-specific class of protein kinases.
Alignment of the human JAK1 PTK domain (255 amino acids), with
other members of the PTK class of proteins revealed homology with
other functional PTKs (for example, 28% identity with c-fes (Wilks
and Kurban, 1988) and 37% homology to TRK (Kozma et al, 1988). The
JH1 domains of each of the JAK family members possess a interesting
idiosyncrasy within the highly conserved sub-domain VIII motif
(residues 1015 to 1027 in JAK2, SEQ. ID NO 1) that is believed to
lie close to the active site, and define substrate specificity. The
phenylalanine and tyrosine residues flanking the conserved
tryptophan in this motif are unique to the JAK family of PTKs (see
Table 4). Aside from this element, the JH1 domains of each of the
members of the JAK family are typical PTK domains.
4TABLE 4 Motif VIII of the JAK family of PTKs bears a conserved
tyrosine Motif VIII JAX1 DSPVFWYAPECLI (SEQ. ID NO 2) JAK2
ESPIFWYAPESLT (SEQ. ID NO 3) Tyk2 DSPVFWYAPECLK (SEQ. ID NO 4) JAK3
QSPIFWYAPESLS (SEQ. ID NO 5) EGF-R KVPIKWMALESIL (SEQ. ID NO 6)
c-SRC KFPIKWTAPEAAL (SEQ. ID NO 7)
The Kinase-Like Domain (KLD or JH2 Domain)
[0023] Based upon cladograms generated using programmes such as
Pile Up, the second kinase-like domain (KM or JH2 Domain) is
clearly ancestrally related to the broader family of kinase
domains, by virtue of the presence of most of the key kinase motifs
defined by Hanks, Quinn and Hunter (Hanks et al, 1988; Hanks &
Quinn 1991). However, in order to distinguish the KLD domain motifs
from the PTK domain motifs, they have been assigned a subscript a,
(eg. I.sub.a, II.sub.a, III.sub.a etc) with respect to their
similarity to the sub-domains described by Hanks and co-workers
(Hanks et al, 1988).
[0024] The overall sequence similarity of this domain to the kinase
domains of both the PTK and serine/threonine kinase families
implies that this region of the protein might also function as a
protein kinase. There are, however, significant differences in the
sequences of key motifs within this domain which suggest that the
catalytic activity of the I(L domain may be something other than
serine/threonine or tyrosine phosphorylation or indeed may not be
kinase related. For example, comparison of sub-domain VI.sub.a of
the KLD domain with sub-domain VI of members of the PTK and
Serine/Threonine families shows the replacement of a conserved
acidic amino acid (aspartic acid) with a neutral amino acid
(asparagine). For example,
5TABLE 5 Motif VIb of the KLD of the JAK family of PTK1 bears a
conserved Asparagine residue. Motif VIb JAK1 (KLD) VHGNVCTKNLL
(SEQ. ID NO 8) JAK2 (KLD) IHGNVCAKNIL (SEQ. ID NO 9) Tyk2 (KLD)
VHGNVCGRNIL (SEQ. ID NO 10) JAK3 (KLD) PNGNVSARKVL (SEQ. ID NO 11)
JAK1 (JH1) VHRDLAARNVL (SEQ. ID NO 12) EGF-R VHRDLAARNVL (SEQ. ID
NO 13) cAMPk.alpha. IYRDLKPENLL (SEQ. ID NO 14)
[0025] Further, while there is conservation of subdomain VII.sub.a
with respect to the equivalent motif in the other kinase families,
the normally invariant D-F-G sequence of the PTK and
serine/threonine families (motif VII) is replaced by the sequence
D-P-G in motif VII.sub.a of the JH2/KLD domain. The conservation of
the precise sequence of subdomain VI in the protein kinase
sub-families appears to correlate with the substrate specificity of
the kinase, and thus it is possible that this domain within the
members of the JAK family of PTKs, may exhibit a substrate
specificity other than that previously observed for other protein
kinases.
[0026] A further sequence anomaly that may suggest a substrate
variation lies within the putative ATP-binding site in the
kinase-related domain (sub-domain I.sub.a). This domain consists of
the absolutely conserved -GXGXXG- in all PTKs described to date.
However, in all known JAK family members, sub-domain I.sub.a is
replaced with -GXGXXT-. This glycine motif has now been defined as
the ATP-binding site, with the first two glycine residues thought
to bend around the nucleotide with the third glycine residue
forming part of this loop. Substitution of the small side chain of
glycine with the slightly larger threonine residue may disrupt the
ATP-specific recognition, and confer some other substrate
recognition. A viral mutation of the third glycine residue to a
lysine in v-SRC abolishes the transformation and catalytic activity
of this oncogene (Verdaane and Varmus 1994). It is also noteworthy,
that this glycine interacts sterically with the conserved
phenylalanine and glycine of the sub-domain VII motif Asp-Phe-Gly
in the catalytic domain of the Insulin Receptor (Hubbard et al,
1994). This conformation is involved in maintaining an open
structure between the two lobes of the catalytic domain, and
perhaps the altered glycine to threonine and phenylalanine to
proline in KLD suggests an alternate structural requirement.
[0027] Certain other subtle differences exist in the normally
consistent spacing between key motifs in KLD as compared with a PTK
domain. For example, the spacing between both components of the
ATP-binding site (I.sub.a and II.sub.a) is different for JAK1,
JAK2, JAK3 and Tyk2 when compared with the broader protein kinase
family. In JAK1 this spacing contains an extra 7 amino acids, JAK2
and JAK3 an extra 3 amino acids, and Tyk, an extra 21 amino acids.
Moreover, for JAK1, JAK2, Tyk2 and JAK3, the spacing between
sub-domains VI.sub.a and VII.sub.a in this region is also longer.
Conversely, the distance between sub-domains VII.sub.a and IX.sub.a
in JAK1, Tyk2 and JAK3 is seven amino acids shorter that the
corresponding region in the JH1 domain. It is worth noting that
this sub-domain in the PTK domain contains the putative
autophosphorylation tyrosine residue, while in each JAK family
member, this tyrosine is not present in the KLD domain. The overall
structure of this domain may be expected to be somewhat different
from the catalytic domains of other members of the PTK and
threonine/serine kinase families.
Regulation of the PTK Activity of JAK Kinases
[0028] Role of the KLD Domain
[0029] The tandem array of kinase domain and kinase-like domain is
a defining feature of all members of the JAK family of PTKs. This
fact, coupled with the high degree of conservation of the primary
amino acid sequence of all members of this family, suggests that
the role played by the KLD in the function of the JAK family of
kinases is an important and evolutionarily conserved one. The
presence of amino acid substitutions in key motifs within the KLD
suggest that it is unlikely that this domain is a functional
protein kinase. Indeed, attempts to demonstrate kinase activity
form isolated purified KLD have so far proved to be impossible
(Wilks et al, 1991) and it is often alternatively referred to as a
pseudoidnase domain.
