U.S. patent application number 11/624686 was filed with the patent office on 2007-08-23 for methods for measuring real time kinase activity.
This patent application is currently assigned to INVITROGEN CORPORATION. Invention is credited to Kyle R. Gee, Min Li, Xiao-Dong Qian, Erik Michael Schaefer.
Application Number | 20070196860 11/624686 |
Document ID | / |
Family ID | 38919529 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196860 |
Kind Code |
A1 |
Gee; Kyle R. ; et
al. |
August 23, 2007 |
Methods for Measuring Real Time Kinase Activity
Abstract
The present invention relates to methods for detecting and/or
measuring the activity of a specific kinase, with the methods
comprising contacting one or more kinases with a binding agent to
isolate a specific kinase of interest. The isolated kinase is then
contacted with a kinase activity sensor, where the kinase activity
sensor is comprised of a kinase recognition motif that is capable
of being recognized by the isolated kinase, and at least one
phosphorylation site. The isolated kinase phosphorylates the amino
acid target of the kinase activity sensor and levels of the
phosphorylated target amino acid can then be quantified.
Inventors: |
Gee; Kyle R.; (Springfield,
OR) ; Li; Min; (Brookline, MA) ; Qian;
Xiao-Dong; (Wellesley, MA) ; Schaefer; Erik
Michael; (Hopkinton, MA) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
INVITROGEN CORPORATION
Carlsbad
CA
|
Family ID: |
38919529 |
Appl. No.: |
11/624686 |
Filed: |
January 18, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60759919 |
Jan 18, 2006 |
|
|
|
60819432 |
Jul 7, 2006 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
530/391.1; 530/409 |
Current CPC
Class: |
C12Q 1/485 20130101;
G01N 33/573 20130101 |
Class at
Publication: |
435/007.1 ;
530/391.1; 530/409 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/46 20060101 C07K016/46 |
Claims
1. A method of measuring the activity of a specific kinase, said
method comprising: providing a solid or semi-solid support
comprising an immobilized binding agent; contacting the immobilized
binding agent with a sample comprising the specific kinase to form
an immobilized specific kinase; contacting the immobilized specific
kinase with a kinase activity sensor to form a contacted specific
kinase, wherein the kinase activity sensor comprises at least one
peptide capable of being phosphorylated and a chelator; incubating
the contacted specific kinase for a sufficient amount of time for
the kinase to phosphorylate the peptide in the presence of a
phosphate source and a metal ion, wherein the kinase activity
sensor forms a ternary complex with the metal ion and
phosphorylated peptide to generate a detectable signal; and
detecting the signal whereby the activity of the specific kinase is
measured.
2. The method of claim 1, wherein the binding agent comprises an
antibody or a functional fragment thereof.
3. The method of claim 1, wherein the support is glass, plastic,
metal, polymeric particle, polymeric gel or a polymeric
membrane.
4. The method according to claim 1, wherein the kinase activity
sensor has an increased fluorescence signal when complexed with the
metal ion.
5. The method of claim 1, wherein the chelator comprises a
signaling moiety.
6. The method according to claim 5, wherein the signaling moiety
comprises a coumarin, cyanine, benzofuran, a quinoline, a
quinazolinone, an indole, a benzazole, a borapolyazaindacene or a
xanthene.
7. The method according to claim 1, wherein the chelator comprises
a quinoline or a derivative thereof, phenanthrolines or derivatives
thereof, BAPTA, IDA, DTPA and derivatives thereof.
8. The method according to claim 1, wherein the kinase activity
sensor further comprises a signaling moiety that is separate from
the chelator.
9. The method according to claim 8, wherein the signaling moiety
comprises a coumarin, cyanine, benzofuran, a quinoline, a
quinazolinone, an indole, a benzazole, a borapolyazaindacene or a
xanthene.
10. The method according to claim 1, wherein the method further
comprises contacting the immobilized specific kinase with ATP.
11. The method according to claim 1, wherein the metal ion is
Ca.sup.2+, Zn.sup.2+, Mg.sup.2+, Ga.sup.3+, Tb.sup.3-, La.sup.3+,
Pb.sup.2+, Hg.sup.2+, Cd.sup.2+, Cu.sup.2+, Ni.sup.2+, Co.sup.2+,
Fe.sup.2+, Mn.sup.2+, Ba.sup.2+, or Sr.sup.2+.
12. The method of claim 1, wherein the peptide comprises an amino
acid that when phosphorylated complexes magnesium.
13. The method according to claim 1, wherein the kinase activity
sensor comprises the formula: ##STR12## where at least one R group
is --SO.sub.2X, where X is --OR'' or --NR''R'''; R' is hydroxy,
amino, or thiol; R'' is C.sub.1-6 alkyl; R''' is hydrogen or alkyl;
and n is 1, 2 or 3.
14. The method of claim 1, wherein said kinase activity sensor has
the formula: ##STR13## wherein R.sup.1, is H, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,
substituted amino, aminocarbonyl, aminothiocarbonyl,
aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino,
carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, halo, hydroxy, nitro, SO.sub.3H, sulfonyl,
substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio,
substituted alkylthio, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, or substituted heterocyclyl; R.sup.2 is a
fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted
alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,
aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,
hydroxy, nitro, SO.sub.3H, sulfonyl, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted
heterocyclyl; R.sup.3 is a fluorophore, H, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,
substituted amino, aminocarbonyl, aminothiocarbonyl,
aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino,
carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, halo, hydroxy, nitro, SO.sub.3H, sulfonyl,
substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio,
substituted alkylthio, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, or substituted heterocyclyl; R.sup.4 is a
fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted
alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,
aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,
hydroxy, nitro, SO.sub.3H, sulfonyl, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted
heterocyclyl; R.sup.5 is a fluorophore, H, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,
substituted amino, aminocarbonyl, aminothiocarbonyl,
aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino,
carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, halo, hydroxy, nitro, SO.sub.3H, sulfonyl,
substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio,
substituted alkylthio, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, or substituted heterocyclyl; and R.sup.6 is a
fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted
alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,
aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,
hydroxy, nitro, SO.sub.3H, sulfonyl, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted
heterocyclyl; R.sup.7 is H, alkyl, substituted alkyl, carbonyl, or
a protecting group; and R.sup.8 is H, alkyl, substituted alkyl,
carbonyl, or a protecting group; L is a linker; and Y is a peptide;
or a tautomer, stereoisomer, or salt thereof.
15. The method of claim 13, wherein at least one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is a fluorophore.
16. The method according to claim 1, wherein the peptide is at
least one of SEQ ID NO. 1 through SEQ ID NO. 133.
17. A composition comprising: (a) a kinase immobilized by a binding
agent; and (b) a kinase activity sensor comprising at least one
peptide capable of being phosphorylated and a chelator.
18. A kit for detecting the activity of a specific kinase,
comprising a binding agent that binds to the specific kinase and a
kinase activity sensor, wherein said kinase activity sensor
comprises at least one peptide capable of being phosphorylated and
a chelator.
19. The kit according to claim 18, further comprising ATP and a
metal ion that has affinity for both the chelator and the
phosphorylated peptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/759,919, filed Jan. 18, 2006 and U.S.
Provisional Application No. 60/819,432, filed Jul. 7, 2006, the
contents of which are incorporated by reference as if set forth
fully herein.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for detecting
and/or measuring the activity of a specific kinase of interest.
BACKGROUND OF THE INVENTION
[0003] Protein kinases are believed to play a role in the formation
of many different diseases. Therefore, new drug candidates and
treatment protocols are being identified using methods for
understanding and identifying protein kinase activity and molecules
that affect that activity.
[0004] A protein kinase catalyzes the transfer of a phosphate group
from adenosine triphosphate (ATP) to a serine, threonine or
tyrosine residue in a peptide or protein sequence. Protein kinases
are involved in all aspects of regulation within cells. The
traditional method for assaying kinase activity is discontinuous
and requires .sup.32P-labelled ATP, which requires special
handling. Many companies market specialized fluorescence kinase
assay systems, all of which are discontinuous, requiring sampling
of the reaction mixture followed by additional handling steps to
label the product of the reaction with a fluorescent moiety (e.g.,
Promega, Panvera, Calbiochem, Cell Signaling Technology, Molecular
Devices, DiscoveRx, Upstate, PerkinElmer). A continuous
fluorescence assay that can be performed in real time is of great
utility. Currently, few examples of sensors capable of such assays
exist.
[0005] U.S. Pat. No. 6,906,104, which is hereby incorporated by
reference, describes sulfonamide substituted quinoline compounds,
including "SOX", for detection of kinases free in solution. An
article by Lawrence et al. describes real time visualization of
protein kinase activity in living cells (Journal of Biological
Chemistry Vol. 277, No. 13, Issue of March 29, pp. 11527-11532,
2002).
[0006] One way to measure kinases activity in real time is to
capture the kinase using the kinase recognition motif, or amino
acid sequence that a particular kinase recognizes and
phosphorylates. Once captured, ATP can be added to the assay and a
signaling moiety can then generate or alter a signal upon a
phosphorylation event. In general, each kinase may be specific for
a particular kinase recognition motif, however, there are some
sequence similarities in the amino acid sequences of the kinase
recognition motif surrounding the target amino acid among kinases.
Thus, using the kinase recognition motif to capture a particular
kinase may result in capturing a population of different kinases
that all recognize the same or similar kinase recognition motif.
The results of these assays have a propensity to generate false
positive results when trying to identify and/or measure the
activity of a specific kinase. What is needed, therefore, is a
method of improving the specificity of the kinase activity assays
to lower the incidences of false positives, thus adding value to
assay results that assess kinase activity. Currently, there are no
methods in the literature that overcome this problem caused by the
promiscuity of kinase recognition motifs in capturing specific
kinases.
SUMMARY OF THE INVENTION
[0007] The present invention relates to methods for detecting
and/or measuring the activity of a specific kinase, with the
methods comprising contacting one or more kinases with a binding
agent to isolate a specific kinase of interest. The isolated kinase
is then contacted with a kinase activity sensor, where the kinase
activity sensor is comprised of a kinase recognition motif that is
capable of being recognized by the isolated kinase, and at least
one phosphorylation site. The isolated kinase, if present,
phosphorylates the amino acid target of the kinase activity sensor
and levels of the phosphorylated amino acid target can then be
quantified.
[0008] One aspect of the present invention provides a method of
measuring the activity of a specific kinase, said method
comprising: [0009] providing a solid or semi-solid support
comprising an immobilized binding agent; [0010] contacting the
immobilized binding agent with a sample comprising the specific
kinase to form an immobilized specific kinase; [0011] contacting
the immobilized specific kinase with a kinase activity sensor to
form a contacted specific kinase, wherein the kinase activity
sensor comprises at least one peptide capable of being
phosphorylated and a chelator; [0012] incubating the contacted
specific kinase for a sufficient amount of time for the kinase to
phosphorylate the peptide in the presence of a phosphate source and
a metal ion, wherein the kinase activity sensor forms a ternary
complex with the metal ion and phosphorylated peptide to generate a
detectable signal; and [0013] detecting the signal whereby the
activity of the specific kinase is measured.
[0014] In a more particular embodiment, the binding agent comprises
an antibody or a functional fragment thereof.
[0015] In another embodiment, the support is glass, plastic, metal,
polymeric particle, polymeric gel or a polymeric membrane.
[0016] In another embodiment, the kinase activity sensor has an
increased fluorescence signal when complexed with the metal ion. In
another embodiment, the kinase activity sensor further comprises a
signaling moiety that is separate from the chelator. More
particular still, the signaling moiety comprises a coumarin,
cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a
benzazole, a borapolyazaindacene or a xanthene.
[0017] In another embodiment, the chelator comprises a signaling
moiety. More particular still, the signaling moiety comprises a
coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an
indole, a benzazole, a borapolyazaindacene or a xanthene. In
another embodiment, the chelator comprises a quinoline or a
derivative thereof, phenanthrolines or derivatives thereof, BAPTA,
IDA, DTPA and derivatives thereof.
[0018] In another embodiment, the metal ion is Ca.sup.2+,
Zn.sup.2+, Mg.sup.2+, Ga.sup.3+, Tb.sup.3+, La.sup.3+, Pb.sup.2+,
Hg.sup.2+, Cd.sup.2+, Cu.sup.2+, Ni.sup.2+, Co.sup.2+, Fe.sup.2+,
Mn.sup.2+, Ba.sup.2+, or Sr.sup.2+.
[0019] In another embodiment, the peptide comprises an amino acid
that when phosphorylated complexes magnesium.
[0020] In another embodiment the method further comprises
contacting the immobilized specific kinase with ATP.
[0021] In another more particular embodiment, the kinase activity
sensor comprises the formula: ##STR1##
[0022] where at least one R group is --SO.sub.2X, where X is --OR''
or --NR''R''';
[0023] R' is hydroxy, amino, or thiol;
[0024] R'' is C.sub.1-6 alkyl;
[0025] R''' is hydrogen or alkyl; and
[0026] n is 1, 2 or 3.
