U.S. patent application number 11/690002 was filed with the patent office on 2007-08-09 for methods for transferases.
Invention is credited to Robert F. Bulleit, SaidA Goueli.
Application Number | 20070184509 11/690002 |
Document ID | / |
Family ID | 30443456 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070184509 |
Kind Code |
A1 |
Goueli; SaidA ; et
al. |
August 9, 2007 |
METHODS FOR TRANSFERASES
Abstract
Methods for detecting transferase activity of a sample are
provided. Transferase activity detected includes kinase and
phosphatase activity. The method includes providing a substrate
having a reporter compound conjugated thereto. A quantity of the
substrate is added to a solution containing the sample. The sample
is incubated with the substrate under conditions where the sample
is active for a time sufficient for kinase or phosphatase activity
to take place. An exopeptidase is added to the solution containing
the sample. Output of the reporter compound is detected.
Inventors: |
Goueli; SaidA; (Fitchburg,
WI) ; Bulleit; Robert F.; (Verona, WI) |
Correspondence
Address: |
WHYTE HIRSCHBOECK DUDEK S.C.
ONE EAST MAIN STREET
SUITE 300
MADISON
WI
53703-3300
US
|
Family ID: |
30443456 |
Appl. No.: |
11/690002 |
Filed: |
March 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10199970 |
Jul 19, 2002 |
7195884 |
|
|
11690002 |
Mar 22, 2007 |
|
|
|
Current U.S.
Class: |
435/15 ;
435/21 |
Current CPC
Class: |
G01N 33/542 20130101;
C12Q 1/48 20130101; C07K 2319/60 20130101; C07K 7/06 20130101; C12Q
1/485 20130101; C12Q 1/37 20130101; C12Q 1/42 20130101; C07K 7/08
20130101 |
Class at
Publication: |
435/015 ;
435/021 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48; C12Q 1/42 20060101 C12Q001/42 |
Claims
1. A method for detecting kinase or phosphatase activity of a
sample, the method comprising: (A) contacting the sample with a
peptide substrate and at least one of a phosphate group donor and a
phosphate group acceptor, wherein the substrate includes a reporter
compound and amino acids; (B) adding an exopeptidase, wherein
phosphorylation of the substrate affects cleavage by the
exopeptidase, wherein adding the exopeptidase comprises adding an
exopeptidase that hydrolyzes the peptide substrate from a
carboxy-terminal end of the substrate; and (C) detecting output of
the reporter compound, wherein the reporter compound exhibits a
different output property when bound to at least one amino acid of
the substrate when compared to when it is not bound to amino
acids.
2. A method of detecting kinase or phosphatase activity of a sample
comprising: (A) providing a substrate having a reporter compound
conjugated thereto; (B) adding a quantity of the substrate to a
solution containing the sample; (C) incubating the sample with the
substrate under conditions where the sample is active for a time
sufficient for kinase or phosphatase activity to take place; (D)
adding an exopeptidase to the solution containing the sample; (E)
detecting output of the reporter compound.
3. A method of claim 2, wherein in step (A) a substrate having a
fluorescent molecule conjugated thereto is provided.
4. A method of claim 2, wherein in step (A) a substrate having a
luminogenic molecule conjugated thereto is provided.
5. A method of claim 2, further comprising terminating the kinase
or phosphatase reaction.
6. A method of claim 2, further comprising terminating the
exopeptidase reaction.
7. A method of claim 2, further comprising linking the substrate to
a solid support.
8. A method of claim 7, wherein the substrate is linked to the
solid support via a first functional group on the substrate and a
second functional group on the solid support.
9. A method for detecting peptide transferase activity of a sample,
the method comprising: (A) contacting the sample with a peptide
substrate and at least one of a group donor and a group acceptor,
wherein the substrate includes a reporter compound and amino acids;
(B) adding an exopeptidase, wherein transfer of a group from one
substrate to another affects cleavage of the substrate by the
exopeptidase; and (C) detecting output of the reporter
compound.
10. The method of claim 9, wherein the transferase activity
detected is kinase activity.
11. The method of claim 9, wherein the transferase activity
detected is phosphatase activity.
12. A method for detecting peptide transferase activity of a
sample, the method comprising: (A) contacting the sample with a
peptide substrate and at least one of a group donor and a group
acceptor, wherein the substrate includes a reporter compound and
two peptides, each peptide being separately linked to the reporter
compound; and (B) adding an exopeptidase, wherein transfer of a
group from one substrate to another affects cleavage of the
substrate by the exopeptidase; and (C) detecting output of the
reporter compound.
13. The method of claim 12, wherein the transferase activity
detected is kinase activity.
14. The method of claim 12, wherein the transferase activity
detected is phosphatase activity.
15. A method for detecting peptide transferase activity of a
sample, the method comprising: (A) contacting the sample with a
peptide substrate and at least one of a group donor and a group
acceptor, wherein the substrate includes a reporter compound and
amino acids; and (B) adding an exopeptidase, wherein transfer of a
group from one substrate to another affects cleavage of the
substrate by the exopeptidase; and (C) liberating the reporter
compound from the substrate.
16. The method of claim 15, wherein the liberated reporter compound
has increased output when compared to when the reporter compound is
not liberated from the substrate.
17. The method of claim 15, wherein the transferase activity
detected is kinase activity.