SUMMARY OF THE INVENTION
[0030] The present inventors have developed a novel model of JAK
kinase signalling. This model provides a number of target points at
which a chemical entity may regulate JAK activity.
[0031] Accordingly, in a first aspect the present invention
consists in a method of selecting or designing a compound for the
ability to regulate JAK activity, the method comprising assessing
the ability of the compound to modulate the interaction of the
pseudo-substrate loop (PSL) with the kinase like domain (KID) of
JAK.
[0032] In a second aspect the present invention consists in a
method of selecting, or designing, a compound which regulates JAK
activity, the method comprising
[0033] (i) selecting or designing a compound which has a
conformation and polarity such that it interacts with at least one
ligand selected from the group consisting of residues 667-679,
711-726 and 757-765 of human JAK2; and
[0034] (ii) testing the compound for the ability to interfere with
the binding of the PSL with the KLD.
[0035] The numbering of residues is based on the sequence of KID of
JAK 2. It will be understood there are corresponding regions in
each of the other JAK kinase. The reference to these residues is
therefore intended to define the regions of the JAK kinase with
which the compound interacts.
[0036] In a third aspect the present invention consists in a
compound which interacts with the PSL or the KLD such as to
interfere with the binding of the PSL with the KID such that the
activity of the JAK is reduced when compared to that of the JAK in
the absence of the compound.
[0037] In a fourth aspect the present invention consists in a
therapeutic composition comprising an agent which inhibits the
ability of PSL to bind to the KLD of JAK.
[0038] In a preferred embodiment of the fourth aspect the agent is
the compound of the third aspect of the present invention.
[0039] In a fifth aspect the present invention consists in a
compound which regulates JAK, the compound having the same or
similar physico-chemical properties to a peptide comprising
following amino acid sequence:
X1-X2-X3-X4-X5 -X6-X7-X8
[0040] in which X1 is any amino acid, preferably proline or
glycine;
[0041] X2 is any amino acid, preferably alanine, valine, leucine or
isoleucine;
[0042] X3 is any amino acid, preferably glutamate or aspartate;
[0043] X4 is phenylalanine or tyrosine, preferably
phenylalanine;
[0044] X5 is leucine or methionine or isoleucine;
[0045] X6 is arginine or lysine;
[0046] X7 is methionine or leucine or isoleucine, preferably
methionine;
[0047] X8 is isoleucine or leucine or methionine, preferably
isoleucine.
[0048] In a preferred embodiment the compound has the same or
similar physico-chemical properties to a peptide having the amino
acid sequence PAEFMRMI.
[0049] In a further preferred embodiment the compound is a peptide
comprising the following amino acid sequence:
X1-X2-X3-X4-X5-X6-X7-X8
[0050] in which X1 to X8 are as defined above.
[0051] In yet another embodiment the compound is a peptide, the
peptide having the amino acid sequence PAEFMRMI.
[0052] In a sixth aspect the present invention consists in a
compound obtained by the method of the first or second aspect of
the present invention.
[0053] In a seventh aspect the present invention consists in a
ligand which specifically binds the compound of the third, fourth
or sixth aspect of the present invention.
BRIEF DESCRIPTION OF FIGURES
[0054] FIG. 1: Domain Structure of JAK family members.
[0055] The seven JAK homology domains (JH domains) are shown as
shaded boxes. JH1 is the PTK domain, whilst JH2 is the kinase-like
domain, that is characteristic of this class of PTKs. The drawing
is approximately to scale.
[0056] FIG. 2: KLD-mediated Regulation of the PTK domain of members
of the JAK family of PTKs
[0057] The model involves a two step triggering mechanism,
involving a "priming" phase, (panel A), wherein the JAK is
converted to a latent form (panel B), followed by a "triggering"
phase, (panel C), wherein the latent kinase activity of the PTK
domain of the JAK is unleashed, which is dependent upon the
interaction of a given cytokine receptor with its cognate
ligand.
[0058] FIG. 3
[0059] A. Kinase Domains Top View
[0060] Top views (i.e. looking from above the smaller lobe of the
PTK domain) of three PTK domains (from HCK, Fibroblast Growth
Factor Receptor and Insulin Receptor) for which the crystal
structures have been determined, are shown. The molceular model of
the JAK2 PTK domain is shown from the same angle. Noteworthy in
this view of the JAK2 PTK domain is the large loop composed of the
amino acids of the PSL.
[0061] B Kinase Domains Side View
[0062] Side views (i.e. looking from the right hand side of the PTK
domain) of three PTK domains (from HCK, Fibroblast Growth Factor
Receptor and Insulin Receptor) for which the crystal structures
have been determined, are shown. The molecular model of the JAK2
PTK domain is shown from the same angle. Noteworthy in this view of
the JAK2 PTK domain is the large loop composed of the amino acids
of the PSL. The potential serine phosphorylation site in the PSL
sequence has been highlighted.
[0063] FIG. 4. Model of the JAK2 KID showing surface features.
[0064] The alpha carbon chain of the ID is shown as a ribbon
structure, and the light grey patch of amino acids rendered as
spheres corresponds to those amino acids analogous to those
responsible for the binding of substrate peptides in the insulin
receptor.
[0065] FIG. 5. Ba/F3 cell assay
[0066] Inhibition of the growth of the IL-3 dependent growth of
Ba;F3 cells has been brought about by the addition of a peptide
derived from JAK2 PSL. PSL.sub.Short PAEFMRMI and PSL.sub.Control
SPSKFRMPEAMIGND were added at concentrations ranging from 1 .mu.M
to 1001 .mu.M in phosphate buffered saline. Cell number after three
days was measured by an MT assay.
[0067] FIG. 6. FP assay on KLD
[0068] a. Fluoresence polarization measurements (in triplicate)
were taken in presence or absence of purified KLD at 30 seconds, 5
minutes, and 10 minutes following peptide (PSL-1) addition.
[0069] b. Fluoresence polarization measurements (in triplicate)
were taken in presence or absence of purified KLD at 30 seconds, 5
minutes, 10 minutes and 20 minutes following peptide (PSL-1)
addition. Competitor peptides PLC and PLD were added simultaneously
with the fluoresceinated PSL-1 peptide.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The present inventors have developed a novel model of JAK
kinase signalling. This model provides a number of target points at
which a chemical entity may regulate JAK activity.