[0027] Alternatively, the kinase activity sensor has the formula:
##STR2## [0028] wherein R.sup.1, is H, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,
substituted amino, aminocarbonyl, aminothiocarbonyl,
aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino,
carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, halo, hydroxy, nitro, SO.sub.3H, sulfonyl,
substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio,
substituted alkylthio, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, or substituted heterocyclyl; [0029] R.sup.2 is a
fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted
alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,
aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,
hydroxy, nitro, SO.sub.3H, sulfonyl, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted
heterocyclyl; [0030] R.sup.3 is a fluorophore, H, alkyl,
substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro,
SO.sub.3H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl,
thiol, alkylthio, substituted alkylthio, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, or substituted heterocyclyl; [0031]
R.sup.4 is a fluorophore, H, alkyl, substituted alkyl, alkoxy,
substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted
amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,
hydroxy, nitro, SO.sub.3H, sulfonyl, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted
heterocyclyl; [0032] R.sup.5 is a fluorophore, H, alkyl,
substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro,
SO.sub.3H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl,
thiol, alkylthio, substituted alkylthio, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, or substituted heterocyclyl; and [0033]
R.sup.6 is a fluorophore, H, alkyl, substituted alkyl, alkoxy,
substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted
amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,
hydroxy, nitro, SO.sub.3H, sulfonyl, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted
heterocyclyl; [0034] R.sup.7 is H, alkyl, substituted alkyl,
carbonyl, or a protecting group; and [0035] R.sup.3 is H, alkyl,
substituted alkyl, carbonyl, or a protecting group; [0036] L is a
linker; and [0037] Y is a peptide; [0038] or a tautomer,
stereoisomer, or salt thereof.
[0039] More particularly, at least one of R.sup.2, R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 is a fluorophore.
[0040] In another embodiment, the peptide is at least one of SEQ ID
NO. 1 through SEQ ID NO. 133.
[0041] Another aspect of the invention provides a composition
comprising at least one of SEQ ID NO. 1 through SEQ ID NO. 133.
[0042] Another aspect of the invention provides a composition
comprising: [0043] (a) a kinase immobilized by a binding agent; and
[0044] (b) a kinase activity sensor comprising at least one peptide
capable of being phosphorylated and a chelator.
[0045] Another aspect of the invention provides a kit for detecting
the activity of a specific kinase, comprising a binding agent that
binds to the specific kinase and a kinase activity sensor, wherein
said kinase activity sensor comprises at least one peptide capable
of being phosphorylated and a chelator. More particularly, the kit
further comprises ATP and a metal ion that has affinity for both
the chelator and the phosphorylated peptide.
[0046] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 depicts the kinase activity of p38 kinase as assessed
using the methods of the present invention.
[0048] FIG. 2 depicts the kinase activity of Erk1/2 kinase as
assessed using the methods of the present invention.
[0049] FIG. 3 depicts Akt1 Activity in Crude Lysates from
PDGF-Treated or Control NIH3T3 Cells.
[0050] FIG. 4 depicts Akt3 Activity in Crude Lysates from
PDGF-Treated or Control NIH3T3 Cells.
[0051] FIG. 5 depicts ERK1/2 Activity in Crude Lysates from
PDGF-Treated or Control NIH3T3 Cells
[0052] FIG. 6 depicts p70-S6K Activity in Crude Lysates from
PDGF-Treated or Control MCF-7 Cells
[0053] FIG. 7 depicts RSK Activity in Crude Lysates from
PMA-Treated or Control HeLa Cells
[0054] FIG. 8 depicts c-Src Activity in Crude Lysates from
c-Src-Transfected CEF Cells.
[0055] FIG. 9 depicts B-Raf Activity in Crude Lysates from NGF
Treated or Control PC12 Cells.
[0056] FIG. 10 depicts ERK1/2 Activity in Crude Lysates from
PDGF-Treated or Control NIH3T3 Cells.
[0057] FIG. 11 depicts MEK1 Activity in Crude Lysates from
PDGF-Treated or Control NIH3T3 Cells.
[0058] FIG. 12 depicts p38 MAPK Activity in Crude Lysates from
Anisomycin Treated or Control RAW Cells.
[0059] FIG. 13 depicts Omnia Tyrosine Kinase Recombinant Assays
Signal to Noise Ratios for various kinase targets, wherein the
kinase recognition motif for each of the figures are as follows:
FIG. 13A corresponds to SEQ ID NO. 42 (.about.2.8 fold); FIG. 13B
corresponds to SEQ ID NO. 43 (.about.2.2 fold); FIG. 13C
corresponds to SEQ ID NO. 45 (.about.2.4 fold); FIG. 13D
corresponds to SEQ ID NO. 44 (.about.1.8 fold); FIG. 13E
corresponds to SEQ ID NO. 46 (.about.8.3 fold); FIG. 13F
corresponds to SEQ ID NO. 48 (.about.6.8 fold); FIG. 13G
corresponds to SEQ ID NO. 47 (.about.7.9 fold); and FIG. 13H
corresponds to SEQ ID NO. 49 (.about.6.9 fold).
[0060] FIG. 14 depicts a Cell-based (Magnetic Bead Capture) Kinase
Assay for Akt/PKB kinase. Dynabeads M-280 w/Sheep anti-Mouse IgG
were used along with Akt Mouse Monoclonal Antibody (Invitrogen). 2
ug anti-Akt mAb to 400 ug lysate were added and incubated at
4.degree. C. overnight. 20 ul Dynabeads were added to each sample
at 4.degree. C. for 2 hrs and then beads were washed 3.times.. 20
uM Aktide were added with 1 mM ATP and results were read in M5 for
5 hrs. The isolation and kinase activity sensor increased
selectivity and adjustable signal.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0061] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a peptide" includes a plurality of peptides and
reference to "an enzyme" includes a plurality of enzymes and the
like.
[0062] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. The
following terms are defined for purposes of the invention as
described herein.
[0063] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms. This term includes, by way of example, linear and
branched hydrocarbyl groups such as methyl (CH.sub.3--), ethyl
(CH.sub.3CH.sub.2--), n-propyl (CH.sub.3CH.sub.2CH.sub.2--),
isopropyl ((CH.sub.3).sub.2CH--), n-butyl
(CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH.sub.2--).
[0064] "Substituted alkyl" refers to an alkyl group having from 1
to 5, preferably 1 to 3, or more preferably 1 to 2 substituents
selected from the group consisting of alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein.
[0065] "Alkoxy" refers to the group --O-alkyl wherein alkyl is
defined herein. Alkoxy includes, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
[0066] "Substituted alkoxy" refers to the group --O-(substituted
alkyl) wherein substituted alkyl is defined herein.
[0067] "Acyl" refers to the groups H--C(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, alkynyl-C(O)--, substituted alkynyl-C(O)--,
cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
substituted aryl-C(O)--, heteroaryl-C(O)--, substituted
heteroaryl-C(O)--, heterocyclic-C(O)--, and substituted
heterocyclic-C(O)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
Acyl includes the "acetyl" group CH.sub.3C(O)--.
[0068] "Acylamino" refers to the groups --NRC(O)alkyl,
--NRC(O)substituted alkyl, --NRC(O)cycloalkyl, --NRC(O)substituted
cycloalkyl, --NRC(O)cycloalkenyl, --NRC(O)substituted cycloalkenyl,
--NRC(O)alkenyl, --NRC(O)substituted alkenyl, --NRC(O)alkynyl,
--NRC(O)substituted alkynyl, --NRC(O)aryl, --NRC(O)substituted
aryl, --NRC(O)heteroaryl, --NRC(O)substituted heteroaryl,
--NRC(O)heterocyclic, and --NRC(O)substituted heterocyclic wherein
R is hydrogen or alkyl and wherein alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0069] "Acyloxy" refers to the groups alkyl-C(O)O--, substituted
alkyl-C(O)O--, alkenyl-C(O)O--, substituted alkenyl-C(O)O--,
alkynyl-C(O)O--, substituted alkynyl-C(O)O--, aryl-C(O)O--,
substituted aryl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, cycloalkenyl-C(O)O--, substituted
cycloalkenyl-C(O)O--, heteroaryl-C(O)O--, substituted
heteroaryl-C(O)O--, heterocyclic-C(O)O--, and substituted
heterocyclic-C(O)O-- wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0070] "Amino" refers to the group --NH.sub.2.
[0071] "Substituted amino" refers to the group --NR'R'' where R'
and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, substituted
heterocyclic, --SO.sub.2-alkyl, --SO.sub.2-substituted alkyl,
--SO.sub.2-alkenyl, --SO.sub.2-substituted alkenyl,
--SO.sub.2-cycloalkyl, --SO.sub.2-substituted cycloalkyl,
--SO.sub.2-cycloalkenyl, --SO.sub.2-substituted cycloalkenyl,
--SO.sub.2-aryl, --SO.sub.2-substituted aryl,
--SO.sub.2-heteroaryl, --SO.sub.2-substituted heteroaryl,
--SO.sub.2-heterocyclic, and --SO.sub.2-substituted heterocyclic
and wherein R' and R'' are optionally joined, together with the
nitrogen bound thereto to form a heterocyclic or substituted
heterocyclic group, provided that R' and R'' are both not hydrogen,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein. When R' is hydrogen and R'' is
alkyl, the substituted amino group is sometimes referred to herein
as alkylamino. When R' and R'' are alkyl, the substituted amino
group is sometimes referred to herein as dialkylamino. When
referring to a monosubstituted amino, it is meant that either R' or
R'' is hydrogen but not both. When referring to a disubstituted
amino, it is meant that neither R' nor R'' are hydrogen.
[0072] "Aminocarbonyl" refers to the group --C(O)N R.sup.10R.sup.11
where R.sup.10 and R.sup.11 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.10 and
R.sup.11 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0073] "Aminothiocarbonyl" refers to the group
--C(S)NR.sup.10R.sup.11 where R.sup.10 and R.sup.11 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0074] "Aminocarbonylamino" refers to the group
--NRC(O)NR.sup.10R.sup.11 where R is hydrogen or alkyl and R.sup.10
and R.sup.11 are independently selected from the group consisting
of hydrogen, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and
substituted heterocyclic and where R.sup.10 and R.sup.11 are
optionally joined together with the nitrogen bound thereto to form
a heterocyclic or substituted heterocyclic group, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0075] "Aminothiocarbonylamino" refers to the group
--NRC(S)NR.sup.10R.sup.11 where R is hydrogen or alkyl and R.sup.10
and R.sup.11 are independently selected from the group consisting
of hydrogen, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and
substituted heterocyclic and where R.sup.10 and R.sup.11 are
optionally joined together with the nitrogen bound thereto to form
a heterocyclic or substituted heterocyclic group, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0076] "Aminocarbonyloxy" refers to the group
--O--C(O)NR.sup.10R.sup.11 where R.sup.10 and R.sup.11 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0077] "Aminosulfonyl" refers to the group
--SO.sub.2NR.sup.10R.sup.11 where R.sup.10 and R.sup.11 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0078] "Aminosulfonyloxy" refers to the group
--O--SO.sub.2NR.sup.10R.sup.11 where R.sup.10 and R.sup.11 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0079] "Aminosulfonylamino" refers to the group
--NR--SO.sub.2NR.sup.10R.sup.11 where R is hydrogen or alkyl and
R.sup.10 and R.sup.11 are independently selected from the group
consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.10 and
R.sup.11 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0080] "Amidino" refers to the group
--C(.dbd.NR.sup.12)R.sup.10R.sup.11 where R.sup.10, R.sup.11, and
R.sup.12 are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0081] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring (e.g.,
phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)
which condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom. Preferred aryl groups include phenyl and naphthyl.
[0082] "Substituted aryl" refers to aryl groups which are
substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to
2 substituents selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein.
[0083] "Aryloxy" refers to the group --O-aryl, where aryl is as
defined herein, that includes, by way of example, phenoxy and
naphthoxy.
[0084] "Substituted aryloxy" refers to the group --O-(substituted
aryl) where substituted aryl is as defined herein.
[0085] "Arylthio" refers to the group --S-aryl, where aryl is as
defined herein.
[0086] "Substituted arylthio" refers to the group --S-(substituted
aryl), where substituted aryl is as defined herein.
[0087] "Alkenyl" refers to alkenyl groups having from 2 to 6 carbon
atoms and preferably 2 to 4 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkenyl unsaturation. Such groups
are exemplified, for example, by vinyl, allyl, and
but-3-en-1-yl.
[0088] "Substituted alkenyl" refers to alkenyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein and with the proviso
that any hydroxy substitution is not attached to a vinyl
(unsaturated) carbon atom.
[0089] "Alkynyl" refers to alkynyl groups having from 2 to 6 carbon
atoms and preferably 2 to 3 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkynyl unsaturation.
[0090] "Substituted alkynyl" refers to alkynyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein and with the proviso
that any hydroxy substitution is not attached to an acetylenic
carbon atom.
[0091] "Carbonyl" refers to the divalent group --C(O)-- which is
equivalent to --C(.dbd.O)--.
[0092] "Carboxyl" or "carboxy" refers to --COOH or salts
thereof.