18. The method of claim 15, wherein the transferase activity
detected is phosphatase activity.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/199,970, filed Jul. 19, 2002 and issuing
Mar. 27, 2007 as U.S. Pat. No. 7,195,884, which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to enzyme assays. More specifically,
the invention relates to the detection of transferase activity,
such as kinase activity and phosphatase activity.
DESCRIPTION OF THE RELATED ART
[0003] Enzymes are classified into groups according to the general
kind of reaction they catalyze. Transferases catalyze the transfer
of a group from one substrate to another and include kinases and
phosphatases. Protein kinases transfer a phosphomoiety from a donor
such as adenosine triphosphate (ATP) or guanosine triphosphate
(GTP) to an acceptor such as a peptide or protein to yield a
phosphorylated peptide or protein and adenosine diphosphate (ADP)
or guanosine diphosphate (GDP), respectively. Protein phosphatases
are enzymes that transfer a phosphate group from a phosphopeptide
or a phosphoprotein donor to an acceptor such as water.
[0004] About two to five percent of the eukaryotic genome encodes
for protein kinases and protein phosphatases. Although
approximately 870 different protein kinases have been identified in
the human genome, there may be many thousands of distinct and
separate enzymes. In addition, protein substrates for these enzymes
may amount to one-third of all cellular proteins. An understanding
of these enzymes and their targets is crucial to understanding
cellular regulation and cellular pathology.
[0005] Protein kinases are often divided into two major groups
based on the amino acid residue that is phosphorylated. The first
group is serine/threonine kinases, which includes cyclic
AMP-dependent protein kinases (PKA), cyclic GMP-dependent protein
kinases (PKG), calcium and phospholipid dependent protein kinases
(PKC), calcium and calmodulin-dependent protein kinases (CaMK),
casein kinases, cell cycle protein kinases (cdc or cdk), protein
kinase B (Akt), and others. These kinases are usually cytoplasmic
or associated with the particulate fractions of cells, possibly by
anchoring proteins. Protein serine/threonine kinases are the most
common type of cytosolic kinases, and are thought to be responsible
for the majority of phosphorylation events in the cell. In
addition, there are some receptor kinases of the serine/threonine
type, such as transforming growth factor beta (TGF-.beta.).
Overall, serine/threonine kinases represent over 70% of cellular
protein kinases.
[0006] The second group of kinases, called tyrosine kinases,
phosphorylate tyrosine residues. Overall, over 10% of kinases are
tyrosine kinases. There are fewer tyrosine kinases, but they play
an equally important role in cell regulation. Studies have
indicated that many tyrosine kinases are transmembrane proteins
with their receptor domains located on the outside of the cell and
their kinase domains on the inside of the cell. More than 50
receptor tyrosine kinases are known. These kinases include several
receptors for molecules such as growth factors and hormones,
cytokines, and neurotransmitters. Examples of these include
epidermal growth factor receptor (EGFR), insulin receptor (IR) and
platelet derived growth factor receptor (PDGFR). There are also
cytosolic tyrosine kinases, such as src, src-N1, fyn, lyk, lynA,
lck. In addition, other kinases phosphorylate proteins or peptides
containing histidine or aspartic acid residues.
[0007] Protein phosphatases are enzymes that catalyze the removal
of phosphate moieties from proteins or peptides that contain such
modifications. As with kinases, classes of phosphatases are
distinguished by their substrate specificity and dependence on
other molecules for activation. Three major classes of phosphatases
have been identified. The first class includes type 1 protein
phosphatase (protein phosphatase-1 or PP1) and type 2 protein
phosphatases (PP2A, PP2B, and PP2C). The second class includes
tyrosine phosphatases such as PTP-1B, and YOP-51. Some phosphatases
in this class are soluble but others comprise parts of a larger
molecule, such as the receptor CD45. The third major class of
phosphatases includes dual-specificity protein phosphatases that
remove phosphate groups from both phosphoserine/phosphothreonine
and phosphotyrosine.
[0008] Protein kinases and protein phosphatases play very important
roles in many cell functions, including, but not limited to,
cellular metabolism, signal transduction, transcriptional
regulation, cell motility, cell division, cellular signaling
processes, cellular proliferation, cellular differentiation,
apoptosis, and secretion. These processes are mediated by
phosphorylation or dephosphorylation of enzymes, protein
substrates, transcription factors, hormone or growth factor
receptors, and other cellular proteins.
[0009] In addition, protein kinases and protein phosphatases are
involved in mediating the response to naturally occurring toxins
and pathogens, which alter the phosphorylation states of proteins.
Additionally, protein kinases are related to many epidemiologically
relevant oncogenes and tumor suppressor genes.
[0010] Notably, there are over 400 human diseases in which kinases
are implicated. Examples include neurodegenerative diseases such as
amyotrophic lateral sclerosis and Alzheimer's disease. In myotonic
dystrophy, a genetic defect in one form of the disorder is
characterized by an amplified trinucleotide repeat in the 3'
untranslated region of a protein kinase gene on chromosome 19.
These modifications may someday elucidate many of the unusual
features of the disorder.