[0071] The model developed by the present inventors involves a two
step triggering mechanism, involving a "priming" or "cocking"
phase, wherein the JAK is converted to a latent form, followed by a
"triggering" phase, (wherein the latent kinase activity of the PTK
domain of the JAK is unleashed), which is dependent upon the
interaction of a given cytokine receptor with its cognate ligand.
See FIG. 2 for details.
[0072] Phase 1: Priming
[0073] The JAK molecule is synthesized in an open conformation
(FIG. 2 panel A), and is converted to a primed or cocked
conformation, perhaps by the agency of a Ser/Thr kinase or
phosphatase. This cocking mechanism may involve ATP, either as a
co-factor for the putative Ser/Thr kinase- or phosphatase-dependent
enzymes involved, or as a co-factor for binding into the ATP
binding site of the KLD. The PSL of the PTK domain is loaded into
its docking site in the KLD, whereupon the access of ATP to the PTK
domain's ATP binding site is restricted. In this form the JAK PTK
domain could be described as being inactive or latent. The PSL
binds into the substrate-binding site of the kinase-like domain,
indicating that the PSL may be a pseudo-substrate for a
pseudo-kinase domain. The cocked JAK is loaded onto the cytokine
receptor either immediately (as has been described for a number of
cytokine receptors) or following cytokine stimulation of a given
receptor (as has been described for Growth Hormone Receptor).
[0074] Phase 2: Triggering
[0075] Following ligand mediated binding to the cytokine receptor,
the PSL is released from the KLD by a mechanism that may involve a
additional kinase or phosphatase, thereby releasing the latent PTK
activity in the JAK kinase domain. Ultimately the activated JAK is
inactivated by binding of SOCS1 to a tyrosine in the PTK domain,
and the molecule is targeted to the proteosome.
[0076] The model developed by the present inventors reveals a
number of points at which a compound may interact to regulate the
JAK activity. For example a compound may regulate JAK activity by
interacting with the JAK at the level of the PTK domain; at the
level of the loading of the PSL to the KLD (the cocking mechanism);
by inhibiting the release of the PSL from the KLD (the triggering
mechanism); or by causing the premature or inappropriate release of
the PSL from the KLD.
[0077] Accordingly, in a first aspect the present invention
consists in a method of selecting or designing a compound for the
ability to regulate JAK activity, the method comprising assessing
the ability of the compound to modulate the interaction of the
pseudo-substrate loop (PSL) with the kinase like domain (KLD) of
JAK.
[0078] In a second aspect the present invention consists in a
method of selecting, or designing, a compound which regulates JAK
activity, the method comprising
[0079] (i) selecting or designing a compound which has a
conformation and polarity such that it interacts with at least one
ligand selected from the group consisting of residues 667-679,
711-726 and 757-765 of human JAK2; and
[0080] (ii) testing the compound for the ability to interfere with
the binding of the PSL with the KLD.
[0081] In a preferred embodiment of the second aspect, the method
comprises selecting or designing a compound which has a
conformation and polarity such that it interacts with at least one,
preferably at least two, more preferably at least three and most
preferably at least four ligands selected from the group consisting
of residues 673, 677, 711-715, 718, 724, 759 and 760 of human
JAK2.
[0082] The numbering of residues is based on the sequence of KLD of
JAK 2. It will be understood there are corresponding regions in
each of the other JAK kinases. The reference to these residues is
therefore intended to define the regions of the JAK kinase with
which the compound interacts.
[0083] As will be appreciated the method of selection or design may
be conducted in a number of ways. Without limiting the general
applicability of the present invention the following non-limiting
examples of how the screening may be conducted are provided.
[0084] By computer modelling techniques the three dimensional
structure of the PSL may be approximated. In combination with
knowledge of the amino acid sequence of the PSL a compound may be
designed which mimics the PSL or a region thereof in respect of
physical characteristics such as shape, size, charge, polarity,
etc. The compound would then be tested for its ability to
bind/interact with KLD, for example by protein binding studies.
Assuming that the compound demonstrated the ability to interact
with KLD the compound would then be tested for biological activity
in a cell based, or other in vitro, assay.
[0085] The ability of a compound to interfere with the interaction
between the PSL and the KLD may also occur as a result of the
compound binding to or altering the conformation of the PSL. Once
again this ability can be screened for by protein binding
studies.
[0086] Using the methods of the present invention the inventors
have developed compounds which regulate JAK activity.
[0087] In a third aspect the present invention consists in a
compound which interacts with the PSL or the KLD such as to
interfere with the binding of the PSL with the KLD such that the
activity of the JAK is reduced when compared to that of the JAK in
the absence of the compound.
[0088] In a preferred embodiment of the third aspect, the compound
binds to the substrate-binding cleft of the KLD such as to
interfere with or prevent the binding of the PSL to the KLD.
[0089] In a further preferred embodiment of the third aspect, the
compound has a conformation and polarity such that it binds to at
least ligand selected from the group consisting of residues
667-679, 711-726 and 757-765 of human JAK2.
[0090] In a still further preferred embodiment of the third aspect,
the compound binds to at least one, preferably at least two, more
preferably at least three and most preferably at least four ligands
selected from the group consisting of residues 673, 677, 711-715,
718, 723-724, 759 and 760 of human JAK2.
[0091] In another preferred embodiment the compound is composed at
least in part of amino acids. It is preferred that the amino acids
are derived from the sequence of the PSL. By "derived from" it is
intended that the residues from the PSL which bind to the selected
ligands in the compound are in the same spatial configuration as
they are in the PSL. For example, the compound may comprise three
residues from PSL where the residues are spaced apart by other
amino acid residues or spacer groups such that the three residues
are arranged spatially in the same conformation as in the PSL. As
will be recognised this is analogous to the concept of
conformational epitopes.
[0092] The amino acids may be D or L amino acids. Where the
compound is a peptide it is preferred that the peptide is
cyclic.
[0093] In a fourth aspect the present invention consists in a
therapeutic composition comprising an agent which inhibits the
ability of PSL to bind to the KLD of JAK.
[0094] In a preferred embodiment of the fourth aspect the agent is
the compound of the third aspect of the present invention.
[0095] In a fifth aspect the present invention consists in a
compound which regulates JAK, the compound having the same or
similar physico-chemical properties to a peptide comprising
following amino acid sequence:
X1-X2-X3-X4-X5-X6-X7-X8
[0096] in which X1 is any amino acid, preferably proline or
glycine;
[0097] X2 is any amino acid, preferably alanine, valine, leucine or
isoleucine;
[0098] X3 is any amino acid, preferably glutamate or aspartate;
[0099] X4 is phenylalanine or tyrosine, preferably
phenylalanine;
[0100] X5 is leucine or methionine or isoleucine;
[0101] X6 is arginine or lysine;
[0102] X7 is methionine or leucine or isoleucine, preferably
methionine;
[0103] X8 is isoleucine or leucine or methionine, preferably
isoleucine.