[0093] "Carboxyl ester" or "carboxy ester" refers to the groups
--C(O)O-alkyl, --C(O)O-substituted alkyl, --C(O)O-alkenyl,
--C(O)O-substituted alkenyl, --C(O)O-alkynyl, --C(O)O-substituted
alkynyl, --C(O)O-aryl, --C(O)O-substituted aryl,
--C(O)O-cycloalkyl, --C(O)O-substituted cycloalkyl,
--C(O)O-cycloalkenyl, --C(O)O-substituted cycloalkenyl,
--C(O)O-heteroaryl, --C(O)O-substituted heteroaryl,
--C(O)O-heterocyclic, and --C(O)O-substituted heterocyclic wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0094] "(Carboxyl ester)amino" refers to the group
--NR--C(O)O-alkyl, substituted --NR--C(O)O-alkyl,
--NR--C(O)O-alkenyl, --NR--C(O)O-substituted alkenyl,
--NR--C(O)O-alkynyl, --NR--C(O)O-substituted alkynyl,
--NR--C(O)O-aryl, --NR--C(O)O-substituted aryl,
--NR--C(O)O-cycloalkyl, --NR--C(O)O-substituted cycloalkyl,
--NR--C(O)O-cycloalkenyl, --NR--C(O)O-substituted cycloalkenyl,
--NR--C(O)O-heteroaryl, --NR--C(O)O-substituted heteroaryl,
--NR--C(O)O-heterocyclic, and --NR--C(O)O-substituted heterocyclic
wherein R is alkyl or hydrogen, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0095] "(Carboxyl ester)oxy" refers to the group --O--C(O)O-alkyl,
substituted --O--C(O)O-alkyl, --O--C(O)O-alkenyl,
--O--C(O)O-substituted alkenyl, --O--C(O)O-alkynyl,
--O--C(O)O-substituted alkynyl, --O--C(O)O-aryl,
--O--C(O)O-substituted aryl, --O--C(O)O-cycloalkyl,
--O--C(O)O-substituted cycloalkyl, --O--C(O)O-cycloalkenyl,
--O--C(O)O-substituted cycloalkenyl, --O--C(O)O-heteroaryl,
--O--C(O)O-substituted heteroaryl, --O--C(O)O-heterocyclic, and
--O--C(O)O-substituted heterocyclic wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0096] "Cyano" refers to the group --CN.
[0097] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10
carbon atoms having single or multiple cyclic rings including
fused, bridged, and spiro ring systems. Examples of suitable
cycloalkyl groups include, for instance, adamantyl, cyclopropyl,
cyclobutyl, cyclopentyl, and cyclooctyl.
[0098] "Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of
from 3 to 10 carbon atoms having single or multiple cyclic rings
and having at least one >C.dbd.C< ring unsaturation and
preferably from 1 to 2 sites of >C.dbd.C< ring
unsaturation.
[0099] "Substituted cycloalkyl" and "substituted cycloalkenyl"
refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or
preferably 1 to 3 substituents selected from the group consisting
of oxo, thione, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein.
[0100] "Cycloalkyloxy" refers to --O-cycloalkyl.
[0101] "Substituted cycloalkyloxy refers to --O-(substituted
cycloalkyl).
[0102] "Cycloalkylthio" refers to --S-cycloalkyl.
[0103] "Substituted cycloalkylthio" refers to --S-(substituted
cycloalkyl).
[0104] "Cycloalkenyloxy" refers to --O-cycloalkenyl.
[0105] "Substituted cycloalkenyloxy refers to --O-(substituted
cycloalkenyl).
[0106] "Cycloalkenylthio" refers to --S-cycloalkenyl.
[0107] "Substituted cycloalkenylthio" refers to --S-(substituted
cycloalkenyl).
[0108] "FMOC" refers to a compound having the general formula (or
derivatives thereof): ##STR3##
[0109] "Guanidino" refers to the group --NHC(.dbd.NH)NH.sub.2.
[0110] "Substituted guanidino" refers to
--NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 where each R.sup.13
is independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heterocyclic, and substituted heterocyclic
and two R.sup.13 groups attached to a common guanidino nitrogen
atom are optionally joined together with the nitrogen bound thereto
to form a heterocyclic or substituted heterocyclic group, provided
that at least one R.sup.13 is not hydrogen, and wherein said
substituents are as defined herein.
[0111] "Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo.
[0112] "Hydroxy" or "hydroxyl" refers to the group --OH.
[0113] "Heteroaryl" refers to an aromatic group of from 1 to 10
carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur within the ring. Such
heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)
or multiple condensed rings (e.g., indolizinyl or benzothienyl)
wherein the condensed rings may or may not be aromatic and/or
contain a heteroatom provided that the point of attachment is
through an atom of the aromatic heteroaryl group. In one
embodiment, the nitrogen and/or the sulfur ring atom(s) of the
heteroaryl group are optionally oxidized to provide for the N-oxide
(N.fwdarw.O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls
include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
[0114] "Substituted heteroaryl" refers to heteroaryl groups that
are substituted with from 1 to 5, preferably 1 to 3, or more
preferably 1 to 2 substituents selected from the group consisting
of the same group of substituents defined for substituted aryl.
[0115] "Heteroaryloxy" refers to --O-heteroaryl.
[0116] "Substituted heteroaryloxy refers to the group
--O-(substituted heteroaryl).
[0117] "Heteroarylthio" refers to the group --S-heteroaryl.
[0118] "Substituted heteroarylthio" refers to the group
--S-(substituted heteroaryl).
[0119] "Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or unsaturated group having a
single ring or multiple condensed rings, including fused bridged
and spiro ring systems, from 1 to 10 carbon atoms and from 1 to 4
hetero atoms selected from the group consisting of nitrogen, sulfur
or oxygen within the ring wherein, in fused ring systems, one or
more the rings can be cycloalkyl, aryl or heteroaryl provided that
the point of attachment is through the non-aromatic ring. In one
embodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic
group are optionally oxidized to provide for the N-oxide, sulfinyl,
sulfonyl moieties.
[0120] "Substituted heterocyclic" or "substituted heterocycloalkyl"
or "substituted heterocyclyl" refers to heterocyclyl groups that
are substituted with from 1 to 5 or preferably 1 to 3 of the same
substituents as defined for substituted cycloalkyl.
[0121] "Heterocyclyloxy" refers to the group --O-heterocyclyl.
[0122] "Substituted heterocyclyloxy refers to the group
--O-(substituted heterocyclyl).
[0123] "Heterocyclylthio" refers to the group --S-heterocyclyl.
[0124] "Substituted heterocyclylthio" refers to the group
--S-(substituted heterocyclyl).
[0125] Examples of heterocycle and heteroaryls include, but are not
limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
dihydroindole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, and tetrahydrofuranyl.
[0126] "Nitro" refers to the group --NO.sub.2--.
[0127] "Oxo" refers to the atom (.dbd.O) or (--O.sup.-).
[0128] "Spirocyclyl" refers to divalent saturated cyclic group from
3 to 10 carbon atoms having a cycloalkyl or heterocyclyl ring with
a spiro union (the union formed by a single atom which is the only
common member of the rings) as exemplified by the following
structure: ##STR4##
[0129] "Sulfonyl" refers to the divalent group --S(O).sub.2--.
[0130] "Substituted sulfonyl" refers to the group --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-alkenyl,
--SO.sub.2-substituted alkenyl, --SO.sub.2-cycloalkyl,
--SO.sub.2-substituted cylcoalkyl, --SO.sub.2-cycloalkenyl,
--SO.sub.2-substituted cycloalkenyl, --SO.sub.2-aryl,
--SO.sub.2-substituted aryl, --SO.sub.2-heteroaryl,
--SO.sub.2-substituted heteroaryl, --SO.sub.2-heterocyclic,
--SO.sub.2-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein. Substituted sulfonyl includes groups such as
methyl-SO.sub.2--, phenyl-SO.sub.2--, and
4-methylphenyl-SO.sub.2--.
[0131] "Sulfonyloxy" refers to the group --OSO.sub.2-alkyl,
--OSO.sub.2-substituted alkyl, --OSO.sub.2-alkenyl,
--OSO.sub.2-substituted alkenyl, --OSO.sub.2-cycloalkyl,
--OSO.sub.2-substituted cylcoalkyl, --OSO.sub.2-cycloalkenyl,
--OSO.sub.2-substituted cycloalkenyl, --OSO.sub.2-aryl,
--OSO.sub.2-substituted aryl, --OSO.sub.2-heteroaryl,
--OSO.sub.2-substituted heteroaryl, --OSO.sub.2-heterocyclic,
--OSO.sub.2-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0132] "Thioacyl" refers to the groups H-C(S)--, alkyl-C(S)--,
substituted alkyl-C(S)--, alkenyl-C(S)--, substituted
alkenyl-C(S)--, alkynyl-C(S)--, substituted alkynyl-C(S)--,
cycloalkyl-C(S)--, substituted cycloalkyl-C(S)--,
cycloalkenyl-C(S)--, substituted cycloalkenyl-C(S)--, aryl-C(S)--,
substituted aryl-C(S)--, heteroaryl-C(S)--, substituted
heteroaryl-C(S)--, heterocyclic-C(S)--, and substituted
heterocyclic-C(S)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined
herein.
[0133] "Thiol" refers to the group --SH.
[0134] "Thiocarbonyl" refers to the divalent group --C(S)-- which
is equivalent to --C(.dbd.S)--.
[0135] "Thione" refers to the atom (.dbd.S).
[0136] "Alkylthio" refers to the group --S-alkyl wherein alkyl is
as defined herein.
[0137] "Substituted alkylthio" refers to the group --S-(substituted
alkyl) wherein substituted alkyl is as defined herein.
[0138] The term "protected" or a "protecting group" with respect to
hydroxyl groups, amine groups, and sulfhydryl groups refers to
forms of these functionalities which are protected from undesirable
reaction with a protecting group known to those skilled in the art
such as those set forth in Protective Groups in Organic Synthesis,
Greene, T. W. , Solon Wiley & Sons, New York, N.Y., (1st
Edition, 1981) which can be added or removed using the procedures
set forth therein. Examples of protected hydroxyl groups include,
but are not limited to, silyl ethers such as those obtained by
reaction of a hydroxyl group with a reagent such as, but not
limited to, t-butyldimethyl-chlorosilane, trimethylchlorosilane,
triisopropylchlorosilane, triethylchlorosilane; substituted methyl
and ethyl ethers such as, but not limited to methoxymethyl ether,
methythiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether,
2-methoxyethoxymethyl ether, tetrahydropyranyl ethers,
1-ethoxyethyl ether, allyl ether, benzyl ether; esters such as, but
not limited to, benzoylformate, formate, acetate, trichloroacetate,
and trifluoracetate. Examples of protected amine groups include,
but are not limited to, benzyl or dibenzyl, amides such as,
fonnamide, acetamide, trifluoroacetamide, and benzamide; imides,
such as phthalimide, BOC (tBoc), FMOC, and dithiosuccinimide; and
others. In some embodiments, a protecting group for amines is an
FMOC group. Examples of protected sulfhydryl groups include, but
are not limited to, thioethers such as S-benzyl thioether, and
S-4-picolyl thioether; substituted S-methyl derivatives such as
hemithio, dithio and aminothio acetals; and others.
[0139] "Stereoisomer" or "stereoisomers" refer to compounds that
differ in the chirality of one or more stereocenters. Stereoisomers
include enantiomers and diastereomers.
[0140] "Tautomer" refer to alternate forms of a compound that
differ in the position of a proton, such as enol-keto and
imine-enamine tautomers, or the tautomeric forms of heteroaryl
groups containing a ring atom attached to both a ring --NH-- moiety
and a ring .dbd.N-- moeity such as pyrazoles, imidazoles,
benzimidazoles, triazoles, and tetrazoles.
[0141] The term "affinity" as used herein refers to the strength of
the binding interaction of two molecules, such as a metal-chelating
compound and a metal ion.
[0142] The term "detectable response" as used herein refers to an
occurrence of, or a change in, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an optical response resulting
in a change in the wavelength distribution patterns or intensity of
absorbance or fluorescence or a change in light scatter,
fluorescence lifetime, fluorescence polarization, or a combination
of these parameters. Alternatively, the detectable response is an
occurrence of a signal wherein the dye is inherently fluorescent
and does not produce a change in signal upon binding to a metal ion
or phosphorylated target molecule. Alternatively, the detectable
response is the result of a signal, such as color, fluorescence,
radioactivity or another physical property of the detectable label
becoming spatially localized in a subset of a sample such as in a
gel, on a blot, or an array, in a well of a micoplate, in a
microfluidic chamber, or on a microparticle as the result of
formation of a ternary complex of the invention that comprises a
phosphorylated target molecule.
[0143] The term "enzyme" as used herein refers to a protein
molecule produced by living organisms, or through chemical
modification of a natural protein molecule, that catalyzes chemical
reaction of other substances without itself being destroyed or
altered upon completion of the reactions. Examples of other
substances, include, but are not limited to chemiluminescent,
chromogenic and fluorogenic substances or protein-based
substrates.
[0144] A "kinase" is an enzyme capable of phosphorylating a
substrate. Due to the extreme versatility of the methods and
components described herein, the present invention finds use in the
detection of activity from all kinases. Preferred kinases for use
in the present invention include but are not limited to: LCK, IRK
(=INSR=Insulin receptor), IGF-1 receptor, SYK, ZAP-70, IRAK1, BLK,
BMX, BTK, FRK, FGR, FYN, HCK, ITK, LYN, TEC, TXK, YES, ABL, SRC,
EGF-R (=ErbB-1), ErbB-2 (=NEU=HER.sup.2), ErbB-4, FAK, FGF1R
(=FGR-1), FGF2R (=FGR-2), IKK-1 (=IKK-ALPHA=CHUK), IKK-2
(=IKK-BETA), MET (=c-MET), NIK, PDGF receptor ALPHA, PDGF receptor
BETA, TIE1, TIE2 (=TEK), VEGFR1 (=FLT-1), VEGFR2 (=KDR), FLT-3,
FLT-4, KIT, CSK, JAK1, JAK2, JAK3, TYK2, RIP, RIP-2, LOK, TAKI,
RET, ALK, MLK3, COT, TRKA, PYK2, Activin-like Kinases (Alk1-7),
EPHA(1-8), EPHB(1-6), RON, GSK3(A and B), Ilk, PDK1, SGK, Fcs, Fer,
MatK, Ark(1-3), Plk(1-3), LimK(1 and 2), RhoK, Pak (1-3), Raf(A, B,
and C), PknB, CDK(1-10), Chk(1 and 2), CamK(I-IV), CamKK, CK1, CK2,
PKR, Jnk(1-3), EPHB4, UL13, ORF47, ATM, PKA (.alpha., .beta. and
.gamma.), P38(.alpha., .beta., and .gamma.), Erk(1-3), PKB
(including all PKB subtypes) (=AKT-1, AKT-2, AKT-3), and PKC
(including all PKC subtypes) and all subtypes of these kinases.