[0011] Because of this role of kinases and phosphatases in human
pathology, modulators of kinases and phosphatases are potential
drug targets. Currently, many inhibitors of kinases and
phosphatases are available for treating a variety of diseases,
while others are being tested for such use. One such inhibitor is
Gleevec.TM. (Imatinib mesylate) (Novartis, Basel, Switzerland),
which is a protein tyrosine kinase inhibitor of the Bcr-Abl
tyrosine kinase. The abnormal constitutive expression of this
tyrosine kinase is created by the "Philadelphia chromosome"
abnormality in chronic myelogenous leukemia (CML). Gleevec.TM.
inhibits proliferation and induces apoptosis in Bcr-Abl positive
cell lines as well as fresh leukemic cells from Philadelphia
chromosome positive CML patients.
[0012] Fasudil (Eril.RTM. Injection S, Asahi Kasei Corp.) is potent
inhibitor of Rho-kinase. Eril.RTM. has been approved in Japan for
the treatment of cerebral vasospasm and an oral formulation is now
is in clinical trials for the treatment of angina.
[0013] An exemplary inhibitor of a clinically relevant phosphatase
is cyclosporine A (CSA), which is used to prevent and treat ongoing
acute rejection of transplanted organs. CSA inhibits the production
of interleukin IL-2 by helper T-cells, thereby blocking T cell
activation and proliferation (and inhibiting amplification of the
immune response). The current model for the mechanism of action of
CSA suggests that it blocks a phosphatase called calcineurin
(PP2B).
[0014] Further, phosphotyrosine phosphatase (PTP-1B) is currently
under investigation as a target for the treatment of type II
diabetes.
[0015] These examples illustrate the importance of modulating
kinases and phosphatases for clinically relevant circumstances.
[0016] Current types of assays used to measure kinase and
phosphatase activity and to detect potential kinase and phosphatase
inhibitors and activators include Fluorescence Resonance Energy
Transfer (FRET) assays, Fluorescent Polarization (FP) assays, and
assays based on radioactivity such as Scintillation Proximity Assay
(SPA). FRET assays used to detect kinase activity utilize a protein
substrate that has two linked fluorescent molecules. The two
molecules are in close proximity, separated by a fixed distance.
The energy of an excited electron in one molecule (the donor) is
passed to an adjacent molecule (the acceptor) through resonance.
The ability of a higher energy donor fluorophore to transfer energy
directly to a lower energy acceptor molecule causes sensitized
fluorescence of the acceptor molecule and simultaneously quenches
the donor fluorescence. In this case, the fluorescence of the donor
is "quenched" by the proximity to the acceptor and the energy of
the donor is transferred to the acceptor in a non-radiative manner.
The efficiency of energy transfer is dependent on the distance
between the donor and acceptor chromophores according to the
Forster equation. In most cases, no FRET is observed at distances
greater than 100 angstroms and thus the presence of FRET is a good
indicator of close proximity.
[0017] In order for FRET to be useful, the fluorescence of the
acceptor molecule must be significantly different from the
fluorescence of the donor. A useful FRET based protein substrate
may include a separation of the two fluorescent molecules via a
peptide linker that maintains specificity for an endopeptidase that
is capable of cleaving the peptide linker between the two
fluorophores. If the peptide is phosphorylated, then the enzyme may
not cleave the protein or may cleave it at a reduced rate, keeping
the fluorescent molecules in close proximity such that quenching
occurs. On the other hand, if the protein is not phosphorylated,
then the endopeptidase cleaves the protein substrate, releasing the
two fluorescent molecules such that the quenching is alleviated,
and the two fluorescent molecules fluoresce independently. The FRET
assay requires peptide substrates that must be carefully engineered
to meet these requirements. That is, the peptide substrates must
contain the enzyme recognition site required for the endopeptidase,
the distance between the two fluorophores must be within the range
to allow FRET to occur and the fluorescent molecules must be paired
in such a way that donor fluorescence is significantly quenched,
minimizing background fluorescence from the donor. Furthermore, the
fluorescence of the starting material (the "quenched" substrate)
must be significantly different from the product (the "released"
non-quenched product). These requirements make a FRET based assay
cumbersome and costly.
[0018] FP assays are based on binding of a high affinity binding
reagent, such as an antibody, a chelating agent, or the like, to a
fluorescently labeled molecule. For example, an antibody that binds
to a phosphorylated fluorescently labeled peptide but not to a
non-phosphorylated fluorescently labeled peptide can be used for a
kinase assay. When the fluorescent label is excited with plane
polarized light, it emits light in the same polarized plane as long
as the fluorescent label remains stationary throughout the excited
state (duration of the excited state varies with fluorophore, and
is 4 nanoseconds for fluoroscein). However, if the excited
fluorescent label rotates or tumbles out of the plane of
polarization during the excited state, then light is emitted in a
different plane from that of the initial excitation state. If
polarized light is used to excite the fluorophore, the emission
light intensity can be monitored in both the plane parallel to the
plane of polarization (the excitation plane) and in the plane
perpendicular to the plane of polarization. The degree to which the
emission intensity moves from the parallel to the perpendicular
plane is related to the mobility of the fluorescently labeled
molecule. If the fluorescently labeled molecules are large, such as
when they are bound to the binding reagent, the fluorescently
labeled molecules move little during the excited state interval,
and emitted light remains highly polarized with respect to the
excitation plane. If the fluorescently labeled molecules are small,
such as when no binding reagent is bound to the fluorescently
labeled molecules, the fluorescently labeled molecules rotate or
tumble faster, and the resulting emitted light is depolarized
relative to the excitation plane. Thus, an FP assay requires a high
affinity binding reagent, e.g., an antibody, capable of binding
with high specificity to the fluorescently labeled molecule. The
time consuming and costly optimization of antibody binding with the
specific fluorescently labeled molecules such as peptides is
required where antibodies are used. Additionally, with FP assay
there is the potential for a phosphorylated protein and other
reaction components, e.g., lipids and detergents, to interfere with
the polarization.