[0104] In a preferred embodiment the compound has the same or
similar physico-chemical properties to a peptide having the amino
acid sequence PAEFMRMI.
[0105] In a further preferred embodiment the compound is a peptide
comprising the following amino acid sequence:
X1-X2-X3-X4-X5-X6-X7-X8
[0106] in which X1 to X8 are as defined above.
[0107] In yet another embodiment the compound is a peptide, the
peptide having the amino acid sequence PAEFMRMI.
[0108] In a sixth aspect the present invention consists in a
compound obtained by the method of the first or second aspect of
the present invention.
[0109] In a seventh aspect the present invention consists in a
ligand which specifically binds the compound of the third, fourth
or sixth aspect of the present invention.
[0110] As will be readily understood by persons skilled in this
field the methods of the present invention provide a rational
method for designing and selecting compounds which interact with
JAK. In the majority of cases these compounds will require further
development in order to increase activity. Such further development
is routine in this field and will be assisted by the information
and screening methods provided in this application. It is intended
that in particular embodiments the methods of the present invention
includes such further developmental steps.
[0111] Accordingly, in another aspect the present invention
consists in a method of designing or selecting a compound which
modulates JAK activity, the method comprising subjecting a compound
obtained by a method according to any one of the previous aspects
of the present invention to biological screens and assessing the
ability of the compound to modulate JAK activity.
[0112] In a further aspect the present invention consists in a
method of treating a subject suffering from a JAK-associated
disease state, the method comprising administering to the subject a
compound of the present invention.
[0113] It is preferred that the JAK-associated disease state is
selected from the group consisting of Asthma, Eczema, Food Allergy,
Inflammatory Bowel Disease, Crohn's Disease, Leukaemia, Lymphoma,
Cutaneous Inflammation, Immune Suppression By Solid Tumour and
Prostate Cancer.
[0114] As used herein the term "JAK", "JAK kinase" or "JAK family"
refers to protein tyrosine kinases which possess the characterizing
features of JAK1, JAK2, JAK3 and TYK as described herein.
[0115] As used herein the term "JAK-associated disease state"
refers to those disorders which result from aberrant JAK activity,
and/or which are alleviated by inhibition of one or more of these
enzymes.
[0116] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0117] All publications mentioned in this specification are herein
incorporated by reference.
[0118] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed in Australia before the priority date of
each claim of this application.
[0119] In order that the nature of the present invention may be
more clearly understood preferred forms thereof will now be
described with references to the following non-limiting
examples.
A Model for the Priming and Activation of the JAK Family of
PTKS
[0120] Modelling the JH1 Domain
[0121] No crystal structure of a JAK family kinase domain has so
far been produced. However, the kinase domains of a number of
protein tyrosine kinases and serine/threonine kinases have been
crystalized (e.g Hubbard et al, 1994; Overduin M, et al (1992);
Schindler, et al, 2000; inter alia) Using the coordinate of JAK
structures it has been possible to generate a model of the human
JAK2 kinase domain.
[0122] Using PsiBlast (sequence similarity search), ProCeryon and
GeneThreader (both threading algorithms) the protein structure
database (PDB) was searched for the template most similar to the
human Jak2 kinase domain. The FGF receptor kinase, insulin and SRC
receptor kinase were found to be the most similar sequences
available. In particular the FGFR Kinase (FGFRK) was found to have
a sequence identity greater 30-35%, which is high enough to create
reliable models.
[0123] The sequence alignments show a sequence id (similarity)
between FGFRK and Jak2K of 35% (46%). The region between 1050-1071
of human JAK2 (SEQ ID NO 1) is a long loop insert, which could not
be aligned properly with any loops in the templates. The
conformation and structure of this insert therefore could not be
predicted reliably. This insert, however, did not interact with the
ATP binding site and should not influence the reliability of the
model in the vicinity of the ATP binding site.
[0124] Structural Conservation
[0125] The templates were overlaid to investigate the structural
conservation of the N- and C-terminal domains of the kinase fold.
While the overall fold appeared well conserved, there were some
larger deviations, in particular in loop regions and the
orientation of the N- and C-terminal regions. The structure of the
ATP binding site was well conserved, however, smaller deviations in
ligand orientations and side chain conformations can be
observed.
[0126] Homology Modelling
[0127] JAK2 Protein Tyrosine Kinase Domain
[0128] Homology models were created based on the sequence
alignments using Andrej Sali's Modeller program, however,
initially, only the AGW FGFRK was used as template because of the
above mentioned deviations in the kinase fold. Although AGW
contains an inhibitor, the ligand was not modeled at this point.
The AGW inhibitor shows a rather different binding mode to other
more ATP-like analogues and we did not want to bias the binding
site for a particular class of inhibitors too much. Homology models
and sequences alignments were iteratively refined during the
modelling process in 6 steps. In each step 200 models were created
(based on the particular alignment) and evaluated. The sequence
alignment was modified according to the evaluation. In a last step,
using the sequence alignment (Table 6), a model with an ATP
analogue based on the AGW and SRC templates were created. All
together, 1400 models were created with Modeller and evaluated. It
should be noted here that the conformation of the loop residue
number 1050-1071 was extremely difficult to predict given the lack
of a template in this area and the length of the loop.
[0129] Model Evaluation
[0130] Models were evaluated using the internal Modeller energy and
the ProsaII and Profiles3D evaluation procedures of six final
models were selected (3 based on AGW alone, 3 based on AGW and SRC)
in order to show the range of possible side chain and backbone
modifications. Models based only on AGW show a better quality
(average ProsaII score: -8.14) than the models created with two
templates (average score -7.73), but both classes of models seem to
result in reasonable protein structures. As expected the overall
structure and fold is very similar in all models, however,
uncertainties are observed in side conformations.
[0131] The Pseudosubstrate Loop
[0132] Whilst the JAK2 kinase domain appeared to conform in most
respects to the structures of other PTKs, the loop structure
located between amino acids 1050-1071 did not resemble any feature
observed in any other kinase. Nonetheless, this loop was a highly
conserved feature of the JAK family of PTKs (Table 7 & FIG. 3)
and it most likely plays an important role in the function of the
JAK kinase family.