Preferred kinases of the present invention are tyrosine and/or
serine/threonine kinases.
[0145] The term "kinase activity sensor" refers to a compound or
composition comprising at least one peptide substrate capable of
being phosphorylated, a chelator and a detection moiety, wherein
the detection moiety is the chelator per se or includes an
additional detection moiety, such as a fluorophore. A preferred
kinase activity sensor of the present invention has the formula:
##STR5##
[0146] wherein the variables are described herein.
[0147] The term "fluorophore" as used herein refers to a
composition that is inherently fluorescent or demonstrates a change
in fluorescence upon binding to a biological compound or metal ion,
i.e., fluorogenic. Fluorophores may contain substitutents that
alter the solubility, spectral properties or physical properties of
the fluorophore. Numerous fluorophores are known to those skilled
in the art and include, but are not limited to coumarin, cyanine,
benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a
borapolyazaindacene and xanthenes including fluoroscein, rhodamine
and rhodol as well as semiconductor nanocrystals and other
fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES
HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (10.sup.th
edition, 2005).
[0148] The term "linker" refers to a divalent moiety capable of
linking two particles, either as a single covalent bond or a series
of stable covalent bonds incorporating 1-30 nonhydrogen atoms
selected from the group consisting of C, N, O, S and P that
covalently attach the fluorophore, quinoline group and amino acids
to form the present kinase activity sensor. Exemplary linking
members include a moiety that includes --C(O)NH--, --C(O)O--,
--NH--, --S--, --O--, and the like. Exemplary linkers include
covalent bonds, amino acids, succinimidyl derivatives, methines,
and alkenyl groups, alkyl and substituted alkyl groups, ethyleyene,
proypylene, polyethylene, and polypropylene glycols, esters,
ethers, amides, carbamates, and carbonyl containing moieties,
diones, squarate, adipic acid as well as other groups, such as
those described in Chemistry of Protein Conjugation and
Cross-Linking by Susan Wong.
[0149] A "cleavable linker" is a linker that has one or more
cleavable groups that may be broken by the result of a reaction or
condition. The term "cleavable group" refers to a moiety that
allows for release of a portion, e.g., a reporter molecule, carrier
molecule or solid support, of a conjugate from the remainder of the
conjugate by cleaving a bond linking the released moiety to the
remainder of the conjugate. Such cleavage is either chemical in
nature, or enzymatically mediated. Exemplary enzymatically
cleavable groups include natural amino acids or peptide sequences
that end with a natural amino acid.
[0150] The present invention relates to methods for detecting
and/or measuring the activity of a specific kinase. "Specific
kinase" is used to indicate a known kinase of interest, where at
least some information about the activity and/or structure is known
to the practitioner prior to implementing the methods of the
present invention.
[0151] The methods comprise contacting a sample comprising one or
more kinases with a binding agent to isolate the specific kinase of
interest. The one or more kinases may be present in any environment
including but not limited to, a cell, a tissue, a body fluid, a
buffer solution or another synthetic composition or fluid. Where
more than one type of kinase is present, the collection of kinases
represents a plurality or population of kinases. Not every member
of the population of kinases need be a specific kinase, as
understood herein. Rather, the sample, which could comprise a
population of different kinases, should comprise or may be
suspected to comprise the specific kinase being assayed.
[0152] The specific kinase must then be exposed to or contacted
with a binding agent capable of specifically binding the specific
kinase. As used herein, the term binding agent is used to mean a
composition that binds specifically to the known biomarker.
Examples of binding agents include, but are not limited to,
receptors, antibodies and functional fragments thereof. As used
herein, the term "antibody" is used to mean immunoglobulin
molecules and functional fragments thereof, regardless of the
source or method of producing the fragment. As used herein, a
"functional fragment" of an immunoglobulin is a portion of the
immunoglobulin molecule that specifically binds to a binding
target. Thus, the term "antibody" as used herein encompasses whole
antibodies, such as antibodies with isotypes that include but are
not limited to IgG, IgM, IgA, IgD, IgE and IgY. Whole antibodies
may be monoclonal or polyclonal, and they may be humanized or
chimeric. The term "monoclonal antibody" as used herein is not
limited to antibodies produced through hybridoma technology. Rather
the term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced. The
term "antibody" also encompasses functional fragments of
immunoglobulins, including but not limited to Fab fragments, Fab'
fragments, F(ab').sub.2 fragments and Fd fragments. "Antibody" also
encompasses fragments of immunoglobulins that comprise at least a
portion of a V.sub.L and/or V.sub.II domain, such as single chain
antibodies, a single-chain Fv (scFv), disulfide-linked Fvs and the
like.
[0153] The antibodies used in the present invention may be
monospecific, bispecific, trispecific or of even greater
multispecificity. In addition the antibodies may be monovalent,
bivalent, trivalent or of even greater multivalency. Furthermore,
the antibodies of the invention may be from any animal origin
including, but not limited to, birds and mammals. In specific
embodiments, the antibodies are human, murine, rat, sheep, rabbit,
goat, guinea pig, horse, or chicken. As used herein, "human"
antibodies include antibodies having the amino acid sequence of a
human immunoglobulin and include antibodies isolated from human
immunoglobulin libraries or from animals transgenic for one or more
human immunoglobulin and that do not express endogenous
immunoglobulins, as described in U.S. Pat. No. 5,939,598, which is
herein incorporated by reference.
[0154] The antibodies used in the present invention may be
described or specified in terms of the epitope(s) or portion(s) of
a polypeptide to which they recognize or specifically bind. Or the
antibodies may be described based upon their ability to bind to
specific conformations of the antigen. In one embodiment, a single
antibody used in the methods of the present invention is specific
towards an epitope presented on the specific kinase of
interest.
[0155] The specificity of the antibodies used in present invention
may also be described or specified in terms their binding affinity
towards the antigen (epitope) or of by their cross-reactivity.
Specific examples of binding affinities encompassed in the present
invention include but are not limited to those with a dissociation
constant (Kd) less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M,5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0156] The antibodies used in the invention also include
derivatives that are modified, for example, by covalent attachment
of any type of molecule to the antibody such that covalent
attachment does not prevent the antibody from generating an
anti-idiotypic response. Examples of modifications to antibodies
include but are not limited to, glycosylation, acetylation,
pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other composition, such as a signaling moiety, a
label etc. In addition, the antibodies may be linked or attached to
solid substrates, such as, but not limited to, beads, particles,
glass surfaces, plastic surfaces, ceramic surfaces, metal surfaces.
Methods of attaching an antibody to a surface are described in
Coligan, J E et al, Current Protocols In Immunology, Wiley
Intersciences, (1993) and Harlow et al., Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988), which
are incorporated by reference. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin and the like.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0157] The antibodies used in the present invention may be
generated by any suitable method known in the art. Polyclonal
antibodies can be produced by various procedures well known in the
art. For example, a kinase or an epitope on the kinase can be
administered to various host animals including, but not limited to,
rabbits, mice, rats, to induce the production of sera containing
polyclonal antibodies specific for the antigen. Various adjuvants
may be used to increase the immunological response, depending on
the host species, and include but are not limited to, Freund's
(complete and incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants are also well known in the art. In addition, portions of
the peptide chain making up the kinase may be attached to multiarm
peptides known as multiple antigentic peptides (MAPs). The MAP can
then be administered to an animal to generate an immune response
that is highly tailored to the epitope presented on the arm or arms
of MAP. Methods of generating antibodies using MAP technology are
well-known in the art. Methods of generating antibodies using MAP
technology is described in Tam, J P, Proc. Natl. Acad. Sci. USA 85:
5409, (1988) and Olive, C. et al., Mini Rev. Med. Chem. 1:429,
(2001), which are incorporated by reference.
[0158] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (both of which are incorporated by
reference).
[0159] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
such as, but not limited to, immunizing a mouse. Once an immune
response is detected, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. The hybridoma clones can then be assayed by
methods known in the art for cells that secrete antibodies capable
of binding a biomarker of the present invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0160] The antibodies used in the present invention can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. In a particular embodiment, such phage can
be utilized to display antigen binding domains expressed from a
repertoire or combinatorial antibody library. Phage expressing an
antigen binding domain that binds the antigen of interest can be
selected or identified with the antigen of interest, such as using
a labeled antigen or antigen bound or captured to a solid surface
or bead. The phage used in these methods are typically filamentous
phage including, but not limited to, fd and M13 binding domains
expressed from phage with Fab, Fv or disulfide stabilized Fv
antibody domains recombinantly fused to either the phage gene III
or gene VIII protein. Examples of phage display methods that can be
used to make the antibodies of the present invention include those
disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995);
Ames et al., J. Immunol Methods 184:177-186 (1995); Kettleborough
et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187
9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994); PCT application No. PCT/GB91/01134; PCT publications WO
90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO
95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;
5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and
5,969,108, all of which are incorporated by reference.
[0161] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab').sub.2
fragments). F(ab').sub.2 fragments contain the variable region, the
light chain constant region and the CH1 domain of the heavy
chain.
[0162] Other methods, such as recombinant techniques, may be used
to produce Fab, Fab' and F(ab').sub.2 fragments and are disclosed
in PCT publication WO 92/22324; Mullinax et al., BioTechniques
12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and
Better et al., Science 240:1041-1043 (1988), which are herein
incorporated by reference. After phage selection, for example, the
antibody coding regions from the phage can be isolated and used to
generate whole antibodies, including human antibodies, or any other
desired antigen binding fragment, and expressed in any desired
host, including mammalian cells, insect cells, plant cells, yeast,
and bacteria.
[0163] Examples of techniques which can be used to produce other
types of fragments, such as scFvs and include those described in
U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al, Methods in
Enzymology 203:46-88 (1991); Shu et al., Proc. Nat'l Acad. Sci.
(USA) 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040
(1988), all of which are incorporated by reference. For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol.
Methods 125:191-202(1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and
4,816,397, all of which are herein incorporated by reference.
Humanized antibodies are antibody molecules from non-human species
antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), both of which are herein
incorporated by reference. Antibodies can be humanized using a
variety of techniques known in the art including, for example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat.
Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing
(EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., Proc. Nat'l. Acad. Sci.
91:969-913 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332),
all of which are hereby incorporated by reference.
[0164] Completely human antibodies may be particularly desirable
for therapeutic treatment or diagnosis of human patients. Human
antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See also.
U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO
98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO
96/33735, and WO 91/10741; each of which is incorporated by
reference.
[0165] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol 13:65-93 (1995), which is hereby incorporated by
reference. For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;
European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;
5,916,771; and 5,939,598, which are incorporated by reference.
[0166] Still another approach for generating human antibodies
utilizes a technique referred to as guided selection. In guided
selection, a selected non-human monoclonal antibody, e.g., a mouse
antibody, is used to guide the selection of a completely human
antibody recognizing the same epitope. (Jespers et al.,
Biotechnology 12:899-903 (1988), herein incorporated by
reference).
[0167] The isolated kinase is then contacted with a kinase activity
sensor. As used herein, a kinase activity sensor is a composition
comprising a kinase recognition motif with at least one
phosphorylation site. The phosphorylation site may or may not be
present within the kinase recognition motif of the kinase activity
sensor. In one embodiment, the phosphorylation site of is an amino
acids reside that is capable being phosphorylated. Thus, in aspect
the kinase activity sensor comprises a peptide that is capable of
being phosphroylated. Examples of amino acids capable of being
phosphorylated include, but are not limited to, naturally occurring
hydroxyl-containing amino acid residues, such as serine, threonine
and tyrosine, and non-naturally occuring hydroxyl-containing amino
acid residues.
Kinase Recognition Motif:
[0168] Any kinase recognition motif can be used in accordance with
the present invention. While still functional, kinase recognition
motifs with acidic residues may be less desirable because of the
acidic conditions that these residues may create during the assay.