[0019] Kinase assays that use radioactive labels include SPA. In
SPA, modified ligand-specific or ligand-capturing molecules are
coupled to fluoromicrospheres, which are solid-phase support
particles or beads impregnated with substances that emit energy
when excited by radioactively labeled molecules. When added to a
modified ligand such as radiolabeled phosphopeptide in a mixture
with the nonphosphorylated peptide, only the phosphopeptide is
captured on a fluoromicrosphere, bringing any bound radiolabeled
peptide close enough to allow the radiation energy emitted to
activate the fluoromicrosphere and emit light energy. If the
concentration of fluoromicrospheres is optimized, only the signal
from the radiolabeled ligand bound to the target is detected,
eliminating the need for any separation of bound and free ligand.
The level of the light energy emitted may be measured in a liquid
scintillation counter and is indicative of the extent to which the
ligand is bound to the target. However, a SPA requires radiolabeled
ligands, which have high disposal costs and possible health risks.
In addition, a SPA requires the fluoromicrospheres to settle by
gravity or be centrifuged, adding an additional step and time to
the assay.
[0020] With phosphorylation and dephosphorylation events involved
in so many cell functions and diseases, identifying kinase and
phosphatase activity is tremendously important. Thus, there is a
need for alternative enzyme assays for detecting transferase
activity, such as protein kinase and protein phosphatase activity,
that do not require large amounts of costly or highly specialized
starting materials and that do not require a large amount of time
to complete. Additionally, there is a need for alternative assays
to identify activators and inhibitors of kinases and phosphatases.
In addition, it would also be desirable to provide kits for
carrying out such assays.
SUMMARY OF THE INVENTION
[0021] The invention, which is defined by the claims set out at the
end of this disclosure, is intended to solve at least some of the
problems noted above. For example, in one aspect of the invention,
a method for detecting transferase activity of a sample is
provided. In a preferred embodiment of the method, the sample is
contacted with a substrate and at least one of a phosphate group
donor and a phosphate group acceptor. The substrate includes a
reporter compound and amino acids. A peptidase is added that
cleaves a non-phosphorylated peptide substrate at a first rate and
a phosphorylated peptide substrate at a second rate. The difference
in the two rates is a measure of transferase activity. The output
of the reporter compound is then detected.
[0022] In a preferred embodiment, the method of detecting
transferase activity is used to detect kinase activity. In another
preferred embodiment, the method is used to detect phosphatase
activity.
[0023] Also provided is a method for detecting alteration in a
transferase reaction. In a preferred embodiment of the method, a
test substance is contacted to a substrate including a reporter
compound and amino acids under conditions in which the transferase
is active. The substrate is cleaved with a peptidase that cleaves a
non-phosphorylated peptide substrate at a first rate and a
phosphorylated peptide substrate at a second rate. The output of
the reporter compound is then detected.
[0024] In a preferred embodiment, the method of detecting
alterations in a transferase activity is used to detect alterations
in kinase activity. In another preferred embodiment, the method is
used to detect alterations in phosphatase activity.
[0025] Also provided is a method of detecting transferase activity
of a sample. In a preferred embodiment of the method, a substrate
having a reporter compound conjugated thereto is provided. A
quantity of the substrate is added to a solution containing the
sample. The sample is incubated with the substrate under conditions
where the sample is active for a time sufficient for transferase
activity to take place. A peptidase is added to the solution
containing the sample. Output of the reporter compound is then
detected.
[0026] Peptide substrates for transferases are also provided. In a
preferred embodiment, the peptide substrate includes a reporter
compound and a first transferase substrate linked to the reporter
compound on a first side of the reporter compound.
[0027] Kits that can be used in carrying out the above methods are
also provided. In a preferred embodiment, the kit includes a
substrate that includes a reporter compound, at least one of a
phosphate group donor and a phosphate group acceptor, and a buffer
that supports enzymatic activity of the transferase. Additionally
included is a peptidase that cleaves a non-phosphorylated peptide
substrate at first rate and a phosphorylated peptide substrate at a
second rate.
[0028] The methods described herein are homogeneous, fast,
sensitive, simple, and non-radioactive. The methods are convenient
and can be used with any instrumentation platform. Reagents
required can be designed with relative ease and may be synthesized
readily. The methods provide assays with fast development time and
low cost.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Preferred exemplary embodiments of the invention are
illustrated in the accompanying drawings, in which like reference
numerals represent like parts throughout and in which:
[0030] FIG. 1 is a graph showing detected output from a
serine/threonine protein kinase assay where the kinase added to the
reaction was titrated. Detected output is shown in relative
fluorescence units (RFU).
[0031] FIG. 2 is a graph showing detected output in RFU from a
serine/threonine protein kinase assay in the presence of certain
inhibitors.