6TABLE 6 * 20 * 40 * 60 * 8 AGW
:------SEYELPEDPRWELPRDRLVLGKPLGEGCFGQV-VLAEAIGLDKDKPNRVTKVAVKMLKSDATEKDL-
SDLISEM : IR3 :SSVFVP--------DEWEVSREKITLLRELGQGSFGMVYEGNA-
RDIIK---GEAETRVAVKTVNESASLRERIEFLNEA : FGIA
:---------ELPEDPRWELPRDRLVLGKPLG-----QV-VLAEAIGL----PNRVTKVAVKMLKSDATEDLS-
DLISEM : SRC :--------------DAWEIPRESLRLEVKLGQGCFGEV-WMGTW-
--------NGTTRVAIKTLKP-GTM-SPEAFLQEA : j1h
:------KNQPTEVDP-THFEKRFLKRIRDLGEGHFGKV-ELCRYDPEDN----TGEQVAVKSLKPESGGNHI-
ADLKKEI : j2h :------SGAFEDRDP-TQFEERHLKFLQQLGKGNFGSV-EMCR-
YDPLQD---NTGEVVAVKKL-QHSTEEHLRDFEREI : j3h
:------AQLYACQDP-TIFEERHLKYISQLGKGNFGSV-ELCRYDPLAH---NTGALVAVKQL-QHSGPDQQ-
RDFQREI : 6 LG g fg V VA6K 6 E 0 * 100 * 120 * 140 * 1 AGW
:EMMKMIGKHKNIINLLGA--CTQDG--PLYVIVEYASKGNLREYLQ---------ARRPPGLEYCYNPSHNP-
EEQLSSK IR3 :SVMK-GFTCHHVVRLLGV---VSKGQPTL-VVMELMAHGDLKSYL-
R---------SLRPE------AENNPGRPPPTLQ FGIA
:EMMKMIGKHKNIINLLGA--CTQDG--PLVIVEYASKGNLREYLQ---------ARRPP--------------
-QLSSK SRC :QVMK-KLRHEKLVQLYAV--V-SEE--PIYIVTEYMSKGSLLDFL--
------------KGET----------GKYLRLP j1h
:EILR-NLYHENIVKYKGI--CTEDGGNGIKLIMEFLPSGSLKEYLP---------KNKNKIN-----------
-----LK j2h :EILK-SLQHDNIVKYKGV--CYSAGRRNLKLIMEYLPYGSLRDYL-
Q---------KHKERID---------------HI j3h
:QILK-ALHSDFIVKYRGV--SYGPGRPELRLVMEYLPSGCLRDFLQ---------RHRARLD-----------
-----AS 1664 66 g g 6 66 E G L 5L 60 * 180 * 200 * 220 * AGW
:DLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDNV-
MKIADFGLAR-DIHHIDYYKKTTNGRLPVKWMAPEALFDRI IR3
:EMIQMAAEIADGMAYLNAKKFVHRDLAARNCMVAHDFTVKIGDFGMTRDIETD----RKGGKGLLPVRWMAP-
ESLKDGV FGIA :DLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDNVMKIADF-
GLA--DIHHIDYYKKT-NGRLPVKWMAPEALFDRI SRC
:QLVDMAAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLI--EDNEYTARQGAKFPIKWTAP-
EAALYGR j1h :QQLKYAVQICKGMDYLGSRQYVHRDLAARNVLVESEHQVKIGDFG-
LTKAIETDKEYYTVKDDRDSPVFWYAPECLMQSK j2h
:KLLQYTSQICKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPGESPIFWYAP-
ESLTESK j3h :RLLLYSSQICKGMEYLGSRRCVHRDLAARNILVESE-
AHVKIADFGLAKLLPLDKDYYVVREPGQSPIFWYAPESLSDNI 6 26 GM Y6 6HRDLaarN 6V
K6 DFG6 y P6 W APE 1 240 * 260 * 280 * 300 * AGW
:YTHQSDVWSFGVLLWEIFT-------------------------LGG-
SPYPGVPVEELFKLLKEGHRMDKPSNCTNEL IR3
:FTTSSDMWSFGVVLWE-------------------------ITSLAEQPYQGLSNEQVLKFVMDGGYLDQPD-
NCPERV FGIA :YTHQSDVWSFGVLLWEIFT-------------------------L-
GGSPYPGVPVEELFKLLKEGHRMDKPSNCTNEL SRC
:FTIKSDVWSFGILLTELTT-------------------------KGRVPYPGMVNREVLDQVERGYRMPCPP-
ECPESL j1h :FYIASDVWSFGVTLHELLTYCDSDSSPM-ALFLKMIG-PTHG-----
------QMTVTRLVNTLVNTLKEGKRLPCPPNCPDEV j2h
:FSVASDVWSFGVVLYELFTYIEKSKSPP-AEFMRMIGNDKQG----------QMIVFHYLIELLKNNGRLPR-
PDGCPDEI j3h :FSRQSDVWSFGVVLYELFTYCDKSCSPS-AEFLRMMGCERD----
-------VALCRLLELLEEGQRLPAPPACPAEV 5 SD6WSFG6 L E t 6 6 g r6 P C 6
320 * 340 * 360 * AGW
:YMMMRDCWHAVPSQRPTFKQLVEDLDRIVALT----------------------- SEQ ID NO
21 IR3 :TDLMRMCWQFNPKMRPTFLEIVNLLKDDLHPSFPEVSFFHSEENK-G- DYMNM---
SEQ ID NO 22 FGIA :YMMMRDCWHAVPSQRPTFKQLVEDLDRI-
VALTS---------------------- SEQ ID NO 23 SRC
:HDLMCQCWRKEPEERPTFEYLQAFLEDYF-------------------------- SEQ ID NO
24 j1h :YQLMRKCWEFQPSNRTSFQNLIEGFEALLK------------------------- -
SEQ ID NO 25 j2h :YMIMTECWNNNVNQRPSFRDLALRVDQIRDNMAG---
------------------- SEQ ID NO 26 j3h
:HELMKLCWAPSPQDRPSFSALGPQLDMLWSGSRG--------------------- SEQ ID NO
27 6M CQ p Rp3F 6
[0133]
7 TABLE 7 Kinase Motif IX Pseudosubstrate Loop Kinase Motif X
HumSRC
SDVWSFGILLTELTT---KGRVPYPG-----------------MVNREVLDQVERGYRMPCPPECPESL
SEQ ID NO 28 HumFGFR SDVWSFGVLLWEIFT---LGGSPYPG------------
------VPVEELFKLLKEGHRMDKPSNCTNEL SEQ ID NO 29 HumJAK1
SDVWSFGVTLHELLTYCDSDSSPMALFLMIGPTH----GQMTVTRLVNTLKE--GKRLPCPPNCPDEV
SEQ ID NO 30 HumJAK2 SDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGNDK----
-QGMIVFHLIELLKNN--GRLPRPDGCPDEI SEQ ID NO 31 HumJAK3
SDVWSFGVVLYELFTYCDKSCSPSAEFLRMMGCER---DVPALC-RLLELLEE--GORLPAPPACPAEV
SEQ ID NO 32 HumTYK2 SDVWSFGVTLYELLTHCDSSQSPPTKFLELIGIA-----
QGQMTVLRLTELLER--GERLPRPD SEQ ID NO 33 MusJAK1
SDVWSFGVTLHELLTYCDSDFSPMALFLKMIGPT----HGQMTVTRLVKTLKEG SEQ ID NO 34
MusJAX2 SDVWSFGVVLYELFTYIEKSKSPPVEFMRMIGND---KQGQMIVFHLIELLKS-
--NGRLPRPEGCPDEIYV SEQ ID NO 35 MusJAK3
SDVWSFGVVLYELFTYCDKSCSPSAEFLRMMGPE-----------------------REGPPLCRLLELLA
SEQ ID NO 36 RatJAK2 SDVWSFGVVLYELFTYIEKSKSPPVEFMRMIGND-
---KQGQMIVFHLIELLKNNGRLPRPEGCPDEIYV SEQ ID NO 37 RatJAK3
SDVWSFGVVLYELFTYSDKSCSPSTEFLRMIGPE---REGSPLCHLLELLAE----GRRLPPPS
SEQ ID NO 38 PigJAK1 SDVWSFGVTLHELLTYCDSDSSPMALFLKMIGPT----HGQ-
MTVTRLVNTLKEGKR SEQ ID NO 39 PigJAX2
SDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGND---KQGQMIVFHLIELLKNNGRLPRPDGCPDEIYI
SEQ ID NO 40 GGJAK1 SDVWSFGVTLYELLTYCDSESSPMTEFLKMIGPT----Q-
GQMTVARLVRVLQEEKRLPR SEQ ID NO 41 PuffJAK1