Examples of recognition motifs that can be used in the kinase
activity sensors include but are not limited to, the motifs shown
in Table I: TABLE-US-00001 TABLE I Kinase SEQ ID NO. Kinase
Recognition Motif 1 Protein Kinase C (PKC) TFRRR 2 Protein Kinase C
(PKC) SFRRR 3 Cyclic-AMP Dependent Kinase LRRASL (PKA) 4 Cyclic-AMP
Dependent Kinase LRRATL (PKA) 5 Abelson Kinase (Abl) IYAAPF
[0169] Other isolated kinase recognition motifs include but are not
limited to, the recognition motifs in Table II, Table III and Table
IV. TABLE-US-00002 TABLE II SEQ ID Kinase Recognition NO. Kinase
Name Motif 6 Akt1-3, CHK1, DAPK3, MLK2, ARKRERAYSF(dP) and MSK1/2,
p70S6K, PAK1/2/4, ARKRERAY(pS)F(dP) PKA, PKG, RSK1-3, SGK1-3 7
AKT2, AuroraB, MLK2, MSK1, LRRASL(dP) and PAK1/2/4, PASK, PKA, PKG,
LRRA(pS)L(dP) PRKX, RSK2, SGK1-3 8 CHK1/2, CHK, MK2/3/5, PAK1,
AHLQRQLSI(dP) and PHKG2, PKD AHLQRQL(pS)I(dP) 9 Akt1-3, AuroaB,
MSK1/2, GRPRTSSF(dP) and PKA, PRKX, Rock1/2, RSK1-3,
GRPRTS(pS)F(dP) SGK1-3, MLK2, PAK1-4/7 10 Akt1/2, MLK2, MSK1/2,
GRTGRRNSI(dP) and p70S6K, PAK1-4/7, PKA, PKG, GRTGRRN(pS)I(dP)
PRKX, RSK1-3, SGK1-3 11 Akt1-3, MLK2, MSK1, KKRNRTLSV(dP) and
PAK1/2, PASK, PKG, SGK1-3 KKRNRTL(pS)V(dP) 12 CSF1R, FLT3, c-Met,
PGIYAAPFAKKK and PDGFRa/b; Ab1, JAK2/3, Lck PGI(pY)AAPFAKKK 13 FYN,
Src PGIYGELEA and PGI(pY)GELEA 14 EPHA4, EPHB2, VEGFR2; FAK,
ELEDDYED(dP) and Syk ELEDD(pY)ED(dP) 15 ALK, FLT3, IGF1R, IR, TrkA;
PGAYGWLDF and FER PGA(pY)GWLDF 16 CDK1, 2, 5 PGTPKKAKKL and
PG(pT)PKKAKKL 17 ERK1 & ERK2; p38 gamma PRTPGGRR and
PR(pT)PGGRR 18 p38 beta, gamma, ERK1 PGTPSGEAPNQALLR and
PG(pT)PSGEAPNQALLR 8 ERK1 & ERK2 AHLQRQLSd(dP)and
AHLQRQL(pS)I(dP) 8 p38 alpha (vs. beta, gamma, AHLQRQLSI(dP) and
delta) AHLQRQL(pS)I(dP) 19 PDK1 ARKRERAYSF(dP) and
ARKRERAY(pS)F(dP) 8 MEK1 AHLQRQLSI(dP) and AHLQRQL(pS)I(dP) 8 MEK2
AHLQRQLSI(dP) and AHLQRQL(pS)I(dP) 8 c-Raf AHLQRQLSI(dP) and
AHLQRQL(pS)I(dP) 8 b-Raf AHLQRQLSI(dP) and AHLQRQL(pS)I(dP) 8 MEKK1
AHLQRQLSI(dP) and AHLQRQL(pS)I(dP)
[0170] TABLE-US-00003 TABLE III SEQ ID NO. Sequence 20
Ac-ARKRERAYSFdPSxG-NH2 21 Ac-LRRASLdPSxG-NH2 22
Ac-AHLQRQLSIdPSxG-NH2 23 Ac-GRPRTSSFdPSxG-NH2 24
Ac-GRTGRRNSIdPSxG-NH2 25 Ac-KKRNRTLSVdPSxG-NH2 26
Ac-SxPKTPKKAKKL-NH2 27 Ac-SxPGSFRRR-NH2 28 Ac-SxPRTPGGRR-NH2 29
Ac-SxPGTPSGEAPNQALLR- NH2 30 Ac-ALKLSRYPSFdPSxG-NH2 31
Ac-GDQDYLSLdPSxG-NH2 32 Ac-SxPGSRRPpSYR-NH2 33 Ac-RRRQFSLdPSxG-NH2
34 Ac-ERMRPRKRQGSVdPSxG- NH2 35 Ac-ALRRFSLdPSxG-NH2 36
Ac-SxPLSPGPF-NH2 37 Ac-KRRRLASLdPSxG-NH2 38 Ac-DRHDSGLDSMdPSxG-NH2
39 Ac-DRHDpSGLDSMdPSxG-NH2 40 Ac-HAAIGDDDDAYSIdPSxG- NH2 41
Ac-SxPGSDDDDD-NH2 42 Ac-SxPGIYAAPFAKKK-NH2 43 Ac-SxPGIYGELEA-NH2 44
Ac-ELEDDYEDdPSxG-NH2 45 Ac-SxPGAYGWLDF-NH2 46 Ac-EAEAIYAAdPSxG-NH2
47 Ac-KKGEAIYAAdPSxG-NH2 48 Ac-EEEEYIQdPSxG-NH2 49
Ac-ESSDDYVNdPSxG-NH2
[0171] TABLE-US-00004 TABLE IV SEQ ID NO. Sequence 50
Ac-SxPGLSPGPF-NH2 51 Ac-SxPGTTPITTTYF-NH2 52 Ac-AEEEIYGEdPSxG-NH2
53 Ac-SxPGEIYGELEA-NH2 54 Ac-RFARKGSLdPSxG-NH2 55
Ac-SxPGLTPSGEAPN-NH2 56 Ac-SxPGASFRGHMAR-NH2 57
Ac-RARRRLSFdPSxG-NH2 58 Ac-SxPGSPGPF-NH2 59 Ac-SxPGTPITTYFFFK-NH2
60 Ac-SxPGSFRGHMAR-NH2 61 Ac-SxPGSPGRRRK-NH2 62 Ac-SxPGTPKKAKKL-NH2
63 Ac-SxPGTPGGRR-NH2 64 Ac-EEEYEEdPSxG-NH2 65 Ac-SxPGEYEEEE-NH2 66
Ac-SxPGIYETDYYRKG-NH2 67 Ac-SxPGDIYETDFFRKG-NH2 68
Ac-SxPDIYETDFFRKG-NH2 69 Ac-LMTGDTYTAdPSxG-NH2 70
Ac-VSETDDYAEdPSxG-NH2 71 Ac-SxPGDYAEIIDEED-NH2 72
Ac-SxPGTSDFQKLKRKY-COOH 73 Ac-KKALRRQETVdPSxG-NH2 74
Ac-EVIEASFdPSxG-NH2 75 Ac-SxPGSFAEQEA-NH2 76 Ac-SxPGSPLRGPPK-NH2 77
Ac-SxPGSVPPpSPD-NH2 78 Ac-SxPGSRTPpSLPTPPTREPK-NH2 79
Ac-SxPGSPHQpSEDEEE-NH2 80 Ac-SxPGSPSLpSRHSSPH-NH2 81
Ac-SxPGSLVGpTPYWMAPE-NH2 82 Ac-KKRFSFKKSFdPSxG-NH2 83
Ac-SxPGIYAAPGD-NH2 84 Ac-SxPGIYGVIE-NH2 85 Ac-SxPGIYFELVAK-NH2 86
Ac-SxPGDYVNVPESGEK-NH2 87 Ac-ALKRASLdPSxG-NH2 88
Ac-AKRRRLSSLdPSxG-NH2 89 Ac-EKNGKKARKSLdPSxG-NH2 90
Ac-VPKQKRKSVdPSxG-NH2 91 Ac-AMARAASAdPSxG-NH2 92
Ac-APSSRRTTLdPSxG-NH2 93 Ac-DSDVHVNATYVNdPSxG-NH2 94
Ac-EEEIYFEdPSxG-NH2 95 Ac-EYCPDPLYEVdPSxG-NH2 96
Ac-KKKKEEIYFFdPSxG-NH2 97 Ac-SxPGDYRATFPEDQFP-NH2 98
Ac-SxPGEYVNIEFG-NH2 99 Ac-SxPGHYVHVNATYVNVK-NH2 100
Ac-SxPGLYEVMLKCWHPK-NH2 101 Ac-TEMVSNESVDYRAdPSxG-NH2 102
Ac-ARRRGVTTKTFdPSxG-NH2 103 Ac-GVTTKTFdPSxG-NH2 104
Ac-SxPKTFCGTPEYLAPEVRR-NH2 105 Ac-SxPLSVSSLPGL-NH2 106
Ac-SxPDSGLDpSMKDE-NH2 107 Ac-SxPDSGLDSMKDE-NH2 108
Ac-SxPGSGLDpSMKDE-NH2 109 Ac-SxPGSGLDSMKDE-NH2 110
Ac-RDKYKTLdPSxG-NH2 111 Ac-SxPKTLRQIRQ-NH2 112
Ac-SxPRTGRGRRGIYR-NH2 113 Ac-SxPSSMVARTQTVR-NH2 114
Ac-SxPFSLRRKAK-NH2 115 Ac-PLLRDASTdPSxG-NH2 116 Ac-SxPASTRDRHA-NH2
117 Ac-KKRNRRLSVdPSxG-NH2 118 Ac-PLSRTLSVdPSxG-NH2 119
Ac-SxPASFAEQEAK-NH2 120 Ac-LKKLRRRLSDdPSxG-NH2 121
Ac-TRPRKRQGSFdPSxG-NH2 122 Ac-RRRDDDSDdPSxG-NH2 123
Ac-RRKDLHDDEEDEAMSIdPSxG-NH2 124 Ac-RRRADDSDdPSxG-NH2 125
Ac-SxPDSDDDDD-NH2 126 Ac-SxPLSQEAFADLWKK-NH2 127
Ac-RKKFGESEKTKdPSxG-NH2 128 Ac-SxPKSWAIPNRARK-NH2 129
Ac-KKKALSRQFSVdPSxG-NH2 130 Ac-KLNRVFSVdPSxG-NH2 131
Ac-Biotin-LC-SxPKTPKKAKKL-NH2 132 Ac-KRRRALS(pS)VASLdPSxG-NH2 133
Ac-KVEKIGEGTYGVdPSxG-NH2
[0172] Reference to "Sx" indicates SOX (a kinase activity sensor
described herein), "dP" indicates D-proline, "Ac" indicates acyl,
--NH2 indicates amino.
[0173] A preferred embodiment of the present invention provides a
composition comprising at least one of SEQ ID NO. 1-133. More
particularly, the invention provides a composition comprising or
consisting of SEQ ID NO. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, or SEQ ID NO. 49. More particular still, at least one of
SEQ ID NO. 1-133 is used as a kinase recognition motif described
herein.
[0174] If present, the isolated kinase, will then phosphorylate the
amino acid target of the kinase activity sensor. The inventors have
discovered that kinases isolated and immobilized with binding
agents are still active enzymes. Thus, the active specific kinase
is available to phosphorylate the target amino acid residue or
residues. In one embodiment, adenosine tri-phosphate (ATP) and a
metal ion that has affinity for the chelator of the kinase activity
sensor and the phosphorylated amino acid are added to the assay
either, before, during or after, the isolated kinase is contacted
with the kinase activity sensor. In a particular aspect the metal
ion is magnesium, however any metal ion as described above is part
of the invention.
Kinase Activity Sensors:
[0175] Levels of phosphorylation activity on the phosphorylation
site can then be quantified using various techniques. In one
embodiment, the kinase activity sensor further comprises a
signaling moiety that is capable of generating or altering a signal
in response to a phosphorylation event on the target
phosphorylation site. In a more specific embodiment, the signaling
moiety is capable of generating a fluorescent signal. Examples of a
signaling moiety include but are not limited to Fluorescent
Magnesium Indicators (Molecular Probes, Eugene, Oreg., USA) and
metal-binding amino acids that fluoresce upon a chelation event.
These amino acids are described in U.S. Pat. No. 6,906,194, which
is hereby incorporated by reference.
[0176] As used herein, the term metal chelator is used as it is in
the art. Namely, a metal chelator is a compound that can form two
or more coordination bonds with metal ion. The term "coordination
bond" is also well known in the art and is used to indicate a
coordinate covalent bond between the metal ion and the
chelator.
[0177] The present metal-chelating moieties are moieties that
simultaneously bind metal ions and have affinity for exposed
phosphate groups on serine, threonine, or tyrosine residues of the
kinase substrate, wherein a ternary complex is formed between the
metal-chelating moiety, the metal ion and the phosphorylated
serine, threonine, or tyrosine residues. Metal ions that have been
found to bind phosphate groups include Ca.sup.2+, Zn.sup.2+,
Mg.sup.2+, Ga.sup.3+, Tb.sup.3+, La.sup.3+, Pb.sup.2+, Hg.sup.2+,
Cd.sup.2|, Cu.sup.2|, Ni.sup.2|, Co.sup.2|, Fe.sup.2|, Mn.sup.2|,
Ba.sup.2|, and Sr.sup.2|. A preferred metal ion for chelation by
the kinase activity sensor of the present invention is magnesium.
Thus, the metal-chelating moieties must 1) bind metal ions that
have affinity for phosphate groups, 2) not interfere with the
binding of the metal ion for the phosphate groups and 3) maintain a
stable ternary complex. Metal-chelating moieties that fit these
three criteria include quinoline or a derivative thereof,
phenanthrolines or derivatives thereof, BAPTA, IDA, DTPA and
derivatives thereof.
[0178] In one embodiment the present kinase activity sensor is
represented by the following formula: ##STR6##
[0179] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 are independently a fluorophore, H, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,
substituted amino, aminocarbonyl, aminothiocarbonyl,
aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino,
carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, halo, hydroxy, nitro, SO.sub.3H, sulfonyl,
substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio,
substituted alkylthio, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, or substituted heterocyclyl;
[0180] R.sup.7 and R.sup.8 are independently H, alkyl, substituted
alkyl, carbonyl, or FMOC; and L is a linker; and Y is a peptide or
H; or a tautomer, stereoisomer, or salt thereof.