[0032] FIG. 3 is a graph showing detected output in RFU from a
serine/threonine protein kinase assay using an aminomethyl coumarin
labeled peptide substrate.
[0033] FIG. 4 is a graph showing detected output in RFU from a
protein tyrosine kinase assay.
[0034] FIG. 5 is a graph showing detected output in RFU from a
protein tyrosine protein kinase assay in the presence of certain
inhibitors.
[0035] FIG. 6 is a graph showing detected output in RFU from a
serine/threonine protein phosphatase assay where the phosphatase
added to the reaction was titrated.
[0036] FIG. 7 a graph showing detected output in RFU from a
serine/threonine protein phosphatase assay in the presence of
certain inhibitors.
[0037] FIG. 8 is a graph showing detected output in RFU from a
serine/threonine protein phosphatase assay using a different
aminopeptidase (Aminopeptidase II) than the aminopeptidase used in
FIGS. 1-7.
[0038] FIG. 9 is a graph showing detected output in RFU from a
protein tyrosine phosphatase assay where the phosphatases added to
the reaction were titrated.
[0039] FIG. 10 is a graph showing detected output in RFU from a
protein tyrosine phosphatase assay in the presence of certain
inhibitors.
[0040] Before explaining embodiments of the invention in detail, it
is to be understood that the invention is not limited in its
application to the details of construction and the arrangement of
the components set forth in the following description or
illustrated in the drawings. The invention is capable of other
embodiments or being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
DETAILED DESCRIPTION
Definitions:
[0041] For purposes of the present invention, the following
definitions apply:
[0042] Amino Acid: In keeping with standard polypeptide
nomenclature, J. Biol. Chem., 243:3557-59, (1969), abbreviations
for amino acid residues are as shown in the following Table of
Correspondence: TABLE-US-00001 TABLE OF CORRESPONDENCE SYMBOL FOR
AMINO ACIDS 1-Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly
glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser
serine I Ile isoleucine L Leu leucine T Thr threonine V Val valine
P Pro proline K Lys lysine H His histidine Q Gln glutamine E Glu
glutamic acid W Try tryptophan R Arg arginine D Asp aspartic acid N
Asn asparagine C Cys cysteine
[0043] As used herein, the term "aminoluciferin" refers to
luciferin that has been modified to include an NH.sub.2 group.
[0044] As used herein, the term "background fluorescence" refers to
the fluorescence outputted by the reporter compound when it is
linked to amino acids of the peptide substrate.
[0045] As used herein, the term "bioluminescence" refers to the
light produced in certain organisms as a result of
luciferase-mediated oxidation reactions. The luciferase genes,
e.g., the genes from luminous beetle and, in particular, the
luciferase from Photinus pyralis (the common firefly of North
America), are currently the most commonly used luminescent reporter
genes.
[0046] As used herein, the term "dephosphorylation" refers to the
removal of a phosphate group.
[0047] As used herein, the term "exopeptidase" refers to a
hydrolase enzyme that removes terminal amino acids of a peptide or
protein by cleaving peptide bonds.
[0048] As used herein, the term "luciferase," unless specified
otherwise, refers to a naturally occurring or engineered
Coleopteran luciferase. The luciferase, if naturally occurring, may
be obtained easily by the skilled from the beetle itself, and
particularly the light organ thereof. If the luciferase is one that
occurs naturally or is engineered, which retains activity in the
luciferase-luciferin reaction, of a naturally occurring luciferase,
it can be obtained readily from a culture of bacteria, yeast,
mammalian cells, insect cells, plant cells, or the like,
transformed to express a CDNA encoding the luciferase, or from an
in vitro cell-free system for making the luciferase from a nucleic
acid encoding same.
[0049] As used herein, the term "luciferin" refers to a substrate
of a Coleopteran luciferase enzyme. For example, firefly luciferin
is a polyheterocyclic organic acid,
D-(-)-2-(6'-hydroxy-2'-benzothiazolyl)-.DELTA..sup.2-thiazoline-4-carboxy-
lic acid.
[0050] As used herein, the term "modulator" refers to an agent
identified using assays for transferase activity. Samples are
treated with a candidate agent. If there is a change in transferase
activity between a sample treated with a candidate agent and one
not treated with the candidate agent, this change indicates the
identification of a modulator. A change in activity can be an
increase or decrease.
[0051] As used herein, the term "peptide substrate" refers to a
peptide that is linked to a reporter compound. Preferably, the
peptide substrate includes at least one amino acid linked to at
least one side of the reporter compound.
[0052] As used herein, the term "peptide" refers to a linear series
of at least two amino acid residues connected one to the other by
peptide bonds between the alpha-amino and carboxy groups of
adjacent residues.
[0053] As used herein, the term "phosphorylation" refers to the
addition of a phosphate. An amino acid within a peptide that has
been phosphorylated is indicated herein with either a "p" that
precedes the amino acid or a (PO.sub.3) that follows or are
otherwise attached to the amino acid.
[0054] As used herein, the term "reporter compound" refers to a
compound, the output of which can be detected either directly or
indirectly. Output can be detected directly where the reporter
compound itself has a property that can be detected. Output can be
indirectly detected where, e.g., the reporter compound when acted
on by another substance produces a property that can be
detected.