SDVWSFGVTLYELITYCDSSKSPMTCFLDMIGWT----QGQMTVMRLVKLL SEQ ID NO 42
PuffJAK2 SDVWSFGVVLYELFTHSSRNSSPPTVFMSMMGND---KQGQLIVYHLIELLKSGS-
RLPQPLDC SEQ ID NO 43 PuffJAK3 SDVWSFGVVLYELFSYCDINSNPKR-
LYMQQIGHN---VQTPSISLHLANILKSNWRLPAPPDCPAKV SEQ ID NO 44 PuffTYK2
SDVWSFGVTLTEILTHCDPKQSPRKKFEEMLEPKSLINQVPLIELLEKKMRLPC SEQ ID NO 45
CCJAK1 SDVWSFGVTMYELLTYCDISCSPMSVFL-MIGPT----HGQMTVT- RLVKVLEE SEQ
ID NO 46 CCJAK3 SDIWSFGIVLHELFSYCDISRNPQKIV-
YPEDRKL----CPEVRPWLSIFLIFSKDNWR SEQ ID NO 47 ZDJAK1
SDVWSFGVTMYELLTYCDASCSPMSVFLKLIGPT----HGQMTVTRLV SEQ ID NO 48
ZDJAK2a SDVWSFGVLYELFTYSEKSCSPPAVFMEQMGED---KQGQMIVYHLIDLLKR SEQ ID
NO 49 ZDJAK2b SDVWSFGVLYELFTYSDKLCSPPTVFLSMVGGD---KQGQT-
IVYHLIDLLKR SEQ ID NO 50 Hopscotch
SDVWSYGVTLFEMFSRGEEPNLVPIQTSQEDFLN--RLQSGERLNRPA SEQ ID NO 51
Peptides used: PAEFMRMI PSL.sub.Short SEQ ID NO 15 SPSKFRMPEAMIGND
PSL.sub.Control SEQ ID NO 16
[0134] Three properties of this loop suggest what the function of
this loop might be. Firstly, the level of conservation within the
family (see Table 7) suggested that it might play a role in a
process that only the JAKs participate. Secondly, its location in
the three dimensional model of JAK2 is directly below the ATP
binding site (see FIG. 3), a feature which was important in
formulating the present inventors' hypothesis. Finally, the most
highly conserved amino acids with the loop indicate a glutamate
adjacent to a phenylalanine residue at the centre of the loop,
which could act as a pseudosubstrate loop (PSL) as outlined
below.
[0135] JAK2 Kinase-Like Domain
[0136] Using PsiBlast (sequence similarity search) and the protein
structure database (PDB) was searched for the template most similar
to the Jak2 kinase like domain. The ABL and the SRC receptor kinase
were found to be the most similar sequences available. In
particular the SRC kinase was found to have a sequence identity of
about 23%, which is enough to create a model. Sequences of a number
of the related kinase domains JAK1, JAK2, JAK3, IGF, FGF, SRC, ABL
were aligned manually after an initial alignment with the ClustaIX
program. The "Composer" homology modelling program (Tripos) was
used to create a three-dimensional structure of the kinase-like
domain. The structure was refined using molecular dynamics
simulation and simulated annealing in conjunction with the Tripos
suite of programs. The Whatcheck program was used to evaluate the
quality of the created models during various stages of the
refinement process. FIG. 4 shows a representation of the molecular
model of the human JAK2 KLD, with the residues corresponding to the
putative substrate binding site (by comparison with the location of
the substrate-binding site of the insulin receptor PTK domain,
derived from the co-crystal of the PTK domain and its substrate
{Hubbard et al, 1994} shown in green).
[0137] The Model
[0138] Any model that describes the activation of the JAK family of
PTKs must account for the fact that the alteration or mutation of
the primary sequence of the JAK KLD can have either an inhibitory
effect or a stimulatory effect, depending upon what one is
measuring (e.g. kinase activity or cytokine mediated JAK
activation, for example). The JAK family of kinases is proposed to
have a two step triggering mechanism, involving a "priming" or
"cocking" phase, wherein the JAK is converted to a latent form,
followed by a "triggering" phase, (wherein the latent kinase
activity of the PTK domain of the JAK is unleashed), which is
dependent upon the interaction of a given cytokine receptor with
its cognate ligand. See FIG. 2 for details.
[0139] Phase 1: Priming
[0140] The JAK molecule is synthesized in an open conformation
(FIG. 2 panel B), and is converted to a primed or cocked
conformation, perhaps by the agency of a Ser/Thr kinase or
phosphatase. This cocking mechanism may involve ATP, either as a
co-factor for the putative Ser/Thr kinase- or phosphatase-dependent
enzymes involved, or as a co-factor for binding into the ATP
binding site of the KLD. The PSL of the PTK domain is loaded into
its docking site in the KLD, whereupon the access of ATP to the PTK
domain's ATP binding site is restricted. In this form the JAK PTK
domain could be described as being inactive or latent. The PSL may
or may not bind into the substrate-binding site of the kinase like
domain, indicating that the PSL may be a pseudo-substrate for a
pseudo-kinase domain. The cocked JAK is loaded onto the cytokine
receptor either immediately (as has been described for a number of
cytokine receptors) or following cytokine stimulation of a given
receptor (as has been described for Growth Hormone Receptor).