[0181] In a preferred embodiment the kinase activity sensor has the
formula: ##STR7## wherein,
[0182] R.sup.4 is a sulfonamide group or a fluorophore
[0183] R.sup.7 and R.sup.8 are independently H, alkyl, substituted
alkyl, carbonyl, or FMOC; and L is a linker; and Y is a peptide or
H; or a tautomer, stereoisomer, or salt thereof.
[0184] In another preferred embodiment the kinase activity sensor
has the formula: ##STR8##
[0185] R.sup.7 and R.sup.8 are independently H, alkyl, substituted
alkyl, carbonyl, or FMOC; and L is a linker; and Y is a peptide; or
a tautomer, stereoisomer, or salt thereof.
[0186] In another preferred embodiment, the fluorophore on the
kinase activity sensor has the formula: ##STR9## wherein R.sup.9
and R.sup.10 are each independently a halogen, --SO.sub.3H,
substituted sulfonyl, or H.
[0187] In another preferred embodiment, the kinase activity sensor
has the formula ##STR10##
[0188] wherein R.sup.7 is H, or a protecting group;
[0189] R.sup.9 is a halogen, --SO.sub.3H, substituted sulfonyl, or
H;
[0190] R.sup.10 is a halogen, --SO.sub.3H, substituted sulfonyl, or
H;
[0191] L is a linker; and
[0192] Y is a peptide or H;
[0193] or a tautomer, stereoisomer, or salt thereof.
[0194] In a more particular embodiment of any of the foregoing,
R.sup.4 is a fluorophore. In another embodiment, the fluorophore is
dansyl, xanthene, cyanine, borapolyazaindacene, pyrene,
naphthalene, coumarin, oxazine, or derivatives thereof. In another
embodiment the fluorophore is a coumarin, a xanthene or a
derivative thereof. In another embodiment, R.sup.1 is hydroxy. In
another embodiment, R.sup.2, R.sup.3, R.sup.5 and R.sup.6 are all
H. In another embodiment, R.sup.7 is H or FMOC. In another
embodiment, R.sup.8 is H. In another embodiment, Y is a peptide. In
another embodiment, the peptide comprises a kinase recognition site
and at least one amino acid residue selected from serine,
threonine, or tyrosine that is subject to phosphorylation by a
kinase. In another embodiment, the position indicated with a bond
is in the S configuration; alternatively it is in the
R-configuration; alternatively the kinase activity sensor is a
racemate. In another embodiment, the salt of the kinase activity
sensor is magnesium. In another embodiment, L is a covalent bond.
In another embodiment, Y is H. In another embodiment, R.sup.9 and
R.sup.10 are both fluorine.
[0195] The present fluorophores can be any fluorophore known in the
art that when conjugated to a chelating moiety are fluorogenic or
essentially non-fluorescence. A fluorophore of the present
invention is any chemical moiety that exhibits an absorption
maximum beyond 280 nm, that when part of a kinase activity sensor
compound retains its unique spectral properties to provide a
detectable signal. In one embodiment the present fluorphores are a
separate moiety from the chelating moiety of the kinase activity
sensor. In another embodiment the chelator is also the
fluorophore.
[0196] Examples of fluorophores that can be used in the present
invention include, but are not limited to; a pyrene, an anthracene,
a naphthalene, an acridine, a stilbene, an indole or benzindole, an
oxazole or benzoxazole, a thiazole or benzothiazole, a
4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a carbocyanine
(including any corresponding compounds in U.S. Ser. Nos.
09/557,275; 09/968,401 and 09/969,853 and U.S. Pat. Nos. 6,403,807;
6,348,599; 5,486,616; 5,268,486; 5,569,587; 5,569,766; 5,627,027
and 6,048,982), a carbostyryl, a porphyrin, a salicylate, an
anthranilate, an azulene, a perylene, a pyridine, a quinoline, a
borapolyazaindacene (including any corresponding compounds
disclosed in U.S. Pat. Nos. 4,774,339; 5,187,288; 5,248,782;
5,274,113; and 5,433,896), a xanthene (including any corresponding
compounds disclosed in U.S. Pat. No. 6,162,931; 6,130,101;
6,229,055; 6,339,392; 5,451,343 and U.S. Ser. No. 09/922,333), an
oxazine or a benzoxazine, a carbazine (including any corresponding
compounds disclosed in U.S. Pat. No. 4,810,636), a phenalenone, a
coumarin (including an corresponding compounds disclosed in U.S.
Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), a
benzofuran (including an corresponding compounds disclosed in U.S.
Pat. Nos. 4,603,209 and 4,849,362) and benzphenalenone (including
any corresponding compounds disclosed in U.S. Pat. No. 4,812,409)
and derivatives thereof. As used herein, oxazines include
resorufins (including any corresponding compounds disclosed in U.S.
Pat. No. 5,242,805), aminooxazinones, diaminooxazines, and their
benzo-substituted analogs.
[0197] In particular embodiments, the fluorophore of the present
invention is selected from the group consisting of acridine,
anthracene, benzofuran, indole, dansyl, cyanine,
borapolyazaindacene, pyrene, naphthalene, coumarin, oxazine, boron
dipyromethene difluoride, and xanthenes, including but not limited
to fluorescein, rhodamine, and rhodol, and derivatives thereof
Additional fluorophores that may be used in the present invention
are listed in Richard P. Haugland, Molecular Probes Handbook of
Fluorescent Probes and Research Chemicals (9.sup.th Ed.),. In a
more particular embodiment, the xanthenes are fluorescein, or
rhodamine. The fluorophores may be substituted to adjust
solubility, spectral or other physical properties.
[0198] Where the fluorophore is a xanthene, the fluorophore may,
but need not be, a fluorescein, a rhodol (including any
corresponding compounds disclosed in U.S. Pat. Nos. 5,227,487 and
5,442,045), a rosamine or a rhodamine (including any corresponding
compounds in U.S. Pat. Nos. 5,798,276; 5,846,737; 5,847,162;
6,017,712; 6,025,505; 6,080,852; 6,716,979; 6,562,632). As used
herein, fluorescein includes benzo- or dibenzofluoresceins,
seminaphthofluoresceins, or naphthofluoresceins. Similarly, as used
herein rhodol includes seminaphthorhodafluors (including any
corresponding compounds disclosed in U.S. Pat. No. 4,945,171).
Fluorinated xanthene fluorophores have been described previously as
possessing particularly useful fluorescence properties (Int. Publ.
No. WO 97/39064 and U.S. Pat. No. 6,162,931) including those sold
under the tradename OREGON GREEN.
[0199] In particular the Oregon Green precursor will allow for
strategic placement of a very bright and highly absorbing
pH-insensitive fluorescein derivative in peptide kinase substrates.
The fluorescence will initially be quenched via PET by the
8-hydroxyquinoline moiety, but chelation induced fluorescence
increase, mediated by magnesium(II) ion will be observed upon
phosphorylation of the appropriate Ser/Thr/Tyr residue that is
separated from the dye by a beta-turn dipeptide sequence. The
fluorescence (490 nm excitation/520 nm emission) increase will be
similar in mechanism and magnitude to that observed upon using
Fluo-4 for calcium measurements. Key synthetic steps will be
regioselective formylation of the 8-hyroxyquinoline amino acid,
followed by condensation of 4-fluororesorcinol and dehydrogenative
oxidation. Key to the success of the invention is appropriate
construction of the fluorophore-amino acid moiety, and placement of
the fluorophore-amino acid moiety in three dimensional geometry
such that the chelating moiety and phosphate moiety can be brought
close together by an intervening metal ion. This geometric
optimization is visualized by semi-empirical molecular modeling
energy minimization of the PKCa peptide substrate Ac-Oregon Green
alanine-Pro-Gly-Ser-Phe-Arg-Arg-Arg-NH.sub.2. The
fluorophore-substituted amino acid is placed two amino acid
residues away from the serine to be phosphorylated so that the
metal chelating portion of the Oregon Green alanine derivative can
be oriented toward the phosphoserine moiety. The
hydroxyquinoline-fluorophore moiety is constructed so that the
fluorescence is quenched in the absence of metal ion chelation, and
the hydroxyquinoline-amino acid connection point is also chosen for
optimal geometry.
[0200] Typically the fluorophore will contain one or more aromatic
or heteroaromatic rings, that are optionally substituted one or
more times by a variety of substituents, including without
limitation, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl,
alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or
heteroaryl ring system, benzo, or other substituents typically
present on chromophores or fluorophores known in the art.
[0201] In an exemplary embodiment, the fluorophores are
independently substituted by substituents selected from the group
consisting of hydrogen, halogen, amino, substituted amino, alkyl,
substituted alkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, alkoxy, sulfo, reactive group and carrier molecule. In
another embodiment, the xanthene fluorophores of this invention
comprise both compounds substituted and unsubstituted on the carbon
atom of the central ring of the xanthene by substituents typically
found in the xanthene-based fluorophores such as phenyl and
substituted-phenyl moieties. In still another embodiment, the
fluorophores used in the amino acids and compositions of the
present invention are rhodamine, fluorescein, dansyl, naphthalene
and derivatives thereof. The choice of the fluorophore attached to
the chelating moiety will determine the absorption and fluorescence
emission properties of the amino acids and compositions of the
present invention as well as its live cell properties.
[0202] Selected sulfonated fluorophores also exhibit advantageous
properties, and include sulfonated pyrenes, coumarins,
carbocyanines, and xanthenes (as described in U.S. Pat. Nos.
5,132,432; 5,696,157; 5,268,486; 6,130,101). Sulfonatedpyrenes and
coumarins are typically excited at wavelengths below about 450 nm
(U.S. Pat. Nos. 5,132,432 and 5,696,157).
[0203] In one embodiment, the label is a fluorophore selected from
the group consisting of fluorescein, coumarins, rhodamines, 5-TMRIA
(tetramethylrhodamine-5-iodoacetamide), (9-(2(or
4)-(N-(2-maleimdylethyl)-sulfonamidyl)-4 (or
2)-sulfophenyl)-2,3,6,7,12,13,16,17-octahydro-(1H,5H,11H,15H-xantheno(2,3-
,4-ij:5,6,7-i'j')diquinolizin-18-ium salt) (Texas Red.RTM.),
2-(5-(1-(6-(N-(2-maleimdylethyl)-amino)-6-oxohexyl)-1,3-dihydro-3,3-dimet-
hyl-5-sulfo-2H-indol-2-ylidene)-1,3-propyldienyl)-1-ethyl-3,3-dimethyl-5-s-
ulfo-3H-indolium salt (Cy.TM.3),
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine (IANBD amide), 6-acryloyl-2-dimethylaminonaphthalene
(acrylodan), pyrene,
6-amino-2,3-dihydro-2-(2-((iodoacetyl)amino)ethyl)-1,3-dioxo-1H-benz(de)i-
soquinoline-5,8-disulfonic acid salt (lucifer yellow),
2-(5-(1-(6-(N-(2-maleimdylethyl)-amino)-6-oxohexyl)-1,3-dihydro-3,3-dimet-
hyl-5-sulfo-2H-indol-2-ylidene)-1,3-pentadienyl)-1-ethyl-3,3-dimethyl-5-su-
lfo-3H-indolium salt (Cy.TM.5),
4-(5-(4-dimethylaminophenyl)oxazol-2-yl)phenyl-N-(2-bromoacetamidoethyl)s-
ulfonamide (Dapoxyl.RTM. (2-bromoacetamidoethyl)sulfonamide)),
(N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)i-
odoacetamide (BODIPY.RTM. 507/545 IA),
N-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N-
'-iodoacetylethylenediamine (BODIPY 530/550 IA),
5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid
(1,5-IAEDANS), and carboxy-X-rhodamine, 5/6-iodoacetamide (XRIA
5,6). Another example of a label is BODIPY-FL-hydrazide.
[0204] In an exemplary embodiment, the fluorophore has a Stokes
shift larger than about 50 nm. In particularly useful embodiments,
the fluorophore has a Stokes shift larger than about 100 nm or
larger than about 150 nm. In still more embodiments, the present
compounds have a Stokes shift larger than about 200 nm or larger
than about 250 nm. The term "Stokes shift" as used herein refers to
the difference in wavelength between absorbed and emitted energy.
Specifically, the Stokes shift is the difference (usually in
frequency units) between the spectral positions and the band maxima
(or band origin) of the absorption and luminescence arising from
the same electronic transitions.
[0205] To facilitate correct kinase substrate recognition motifs,
S/T or Y peptide substrate sampler plates with 14 substrates (1
substrate per well and 6 wells per substrate) dried onto the
surface of a 96-well plate are provided. Each substrate is
resuspended in reaction buffer and the kinase(s) of interest are
added to each well, followed by monitoring the increase in
fluorescence in real time.