I. Methods for Assaying Samples for Transferase Activity and for
Alterations in Transferase Activity
[0055] In a preferred embodiment, a method for detecting
transferase activity of a sample involves contacting the sample
with a substrate and at least one of a phosphate group donor and a
phosphate group acceptor. The substrate includes a reporter
compound and amino acids, as are explained in detail below. A
peptidase is added that cleaves a non-phosphorylated peptide
substrate at a first rate and a phosphorylated peptide substrate at
a second rate. For example, the peptidase cleaves a
non-phosphorylated peptide substrate at a faster rate than a
phosphorylated peptide substrate. The output of the reporter
compound is then detected. This general assay can be tailored to
screen for various transferases, including, but not limited to,
kinases and phosphatases. In addition, the general assay can be
used to screen for alterations in transferase activity, such as
kinases and phosphatases. For instance, the assay can be used to
screen for enhancers and inhibitors of transferases (kinases and
phosphatases, etc).
[0056] In a preferred embodiment, a test substance is contacted to
a transferase in the presence of a substrate that includes a
reporter compound and amino acids under conditions in which the
transferase is active. The substrate is cleaved with an
aminopeptidase, which cleaves a non-phosphorylated peptide
substrate at a first rate and a phosphorylated peptide substrate at
a second rate. The output of the reporter compound is then
detected.
[0057] In a preferred embodiment, the reporter compound is not
linked to a solid support such that a kinase (and phosphatase)
reaction and a peptidase reaction can be performed in a
solution-phase reaction.
[0058] In another preferred embodiment, the peptide substrate is
linked to a solid support and the kinase (or phosphatase) reaction
and the peptidase reaction are performed in solid phase.
Additionally, output is detected in solid phase. The peptide
substrate is linked to a solid support via functional groups. A
functional group on a peptide substrate should have the ability to
bind to another functional group attached to or otherwise part of a
solid support. For this, a peptide substrate can be linked to the
solid support by incorporating a functional group on the peptide
substrate and by having a corresponding functional group on the
solid support such that the peptide substrate and the solid support
can be linked together.
[0059] Examples of useful functional groups include those that
contain a carboxy group. Biotin is an example of such a functional
group. The carboxy group of the functional group is linked to an
amino group on a reporter compound or on a peptide. Streptavidin
and avidin are examples of functional groups having amino groups.
The amino group of the functional group is linked to a carboxy
group on a solid support. Biotin has an affinity for both
streptavidin and avidin. Through functional groups, such as biotin
and streptavidin, the peptide substrate can be immobilized on a
solid support.
[0060] The functional group can also be attached to the peptide
substrate through other linkages, such as by a thioether (or
sulfide) linkage. For example, the peptide substrate includes a
free sulfhydryl group and the solid support can be derivatized to
contain a maleimide end group (Pierce Biotechnology, Inc.,
Rockford, Ill.). Other linkages can be used, such as a disulfide
linkage. For example, the peptide substrate includes a free
sulfbydryl group and the substrate includes a free sulfhydroxy
group that oxidizes the free sulfhydryl group of the peptide
substrate to form the disulfide linkage. In addition, an amide
linkage in which the peptide substrate includes a free carboxy
group and a solid support contains an amino group. The free carboxy
group can oxidize the free amino group to form an amide linkage
between the peptide substrate and the solid support. It should be
noted that other types of linkages can also be used and that the
location of the functional groups listed above can be reversed. For
example, a biotin group can be located on a solid support and a
streptavidin or avidin group can be located on the peptide
substrate.
[0061] In a preferred embodiment, a bis-reporter compound, i.e., a
reporter compound having two amino groups, includes a functional
group on a first amino group and a peptide on a second amino group.
In another preferred embodiment, a bis-reporter compound includes a
functional group at a free end of a peptide linked to one of the
free amino groups. In yet another preferred embodiment, the peptide
substrate includes a functional group on the reporter compound
itself. Each of these will now be described in more detail.
[0062] In the case of a bis-substituted reporter compound having
two free amino groups, the reporter compound is linked via a first
amino group to a peptide substrate and a second amino group of the
reporter compound is linked to a functional group. For example, a
biotin group, which includes a carboxy group, can be linked via an
amide bond to the other amino group of the reporter compound. A
solid support can then be derivatized to contain streptavidin or
avidin derivatives, both of which have affinity (or avidity) for
biotin. Additionally, a matrix consisting of avidin or streptavidin
or any of their derivatives can be used with a biotinylated
reporter compound. Examples of solid supports containing
streptavidin include streptavidin linked membranes (SAM.RTM.),
polystyrene linked avidin, streptavidin plates, streptavidin or
avidin coated microtiter plates. For the solid-phase reactions, the
kinase (or phosphatase) and peptidase protocols described herein
for solution-phase reactions can be followed, and the same
detection can be conducted as described earlier for solution-phase
assays.
[0063] Where two amino groups are present on the reporter compound,
a first peptide can be attached to a first amino group and a second
peptide can be attached to a second amino group. A functional group
can be attached at the free end of the second peptide in any of the
manners described above. In this configuration, the peptide
attached to the second group and to the functional group acts as a
linker and does not serve as a substrate for the peptidase. In
addition, the functional group can be linked to the reporter
compound through any other suitable linker, e.g., a series of
carbons, extending from the free amino group and terminating in,
e.g., an amino group. This configuration permits the use of the
peptide on the first amino group to act as a substrate in both the
kinase (or phosphatase) reaction and in the peptidase reaction.