[0141] Phase 2: Triggering
[0142] Following ligand mediated binding to the cytokine receptor,
the PSL is released from the KLD by a mechanism that may (or may
not) involve a additional kinase or phosphatase, thereby releasing
the latent PTK activity in the JAK kinase domain. Ultimately the
activated JAK is inactivated by binding of SOCS1 to a tyrosine in
the PTK domain, and the molecule is targeted to the proteosome.
[0143] Conclusions
[0144] This model suggests a number of possible sites of action
that a potential JAK inhibitor might act to inhibit the cytokine
dependent signal; namely, at the level of the PTK domain; at the
level of the loading of the PSL to the KLD (the cocking mechanism);
by inhibiting the release of the PSL from the KLD (the triggering
mechanism); or by causing the premature or inappropriate release of
the PSL from the KLD.
Experimental Results
[0145] Peptides from the JAK Kinase Loop are Inhibitory for IL3
Signalling
[0146] Following the generation of an alignment of the kinase
domains of all of the available members of the JAK family of PTKs
with the kinase domains of human c-SRC and the human FGF-receptor
(Table 6 see above) revealed the presence of a JAK-family specific
loop (the putative pseudo-substrate loop or PSL) between Hanks
motifs IX and X. Alignment of the PSLs present in the JAKs
demonstrated that the loop structures contained conserved elements,
the consensus of which was:
X--X--S--P-p-X--X--F-.sup.L/.sub.M-.sup.R/.sub.K-M-I-G-p-X--X--
[0147] In particular the serine/proline pair (predicted to be a
site of serine phosphorylation, with a high score (0.994) using
NetPhos 2.0 (Blom et al, 1999)), suggested itself as a possible
site of regulation.
[0148] Two peptides were constructed based upon the PSL sequence.
These were:
8 PSL.sub.short PAEFMRMI SEQ ID NO 15 PSL.sub.control
SPSKFRMPEAMIGND SEQ ID NO 16
[0149] These peptides were tested for biological activity by means
of a proliferation assay using the murine growth factor dependent
hematopoietic cell line Ba/F3. On the basis of our hypothesis of
the role of how the PSL region might work, we reasoned that if
these peptides were to supplant the PSL from the KLD of JAK2, then
the JAK PTK activity would thereby either become unregulated,
resulting in a IL-3 independent growth phenotype, or the normal
IL-3 dependent growth of these cells would be inhibited. The data
presented in FIG. 5 demonstrated that the PSL.sub.short peptide was
able to inhibit growth of wild-type Ba/F3 cells grown upon EL-3,
whereas they were unable to inhibit the growth of Ba/F3 cells
transformed to factor independence by ectopic expression of an
oncogeneic form of JAK2, wherein the PTK domain of JAK2 was fused
to the pointed (PNT) domain of the TEL gene product. The
PSL.sub.short peptide was therefore capable of inhibiting cells
supported by the IL-3 dependent JAK/STAT pathway (i.e. using the
full-length JAK2 protein) but was unable to inhibit cells supported
by the expression of the TEL/JAK2 fusion (containing only the PTK
domain of JAK2). We hypothesize that the activity of the
PSL.sub.short peptide was dependent upon the presence of the JAK2
KLD, and conclude that its mode of action is by displacement of the
PSL of JAK2 from the JAK2 KLD, resulting in unprimed JAK2 molecules
that cannot be triggered by IL-3.
[0150] Demonstration of Binding of Peptides from the PSL to
Purified Preparations of the KLD.
[0151] Our observations that small peptides derived from the PSL
were able to inhibit the IL-3 mediated proliferation of Ba/F3
cells, suggested that the PSL.sub.short inhibited the normal cycle
of priming and triggering of the JAK2 molecule by displacing the
PSL of JAK2 from its binding site in the KLD. In order to
demonstrate this directly we generated highly purified (>95%)
KLD of JAK2 and synthesised two fluorescenated peptides covering
the PSL of JAK2, and attempted to demonstrate binding of the
peptide by the KLD. The two peptides are outlined below:
[0152] PSL-1 Fluorescein-A-Y--I-E-K--S--K--S--P--P-A-E-F-M-(SEQ ID
NO 17)
[0153] Fluorescence Polarization (FP), also known as Fluorescence
Anisotropy (FA), is a means of measurement of peptide protein
binding (Checovich et al, 1995). A measurement of FP is a function
of a particular fluorescenated molecule's rotational relaxation
time, and is empirically a measurement of the time it takes the
molecule to rotate through an angle of 68.5.degree.. The Rotational
relaxation time is related to viscosity (.function.), absolute
temperature (T), molecular volume (V) and the gas constant (R),
according to the formula: 1 FP value Rotational relaxation time = 3
V RT
[0154] Therefore, when temperature and viscosity are both held
constant the FP value is directly related to molecular volume (i.e.
to molecular size). Thus a small fluoresceinated molecule such as a
peptide, will have a lower FP than a larger protein such as an
antibody. Binding of a smaller fluoresceinated peptide to a larger
non-fluoresceinated protein will result in the appropriation of a
higher FP value by the peptide. Thus binding of a peptide to a
receptor can be measured by following the FP value of a
fluoresceinated peptide in the presence or absence of the putative
binding protein for that peptide.
[0155] The binding of the PSL to the JAK2 KLD was tested by means
of an FP assay as follows. 2 pg of the peptide PSL-1 was incubated
in the presence of approximately 2.5 .mu.g of highly purified KLD.
Following a brief incubation at room temperature, a series of FP
measurements were taken using a BMG PolarStar. Comparisons of FP
values in the presence and absence of KLD were compared over time.
These data appear in FIG. 6.
[0156] Assays for the Measurement of the Inhibition for the
Function of the JAK, PSL and KLD
[0157] Cell-Based Assays
[0158] Proliferation Assays
[0159] The model that we have developed for the allosteric
regulation of the protein tyrosine kinase domain of members of the
JAK family by their respective KLDs suggests a number of methods by
which cell-based assays and screens for inhibitors of this
regulation might be brought about. In particular it would be
possible to establish cell based assays by means of the use of
cytokine dependent pathway screens. One example of this would be
the use of a cytokine-dependent cell line such as Ba/F3 and/or
FDCP-1. Each of these cell lines requires the activation of the
JAK/STAT pathway by a cytokine such as interleukin-3 (IL-3) and/or
GMCSF. In the case of Ba/F3, the triggering of the release of the
intrinsic protein tyrosine kinase activity of the JAK2 molecule
depends on the activation of the IL3 receptor by IL-3. In turn, the
priming of the JAK2 molecule by means of the docking of the PSL
into the binding site of the KLD is required. Therefore, in the
presence of an inhibitor of this binding, IL-3 would not be able to
release the kinase activity of JAK2, since the priming step would
not have taken place. Inhibitors of the binding of the PSL into the
KLD would therefore be potential inhibitors of any cytokine that
worked through the JAK kinases, a list of these cytokines appears
below in Table 7. An example of this type of assay is shown in FIG.