[0206] Another embodiment of the present invention provides a
method of detecting kinase activity comprising [0207] measuring the
fluorescence of a kinase activity sensor comprising a metal
chelator, one or more amino acids and a fluorophore, wherein the
chelator comprises a quinoline group and both the fluorophore and
amino acid are conjugated to the quinoline group and, and wherein
the amino acids comprise a kinase recognition site and a
phosphorylation site; [0208] providing a solid or semi-solid
support comprising an immobilized binding agent; [0209] contacting
the immobilized binding agent with a sample comprising the specific
kinase to form an immobilized specific kinase; [0210] contacting
the immobilized specific kinase with a kinase activity sensor to
form a contacted specific kinase, wherein the kinase activity
sensor comprises at least one peptide capable of being
phosphorylated and a chelator; [0211] incubating the contacted
specific kinase for a sufficient amount of time for the kinase to
phosphorylate the peptide in the presence of a phosphate source and
a metal ion, wherein the kinase activity sensor forms a ternary
complex with the metal ion and phosphorylated peptide to generate a
detectable signal; and [0212] detecting the signal whereby the
activity of the specific kinase is measured [0213] determining the
difference in fluorescence of the kinase activity sensor between
the non-contacted and contacted states; [0214] wherein a difference
in fluorescence indicates the presence kinase activity.
[0215] In another more particular embodiment, the measured
fluorescence is selected from the group consisting of intensity,
excitation or emission wavelength, distribution of fluorescence,
fluorescence lifetime, fluorescence polarization, or a combination
thereof.
[0216] Another embodiment of the present invention provides a
method of detecting kinase activity, wherein the method comprises:
[0217] contacting one or more kinases with a binding agent to
isolate said specific kinase, thereby forming an isolated kinase;
[0218] contacting said isolated kinase with a kinase activity
sensor, wherein said kinase activity sensor comprises a kinase
recognition motif that is capable of being recognized by said
isolated kinase, and at least one phosphorylation site; and [0219]
illuminating the incubated sample with an appropriate wavelength to
form an illuminated sample; [0220] observing the illuminated sample
wherein the kinase activity is detected by the presences of an
observable fluorescent signal.
[0221] Scheme 1 provides an illustration of one embodiment of the
present invention employing a kinase activity sensor: ##STR11##
[0222] The magnesium complex modifies the emission and/or
excitation spectrum of the kinase activity sensor, such that
phosphorylation of the peptide can be detected and/or
quantified.
[0223] The present kinase activity sensors comprise one or more
amino acids. In one embodiment, the amino acids form a peptide that
comprise at least one kinase recognition motif, wherein the kinase
recognition motifs comprise at least one phosphorylatable amino
acid residue.
[0224] Any kinase recognition motif can be used in accordance with
the present invention. Recognition sequences with acidic residues
may show a lesser magnitude of fluorescence increase upon
phosphorylation than comparable sequences, as the affinity of the
unphosphorylated peptide for magnesium tends to increase under
acidic conditions. Phosphorylation sites within the kinase
recognition motif accordance with the present invention include,
but are not limited to, amino acids comprising hydroxyl groups.
Examples include, but are not limited to, naturally occurring
hydroxyl-containing amino acid residues, such as serine, threonine
and tyrosine, as well as non-naturally occurring
hydroxyl-containing amino acid residues. Preferred recognition
motifs are listed in Tables I, II, and III.
[0225] Other methods of detecting levels of phosphorylation
activity include but are not limited to the use of phosphorylation
site specific antibodies that can recognize and bind to
phosphorylated amino acid residues. Examples of antibodies that
recognize phosphorylated amino acid residues include those
antibodies described in United Stated Patent Application
Publication No. 2003/0162330, Mandel, J., Phosphorylation
State-Specific Antibodies, Amer. J. Pathol., 163:1687 (2003) and
Glenney, J R et al., Monoclonal antibodies to phosphotyrosin. J.
Immunol. Methods. 109:277 (1988), which are hereby incorporated by
reference.
[0226] The methods of the present invention are useful for drug
discovery settings. In one particular embodiment, the methods of
the present invention comprise exposing a cell or tissue to
compound, such as drug or drug candidate, prior to isolating the
specific kinase of interest. In this regard, the collection of
kinases is present in the cells or tissue and the drug or drug
candidate is administered to the cells or tissue. Once exposed, the
cells tissue can be lysed and the specific kinase of interest can
then be isolated and its activity quantified using the methods of
the present invention.
Solid or Semi-Solid Supports:
[0227] In an exemplary embodiment, the compounds or binding agents
(e.g. antibodies) of the invention are bonded to a solid or
semi-solid support. A support suitable for use in the present
invention is typically substantially insoluble in liquid phases.
Solid or semi-solid supports of the current invention are not
limited to a specific type of support. Rather, a large number of
supports are available and are known to one of ordinary skill in
the art. Thus, useful solid supports include semi-solids, such as
aerogels and hydrogels, resins, beads, biochips (including thin
film coated biochips), multi-well plates (also referred to as
microtitre plates), membranes, conducting and nonconducting metals
and magnetic supports. More specific examples of useful solid
supports include silica gels, polymeric membranes, particles,
derivatized plastic films, glass beads, cotton, plastic beads,
alumina gels, polysaccharides such as Sepharose, poly(acrylate),
polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,
dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan,
inulin, nitrocellulose, diazocellulose, polyvinylchloride,
polypropylene, polyethylene (including poly(ethylene glycol)),
nylon, latex bead, magnetic bead, paramagnetic bead,
superparamagnetic bead (Dynabeads (e.g. M-280)), starch and the
like. Preferred supports include agarose and magnetic beads.
[0228] In some embodiments, the solid or semi-solid support may
include a support reactive functional group, including, but not
limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen,
nitro, cyano, amido, urea, carbonate, carbamate, isocyanate,
sulfone, sulfonate, sulfonamide, sulfoxide, etc., for attaching the
compounds of the invention. In a preferred embodiment, the solid
supports contain a nucleophile (x), such as amino, thiol, or
hydroxyl.
[0229] A suitable solid or semi-solid support can be selected on
the basis of desired end use and suitability for various synthetic
protocols. For example, where amide bond formation is desirable to
attach the compounds of the invention to the solid support, resins
generally useful in peptide synthesis may be employed, such as
polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula
Laboratories, etc.), POLYHIPE.TM. resin (obtained from Aminotech,
Canada), polyamide resin (obtained from Peninsula Laboratories),
polystyrene resin grafted with polyethylene glycol (TentaGel.TM.,
Rapp Polymere, Tubingen, Germany), polydimethyl-acrylamide resin
(available from Milligen/Biosearch, California), or PEGA beads
(obtained from Polymer Laboratories).
Kits:
[0230] Additional embodiments of the present invention include kits
comprising the compositions described herein for use in detection
of kinase activity. In addition, the kits include instructions on
how to best detect the kinase of interest.
[0231] Another aspect of the invention provides a kit for detecting
the activity of a specific kinase, comprising a binding agent that
binds to the specific kinase and a kinase activity sensor, wherein
said kinase activity sensor comprises at least one peptide capable
of being phosphorylated and a chelator. More particularly, the kit
further comprises ATP and a metal ion that has affinity for both
the chelator and the phosphorylated peptide.
[0232] To facilitate correct kinase substrate recognition motifs,
S/T or Y peptide substrate sampler plates with 14 substrates (1
substrate per well and 6 wells per substrate) dried onto the
surface of a 96-well plate are provided. Each substrate is
resuspended in reaction buffer and the kinase(s) of interest are
added to each well, followed by monitoring the increase in
fluorescence in real time.
[0233] A preferred kit of the invention includes 14 Sox-based
peptide substrates that can be used to measure the activity of a
variety of Ser/Thr kinases. The peptides are arranged in sets of 6
replicates with a bottom row of 12 replicates of a control peptide.
These substrates correspond to peptides listed in SEQ ID NOs
20-33.
[0234] Another preferred kit includes 14 Sox-based peptide
substrates (kinase activity sensors) that can be used to measure
the activity of a variety of Ser/Thr kinases. The peptides are
arranged in sets of 6 replicates with a bottom row of 12 replicates
of a control peptide. These substrates correspond to peptides
listed in SEQ ID NOs 4 & 34-36. Also, optionally included in
this kit are additional kinase activity sensors for detection of
Ser/Thr kinases.
[0235] Another preferred kit includes includes 14 different kinase
activity sensors that can be used to measure the activity of a
variety of Tyr kinases. The peptides are arranged in sets of 6
replicates with a bottom row of 12 replicates of a control peptide.
These substrates correspond to peptides listed in SEQ ID NOs 42-49.
Also, optionally included in the kit are additional kinase activity
sensors for the detection of tyrosine kinases.
[0236] Various ancillary materials will frequently be employed in
an assay or kit in accordance with the present invention. In an
exemplary embodiment, buffers and/or stabilizers are present in the
kit components. Further components include gels, beads, supports,
reagents and the like. In another exemplary embodiment, the kits
comprise indicator solutions or indicator "dipsticks", blotters,
culture media, cuvettes, and the like. In yet another exemplary
embodiment, the kits comprise indicator cartridges (where a kit
component is bound to a solid support) for use in an automated
detector. In another exemplary embodiment, the kit further
comprises addititives, wherein said additives are selected from
phosphorylated and non-phosphorylated polypeptides, calcium-binding
and non-calcium binding polypeptides, sulfonated and non-sulfonated
polypeptides, and sialylated and non-sialylated polypeptides. In
another exemplary embodiment, the kit further comprises a member
selected from a fixing solution, a detection reagent, a standard, a
wash solution, and combinations thereof.
Illumination:
[0237] The sample or medium in which the complex of the
phosphorylated amino acid, metal ion and chelator of the kinase
activity sensor of the present invention is present is illuminated
with a wavelength of light selected to give a detectable optical
response, and observed with a means for detecting the optical
response. Equipment that is useful for illuminating the present
compounds and compositions of the invention includes, but is not
limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon
lamps, lasers and laser diodes. These illumination sources are
optically integrated into laser scanners, fluorescence microplate
readers or standard or microfluorometers.
[0238] The the kinase activity sensor of the invention may, at any
time after or during an assay, be illuminated with a wavelength of
light that results in a detectable optical response, and observed
with a means for detecting the optical response. Selected equipment
that is useful for illuminating the kinase activity sensor of the
invention includes, but is not limited to, hand-held ultraviolet
lamps, mercury arc lamps, xenon lamps, argon lasers, laser diodes,
and YAG lasers. These illumination sources are optionally
integrated into laser scanners, fluorescence microplate readers,
standard or mini fluorometers, or chromatographic detectors. This
fluorescence emission is optionally detected by visual inspection,
or by use of any of the following devices: CCD cameras, video
cameras, photographic film, laser scanning devices, fluorometers,
photodiodes, quantum counters, epifluorescence microscopes,
scanning microscopes, flow cytometers, fluorescence microplate
readers, or by means for amplifying the signal such as
photomultiplier tubes.
[0239] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
EXAMPLES
Example 1
Quantification of p38 Kinase Activity
[0240] A mouse monoclonal antibody specific for p38 kinase was
attached to the wells of a 96-well plate. The concentrations of p38
antibody in carbonate buffer were 3 .mu.g/ml, 6 .mu.g/ml and 12
.mu.g/ml and 100 .mu.l of each concentration was used per well.
[0241] Murine macrophage cells (RAW264.7) were exposed to
anisomycine and incubated for about 2 hours in culture. Control
cells were untreated. After incubation, cell culture was removed
from the cells and the cells were washed. Cells were then lysed
using commercially available cell lysis reagents, and the cell
lysate was added to the wells of the coated plate.
[0242] After the binding agent was allowed to capture the specific
kinase of interest (p38), the kinase activity sensor was added to
the wells (50 ng) along with 1 mM ATP. In this instance, the kinase
recognition motif of the activity sensor comprised the amino acid
sequence: AHLQRLSI(dP) (SEQ ID NO. 134), where the serine was the
phosphorylation target site. The kinase activity sensor further
comprised the metal binding amino acid that is described in U.S.
Pat. No. 6,906,194 and referred to as SOX. The SOX amino acid
fluoresces upon chelation of magnesium. FIG. 1 depicts levels
phosphorylation activity that can be solely attributed to p38.
Example 2
Quantification of Erk1/2 Kinase Activity
[0243] A rabbit monoclonal antibody specific for Erk1/2 kinase was
attached to the wells of a 96-well plate. The concentrations of
Erk1/2 antibody in carbonate buffer were 3 .mu.g/ml, 6 .mu.g/ml and
12 .mu.g/ml and 100 .mu.l of each concentration was used per
well.
[0244] Murine embryonic fibroblast cells (3T3) were exposed to
platelet derived growth factor (PDGF) and incubated for about 2
hours in culture. Control cells were untreated. After incubation,
cell culture was removed from the cells and the cells were washed.
Cells were then lysed using commercially available cell lysis
reagents, and the cell lysate was added to the wells of the coated
plate.
[0245] After the binding agent was allowed to capture the specific
kinase of interest (Erk1/2), the kinase activity sensor was added
to the wells (50 ng) along with 1 mM ATP. In this instance, the
kinase recognition motif of the activity sensor comprised the amino
acid sequence: AHLQRLSI(dP) (SEQ ID NO. 134), where the serine was
the phosphorylation target site. The kinase activity sensor further
comprised the metal binding amino acid that is described in U.S.
Pat. No. 6,906,194 and referred to as SOX. The SOX amino acid
fluoresces upon chelation of magnesium. FIG. 2 depicts levels
phosphorylation activity that can be solely attributed to Erk1/2
kinase.
Example 3
Measurement of Akt1 Activity in Crude Lysates from PDGF-Treated or
Control NIH3T3 Cells
[0246] NIH3T3 cells were seeded in 100 mm dishes and grown in DMEM
plus 10% fetal bovine serum until 90% confluent. The cells were
then incubated overnight in serum-free medium to induce quiescence,
followed by treatment with PDGF A/B (50 ng/mL, 10 min) or control
media. Cell lysates were prepared and Akt1 activity was assayed as
described below in the assay procedure.