[0064] The reporter can also be linked to the two peptides (or
phosphopeptides) of interest on both of its amino groups and also
be derivatized on a position of choice on the reporter compound,
such as the benzyl group of Rhodamine 110. In a preferred
embodiment, the functional group is attached directly to the benzyl
group. In another preferred embodiment, the functional group is
attached via a linker, such as C6 or C12, that contains, e.g., an
amino group. This permits the linkage of the same peptide to the
amino groups or different peptides to the amino groups of the
reporter compound. Another advantage of having the functional group
on a location other than an amino group is that a larger increase
in fluorescence is obtained when both amino groups are free, such
as by cleavage of peptide attached to the reporter compound. For
Rhodamine 110, where one amino group is free, there is a 10-fold
increase in fluorescence over where no amino groups are free. For
Rhodamine 110, where to amino groups are free, there is a 100-fold
increase in fluorescence over where no amino groups are free.
Therefore, where the Rhodamine 110 has a function group on a
location other than an amino group, this permits both amino groups
to be freed, which would result in a 100-fold increase in
fluorescence as discussed above. A benefit of having the functional
group on a location other than the amino group(s) is that two
peptides can be attached to the reporter compound.
II. Methods for Assaying Samples for Protein Kinase Activity
[0065] a. In General
[0066] A preferred embodiment of the invention is an assay to
screen for protein kinase activity. Protein kinase activity in a
sample can be determined by contacting a sample with a phosphate
donor and a peptide substrate for a protein kinase. The peptide
substrate includes a reporter compound, amino acids, and a
phosphorylation site for a protein kinase.
[0067] The peptide substrate is incubated with a peptidase that
cleaves a non-phosphorylated peptide substrate at a different rate
than it cleaves a phosphorylated peptide substrate. Preferably, the
peptidase cleaves a non-phosphorylated peptide substrate at a
faster rate than it cleaves a phosphorylated peptide substrate. The
output of the reporter compound is then detected. The reporter
compound exhibits a different output property when bound to at
least one amino acid of the peptide substrate than when it is not
bound to amino acids of the peptide substrate. Where no
phosphorylated amino acids are present, the peptidase can cleave
the amino acids from the substrate to liberate the reporter
compound. When liberated from the peptide substrate, such as
through hydrolysis of the surrounding amino acids, the reporter
compound has increased output when compared to when it is bound to
the peptide substrate. Notably, the presence of a phosphorylated
amino acid blocks or slows removal of amino acids by the peptidase.
When the reporter compound is linked to amino acids of a peptide
substrate, it has no or diminished output. Therefore, the output of
the reporter compound can be used to determine whether a peptide
substrate is phosphorylated.
[0068] The assays of the invention can be performed in a single
tube or well. In addition, the assays of the invention are amenable
to high throughput screening. For example, the assays can be run in
96 well, 384 well, and plates with even more wells.
[0069] A preferred embodiment of the assay to screen for protein
kinase activity can be represented schematically with the following
equations ##STR1##
[0070] In the equations above, RC is the reporter compound,
PO.sub.3 is a phosphate group, M is a metal or a divalent cation,
and NTP is a nucleotide triphosphate.
[0071] b. Kinase Reaction:
[0072] In a preferred embodiment, a kinase reaction includes a
buffer, a source of metal or divalent cation, a nucleotide
triphosphate (NTP), which can act as a phosphate donor, a peptide
substrate, and, optionally, an activator of the kinase. The buffer,
cation, NTP, and peptide substrate are selected based on the
protein kinase under investigation, as is explained below. If
desired, an activator of the kinase, can also be added. The sample
is added to the reaction.
[0073] If the sample contains a protein kinase, the protein kinase
can catalyze the transfer of the phosphate group from the NTP to
phosphorylate the peptide substrate Kinase reactions can be
incubated at a temperature at which the enzyme is active.
Preferably, the temperature is about 21.degree. C. or higher. Also
preferred is a temperature of 37.degree. C. or lower. Incubation
time preferably is 5 seconds or more. Also preferred is an
incubation time of one hour or less. However, the incubation time
may be longer than one hour, as long as the reaction time is not
longer than the transferase remains active under assay conditions.
Incubation time may be optimized depending on, e.g., the incubation
temperature, the stability and amount of kinase under
investigation, and the amount of peptide substrate. The reaction is
instantaneous, so measurement can be taken as soon as is
practicable.
[0074] Buffers useful in a kinase reaction include, but are not
limited to, Tris(hydroxymethyl)aminomethane hydrochloride
(Tris-HCl), N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
(HEPES), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid)
(HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), at
concentrations and pH levels that are optimal for the particular
enzyme under investigation. Preferably, the buffer concentration is
10 mM or higher. Also preferred is a buffer concentration of 100 mM
or lower. The pH of the kinase reaction preferably is 7.0 or
higher. Also preferred is a pH of 9.0 or lower.
[0075] A preferred divalent cation for the kinase reaction is
magnesium. Other divalent cations, such as manganese, calcium,
nickel, and the like, can substitute for magnesium. In addition,
these other divalent cations can be combined with magnesium.