5, where inhibition of the growth of the IL-3 dependent growth of
Ba/F3s has been brought about by the addition of a peptide derived
from the JAK2 PSL. In these experiments this peptide is able to
inhibit the growth of Ba/F3 cells at a concentration of 50
.mu.M.
[0160] Any cell line that is dependent for its continued growth and
proliferation upon the presence of a cytokine has the potential for
screening for inhibitors of the binding of the PSL to the KLD.
[0161] Gene Expression Assays.
[0162] The JAK/STAT pathway is responsible for the regulation of
many cytokine dependent genes. Examples of such genes would include
BCl.sub.XL and MHC Class II genes. The generation of cell lines in
which the expression of an indicator gene, such as
.beta.-galactosidase or a selectable marker, such as the gene for
Neomycin resistance (Neo.sup.R), is regulated by an inducible
promoter element is a common method by which studies of gene
regulation have been undertaken. Cell lines in which indicator
genes such as these were regulated by a cytokine regulated GAS
element therefore offers the potential to screen for compounds
which modulate this regulation, such as inhibitors of the
interaction of the PSL with the JAK KLD.
[0163] In Vitro Assays Using Purified Proteins and/or Peptides.
[0164] Assays Measuring the Binding of the PTK Domain with the
KLD
[0165] We have postulated that the PTK domain is regulated by its
binding or otherwise with the KLD. Therefore, by measuring the
enzymatic activity of the PTK domain in the presence of the KLD, an
indirect measure of the binding of the PSL to the KLD can be
obtained. This can be done in a standard ELISA or Fluorescence
Polarisation assay, such as those that have been described
elsewhere. The presence of an inhibitor of the binding of the PSL
to the KLD would be revealed by an increase in PTK activity.
[0166] Assays Measuring the Binding of Portions of the PSL with
Portions of the KLD
[0167] Binding of the PSL to purified KLD could be established as a
sensitive high-throughput screen for inhibitors of the interaction
between the PSL and the KLD. Any means by which the binding of one
protein or peptide to another could be used. Two examples of this
approach would be the use of a fluorescenated or
radioactively-labeled peptide representing the PSL coupled with its
binding to purified KLD.
[0168] The use of a fluorescence labeled peptide should allow the
development of a fluorescence-based assay such as a fluorescence
polarisation assay or a FRET assay. In either of these cases, the
binding of the PSL to the KLD could be determined in a 96, 384 or
1536 well format as the induction of fluorescence polarisation or
FRET. Therefore, inhibitors that prevent the binding of the PSL to
the KLD could be detected as a consequence of reduction in this
signal. An example of this type of approach in shown in FIG. 6.
wherein purified KLD and a fluorescent peptide representing the PSL
are combined in a 96 well plate format with the result that
fluorescence polarisation is induced as a consequence of the
binding of their mutual association.
[0169] An alternative strategy would be to use a filter-binding
assay using radio-labeled peptide and purified KLD in this case
high throughput format screen could easily be established.
Methods
Cloning of JAK2 Kinase-Like Domain (KLD)
[0170] RNA was prepared from .gamma.-IFN stimulated U937 cells.
cDNA (20 .mu.) was prepared using Superscript kit (Gibco). Using an
oligo dT primer provided in the kit was used to prepare the mRNA.
RT-PCR was performed using the Kinase-like domain specific
primers,
9 HJ2KLF GCG CGC GAA TTC ACC TAT CCT CAT ATT (SEQ ID NO 19)
HJ2KLDRE GCG CGC GAA TTC ATC AGA AAT GAA GAT (SEQ ID NO 20)
[0171] and standard PCR conditions. PCR products were gel-purified
from a 1% TAE agarose gel using Gibco Gel Extraction kit.
[0172] PCR products were digested using EcoR1 for 2 hr at
37.degree. C. and then gel purified as above.
[0173] The Invitrogen bacterial expression vector pBAD/gIII (2.5
.mu.g) was digested with EcoRI for 2 hr at 37.degree. C. and then
treated with Calf Intestinal Phosphatase for 1 hr at 37.degree. C.
After the addition of 2 .mu.l 0.5M EDTA at 70.degree. C. for 10
mins, the vector was applied to a PCR purification column and
eluted in 50 .mu.l TE. Vector was ligated to 10 .mu.l PCR product
using T4 ligase at 14.degree. C. overnight. The ligation mix was
transformed into competent One Shot E. Coli. (Invitrogen).
[0174] (Sequencing of the construct (pBKLD) was performed using Big
Dye Termination Kit (Perkin Elmer) with the primers KLDF, J2KLSEQ1
and J2KLSEQ2.
Expression of Kinase-Like Domain in E. coli
[0175] Recombinant E. coli bearing pBKLD were induced for 4 hours
with Serakinase at a final concentration of 0.2% as described in
the pBAD Protocols book (Invitrogen). Periplasmic Cellular
fractions were prepared as recommended in the Invitrogen pBAD
Protocols book Shock Solutions #1 and #2 were then pooled, protease
inhibitors added and the KLD domain purified as outlined below.
[0176] Dot Blotting using anti-C-Term His Antibody (Invitrogen) was
then performed to confirm the presence of expressed KLD domain.
[0177] Assays
[0178] Cell Based Assay
[0179] The murine hematopoietic cell line Ba/F3 was grown in the
presence of IL-3. Peptides (PSL.sub.Short PAEFMRMI and
PSL.sub.Control SPSKFRMPEAMIGND) were added at concentrations
ranging from 1 .mu.M to 100 .mu.M in phosphate buffered saline.
Cell number after three days was measured by an MTT assay.
[0180] KLD Binding Assay
[0181] For fluoresence polarisation assays 1-5 .mu.g of purified
KLD was incubated in 50 mM HEPES, pH 7.5, 12.5 mM NaCl, 1 mM
MgCl.sub.2, in the presence or absence of 1 mM ATP. Peptide JL1
(FITC-.beta.Ala-YIEKSKSPPAEF- M-NH.sub.2) was added to a
concentration of 1 nM and non-fluoresceinated peptide competitors
JLC and JLD were added at 10 or 100 fold molar excess. Fluorescence
polarisation was measured using a BMG Polarstar.
[0182] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
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