[0247] The PDGF-treated sample showed a reaction rate of 1.13
RFU/sec, whereas the control treated sample had a markedly reduced
rate of 0.12 RFU/sec, resulting in a dramatic signal-to-noise ratio
of 9. Other assay control groups (bead only group or bead and
antibody only group) also had reaction rates less than 0.12
RFU/sec. Activity was assayed as described in the Assay Procedure
section below using SEQ ID NO. 25. Results are shown in FIG. 3.
Assay Procedure:
Reagents:
[0248] Wash Buffer (prepare 1.times. stock): Dilute an appropriate
amount of the 10.times. Wash Buffer Concentrate 10-fold with
ultrapure water (e.g., 5 mL of 10.times. Wash Buffer+45 mL of
ultrapure water).
[0249] Omnia.TM. Kinase Reaction Buffer (prepare 1.times. stock):
Dilute an appropriate amount of the 10.times. Omnia.TM. Kinase
Reaction Buffer 10-fold with ultrapure water and add DTT (provided)
to a final concentration of 0.2 mM (e.g., 500 .mu.L of 10.times.
Omnia.TM. Kinase Reaction Buffer+10 .mu.L of 100 mM DTT
solution+4490 .mu.L ultrapure water).
[0250] We have found that a useful reaction buffer for
phosphorylation Ser/Thr kinases comprises: 20 mM Tris, pH 7.5; 15
mM MgCl.sub.2; 1 mM EGTA; 5 mM beta-glycerophosphate; 5% glycerol;
1 mM ATP (added from separate stock); 0.2 mM DTT (added from
separate stock; 1.5 mM CaCl.sub.2 (PKC only; added from separate
stock); 2.5 ug/mL phosphatidylserine (PKC only; added from separate
stock); and 0.5 ug/mL diacylglycerol (PKC only; added from separate
stock).
[0251] Alterantively, we have found that a useful reaction buffer
for phosphorylation of Tyr kinases comprises: 20 mM Tris, pH 7.5; 5
mM MgCl.sub.2; 1 mM EGTA; 5 mM beta-glycerophosphate; 5% glycerol;
1 mM ATP (added from separate stock); and 0.2 mM DTT (added from
separate stock).
[0252] Kinase Activity Sensor (prepare 100 .mu.M stock): Dilute an
appropriate amount of the peptide substrate solution (1 mM) 10-fold
with 1.times. Omnia.TM. Kinase Reaction Buffer (e.g., 10 .mu.L of 1
mM peptide+90 .mu.L of 1.times. Omnia.TM. Kinase Reaction
Buffer).
[0253] ATP Solution (prepare 2 mM stock): Dilute an appropriate
amount of 100 mM ATP solution 50-fold with 1.times. Omnia.TM.
Kinase Reaction Buffer (e.g., 10 .mu.L of 100 mM ATP+490 .mu.L of
1.times. Omnia.TM. Kinase Reaction Buffer).
[0254] Cell lysates: Dilute the lysate to 0.5 to 1 mg/mL total
protein with Omnia.TM. Cell Lysis Buffer. The amount of cell lysate
protein used in the assay varies depending on the quantity and
activity of kinase enzyme in the individual cell line.
[0255] We have found that a useful buffer for preserving the kinase
activity of kinases from crude cell or tissue lysates, comprises:
50 mM Tris, pH 7.5; 150 mM NaCl; 2 mM EGTA; 30 mM NaF; 10 mM
Na.sub.4P.sub.2O.sub.7; 100 mM Na.sub.3VO.sub.4; 1% Triton X-100;
50 mM b-glycerophosphate; 1 mM DTT; Sigma Protease Inhibitor
cocktail (Cat. # P8340) and Sigma Phosphatase Inhibitor cocktail 1
(Cat. # P2850).
[0256] Kinase Mix (prepare 1.times. stock): Dilute an appropriate
amount of the Kinase solution with 1.times. Omnia.TM. Kinase
Reaction Buffer (e.g., 10 .mu.L of Kinase Mix+290 .mu.L of 1.times.
Kinase Reaction Buffer).
[0257] Kinase control: Recombinant kinase enzyme can be used as a
positive control to quantify the activity of kinase in the cell
lysates.
Procedure:
[0258] 100 .mu.L of cell lysate containing 25-100 .mu.g of total
protein prepared in Omnia.TM. Cell Lysis Buffer is added to each
sample well in the antibody coated plate strips. The plates are
sealed with Adhesive Plate Cover Strips and incubated at room
temperature for 2 hours. Cell lysate is removed and discarded and
wells and plates are washed multiple times with 200 .mu.L of Wash
Buffer. Wash buffer is aspirated or decanted and any residual
liquid is removed between washes by tapping the plate firmly on
absorbent paper.
[0259] 30 .mu.L of the kinase mix is added to 20 .mu.L of the
kinase activity sensor solution (100 .mu.M) and 50 .mu.L of ATP (2
mM). The final concentration of kinase activity sensor in the
reaction mixture is 20 .mu.M and the final concentration of ATP is
1 mM. The final volume of the reaction is 100 .mu.L.
[0260] The solution is incubated at 30.degree. C. for 1 hour. The
plate is transferred to a fluorescence plate reader (such as
SpectraMax M5.RTM. by Molecular Devices or comparable instrument).
The fluorescence values are read from each well every 30 seconds at
an excitation wavelength of 360 nm and an emission wavelength of
485 nm for up to 5 hours at 30.degree. C. in a kinetic mode.
[0261] The Assay Procedure is also described in the IP Kinase
Activity Assay Kit, Catalog # KNZ7011, Omnia.TM., Plate IP Kit for
B-Raf, which is hereby incorporated by reference.
Example 4
Measurement of Akt3 Activity in Crude Lysates from PDGF-Treated or
Control NIH3T3 Cells
[0262] NIH3T3 cells were seeded in 100 mm dishes and grown in DMEM
plus 10% fetal bovine serum until 90% confluent. The cells were
then incubated overnight in serum-free medium to induce quiescence,
followed by treatment with PDGF A/B (50 ng/mL, 10 min) or control
media. Cell lysates were prepared and Akt3 activity was assayed as
described in the Assay Procedure of Example 3. The PDGF-treated
sample showed a reaction rate of 1.24 RFU/sec, whereas the control
treated sample had a markedly reduced rate of 0.28 RFU/sec,
resulting in a dramatic signal-to-noise ratio of 4.42. Other assay
control groups (bead only group or bead and antibody only group)
also had reaction rates less than 0.28 RFU/sec. Activity was
assayed as described in the Assay Procedure section in Example 3
using SEQ ID NO. 25. Results are shown in FIG. 4.
Example 5
Measurement of ERK1/2 Activity in Crude Lysates from PDGF-Treated
or Control NIH3T3 Cells
[0263] NIH3T3 cells were seeded in 100 mm dishes and grown in DMEM
plus 10% fetal bovine serum until 90% confluent. The cells were
then incubated overnight in serum-free medium to induce quiescence,
followed by treatment with PDGF A/B (50 ng/mL, 10 min) or control
media. Cell lysates were prepared and ERK1/2 activity was assayed
as described in the Assay Procedure of Example 3. The PDGF-treated
sample showed a reaction rate of 1.55 RFU/sec, whereas the control
treated sample had a markedly reduced rate of 0.27 RFU/sec,
resulting in a dramatic signal-to-noise ratio of 5.7. Other assay
control groups (bead only group or bead and antibody only group)
also had reaction rates less than 0.27 RFU/sec. Activity was
assayed as described in the Assay Procedure section in Example 3
using SEQ ID NO. 41. Results are shown in FIG. 5.
Example 6
Measurement of p70-S6K Activity in Crude Lysates from PDGF-Treated
or Control MCF-7 Cells
[0264] MCF-7 cells were seeded in 100 mm dishes and grown in DMEM
plus 10% fetal bovine serum until 90% confluent. The cells were
then incubated overnight in serum-free medium to induce quiescence,
followed by treatment with insulin (100 nM, 10 min) or control
media. Cell lysates were prepared and p70-S6K activity was assayed
as described in the Assay Procedure of Example 3. The
insulin-treated sample showed a reaction rate of 1.13 RFU/sec,
whereas the control treated sample had a markedly reduced rate of
0.12 RFU/sec, resulting in a dramatic signal-to-noise ratio of 9.4.
Other assay control groups (bead only group or bead and antibody
only group) also had reaction rates less than 0.12 RFU/sec.
Activity was assayed as described in the Assay Procedure section
using SEQ ID NO. 42.
Example 7
Measurement of RSK Activity in Crude Lysates from PMA-Treated or
Control HeLa Cells
[0265] HeLa cells were seeded in 100 mm dishes and grown in DMEM
plus 10% fetal bovine serum until 90% confluent. The cells were
then incubated overnight in serum-free medium to induce quiescence,
followed by treatment with PMA(200 nM, 30 min) or control media.
Cell lysates were prepared and RSK activity was assayed as
described in the Assay Procedure of Example 3. The PMA-treated
sample showed a reaction rate of 3.17 RFU/sec, whereas the control
treated sample had a markedly reduced rate of 0.64 RFU/sec,
resulting in a dramatic signal-to-noise ratio of 4.95. Other assay
control groups (bead only group or bead and antibody only group)
also had reaction rates less than 0.64 RFU/sec. Activity was
assayed as described in the Assay Procedure section using SEQ ID
NO. 30. Results are shown in FIG. 7.
Example 8
Measurement of c-Src Activity in Crude Lysates from
c-Src-Transfected CEF Cells
[0266] C-Src transfected and mock transfected chicken embryo
fibroblast (CEF) cell lysates were prepared as described (Thomas,
et al., (1999) J. Biol. Chem., 74:36684-36692). The c-Src
transfected CEF sample showed a reaction rate of 50 RFU/sec,
whereas the control treated sample had a markedly reduced rate of
0.048 RFU/sec, resulting in a dramatic signal-to-noise ratio of
10.5. Other assay control groups (bead only group or bead and
antibody only group) also had reaction rates less than 0.05
RFU/sec. Activity was assayed as described in the Assay Procedure
of Example 3 using SEQ ID NO. 48. Results are shown in FIG. 8.
Example 9
Measurement of B-Raf Activity in Crude Lysates from NGF Treated or
Control PC12 Cells
[0267] PC12 cells were seeded in 100 mm Petri dishes and grown in
DMEM plus 2.5% fetal bovine serum and 15% horse serum. Cells were
then treated with NGF (50 ng/mL, 15 minutes) or with control media.
Cell lysates were prepared and B-Raf activity was assayed as
described in Example 3. The NGF-treated sample produced
fluorescence signals that reached a plateau at 150 minutes of
incubation (3200 RFU), while the control group reached a plateau at
240 minutes after the reaction started. Activity was assayed as
described in the Assay Procedure section using a cascade reaction
with SEQ ID NO. 27, used to measure the level of activated MK2
enzyme. Results are shown in FIG. 9.
Example 10
Measurement of ERK1/2 Activity in Crude Lysates from PDGF-Treated
or Control NIH3T3 Cells
[0268] NIH3T3 cells were seeded in 100 mm Petri dishes and grown in
DMEM plus 10% fetal bovine serum. Cells then were treated with PDGF
(50 ng/mL, 15 minutes) or with control media. Cell lysates then
were prepared and ERK1/2 activity was assayed as described in
Example 1. The PDGF-treated sample produced a fluorescence signal
of 3000 RFU that reached a plateau at 335 minutes of incubation,
while the control group produced fluorescence signal of 845 RFU.
Activity was assayed as described in the Example 3 using a cascade
reaction with SEQ ID NO. 27 used to measure the level of activated
MK2 enzyme. Results are shown in FIG. 10.
Example 11
Measurement of MEK1 Activity in Crude Lysates from PDGF-Treated or
Control NIH3T3 Cells
[0269] NIH3T3 cells were seeded in 100 mm Petri dishes and grown in
DMEM plus 10% fetal bovine serum. Cells then were treated with PDGF
(50 ng/mL, 15 minutes) or with control media. Cell lysates then
were prepared and MEK1 activity was assayed as described in the
Assay Procedure of Example 3. The PDGF-treated sample produced
fluorescence signal of 3650 RFU that reached a plateau at 250
minutes of incubation, while the control group produced
fluorescence signal of 1050 RFU. Activity was assayed as described
in the Assay Procedure section using a cascade reaction with SEQ ID
NO. 27 used to measure the level of activated MK2 enzyme. Results
are shown in FIG. 11.
Example 12
Measurement of p38 MAPK Activity in Crude Lysates from Anisomycin
Treated or Control RAW Cells
[0270] RAW cells were seeded in 100 mm Petri dishes and grown in
DMEM plus 10% fetal bovine serum. Cells then were treated with
anisomycin (10 .mu.g/mL, 15 minutes) or with control media. Cell
lysates then were prepared and p38 MAPK activity was assayed as
described in the Assay Procedures of Example 3. The anisomycin
treated sample produced fluorescence signal of 3350 RFU that
reached a plateau at 60 minutes of incubation, while the control
group produced fluorescence signal of 1250 RFU. Activity was
assayed as described in the Assay Procedure section using a cascade
reaction with SEQ ID NO. 27 used to measure the level of activated
MK2 enzyme. Results are shown in FIG. 12.
[0271] Each of the aforementioned references are hereby
incorporated by reference as if set forth fully herein.
* * * * *