Notably, some of the other divalent cations can be added for
optimal activity of the kinase. Preferably, the divalent cation is
added at a 1 mM or higher concentration. Also preferred is adding
magnesium at a concentration 50 mM or lower concentration. Other
divalent cations can be added in the micromolar to millimolar
ranges.
[0076] The NTP added to the kinase reaction typically is ATP or
GTP. As is known in the art, the choice of which NTP is added to
the kinase reaction depends on the kinase used in the assay. A
preferred concentration of NTP in a kinase reaction is about 1 uM
or higher, and is also preferred at 1 mM or lower, and more
preferably is 100 uM.
[0077] The peptide substrate for the kinase is one that can be
phosphorylated by the kinase. That is, a potential peptide
substrate for protein kinase must have an amino acid that can act
as a phosphate group acceptor. For example, a peptide substrate for
a serine/threonine kinase has a serine or threonine. Consensus
sequences for various protein kinases are known. (Methods in
Enzymology 200: 62-81 (1991)). Table 1 shows consensus
phosphorylation site motifs for various protein kinases. An
asterisk indicates the phosphorylable residue. An "X" indicates any
amino acid. TABLE-US-00002 TABLE 1 Protein Kinase Consensus Motifs
Calmodulin- XRXXS*/T* (SEQ. ID. NO:7); dependent protein XRXXS*/T*V
(SEQ. ID. NO:8) kinase II Casein kinase I S(PO.sub.3)XXS*/T* (SEQ.
ID. NO:9) Casein kinase II S*/T*XXEX (SEQ. ID. NO:10); S*/T*XXDX
(SEQ. ID. NO:11) c-AMP-dependent RXS* (SEQ. ID. NO:12); protein
kinase RRXS* (SEQ. ID. NO:13); RXXS* (SEQ. ID. NO:14); KRXXS* (SEQ.
ID. NO:15) c-GMP-dependent R/KXS*/T* (SEQ. ID. NO:16); protein
kinase R/KXXS*/T* (SEQ. ID. NO:17); R/KR/KXS*/T* (SEQ. ID. NO:18);
R/KXXXS*/T* (SEQ. ID. NO:19); S*/T*XR/K (SEQ. ID. NO:20) Glycogen
synthase S*XXXS(PO.sub.3) (SEQ. ID. NO:21) kinase-3
Growth-associated S*/T*PXK/R (SEQ. ID. NO:22); histone H1 kinase
K/RS*/T*P (SEQ. ID. NO:23); (MPF, cdc2.sup.+/CDC28 S*/T*PK/R (SEQ.
ID. NO:24) protein kinases) Phosphorylase K/RXXS*V/I (SEQ. ID.
NO:25) kinase Protein kinase C S*/T*XKR (SEQ. ID. NO:26); K/RXX
S*/T* (SEQ. ID. NO:27); K/RXXS*/T*XK/R (SEQ. ID. NO:28); K/RXS*/T*
(SEQ. ID. NO:29); K/RXS*/T*XK/R (SEQ. ID. NO:30) Tyrosine kinase/
XE/DY*X (SEQ. ID. NO:31); EGF-receptor XE/DY*I/L/V (SEQ. ID. NO:32)
kinase
[0078] The utility of a potential peptide substrate for the assay
can be determined by incubating the potential peptide substrate
with the kinase under conditions where the kinase is known to be
active. Preferred peptide substrates for the kinase assays include
a peptide substrate that includes a reporter compound and at least
one amino acid and that is useful in a kinase reaction, a reporter
compound and at least two amino acids and that is useful in a
kinase reaction, and a reporter compound and at least four amino
acids and that is useful in a kinase reaction. Those peptide
substrates that are useful in a kinase reaction are those that can
be phosphorylated by a kinase of interest. Other preferred peptide
substrates are listed in the Examples.
[0079] The reporter compound in the peptide substrate is any
compound that, when released, has a property that is detectably
outputted or that is a substrate in a reaction that produces a
property that is detectably outputted. For example, when a
fluorogenic reporter compound is used, and output is a detectable
fluorescence. The fluorogenic reporter compound preferably has no
or diminished fluorescence when linked to the amino acids of the
peptide substrate. However, when it is liberated from the peptide
substrate, the fluorogenic reporter compound has increased
fluorescence.
[0080] The reporter compound may be a fluorgenic compound, such as
aminomethylcoumarin (AMC) or Rhodamine 110 (R-110) or any other
fluorogenic compound that can be linked to a peptide without
interfering with the recognition site for the kinase or the
phosphatase under investigation. Rhodamine 110 is a preferred
fluorogenic substrate with a proven utility in high throughput
screening applications.
[0081] In a preferred embodiment, the reporter compound is
covalently linked to the peptide substrate through an amide bond.
AMC has a single site at which amino acid chain can be linked,
whereas Rhodamine 110 has two. Where Rhodamine 110 is used as a
reporter compound, one of the two linkage sites can be blocked with
a suitable blocking compound such that only a single site is
available for linkage to a peptide. Additionally, where both sites
are available on Rhodamine 110, the same peptide can be linked
thereto or different peptides can be linked thereto. Where
different peptides are used, two different kinases can be assayed
using the same modified peptide substrate.
[0082] In another preferred embodiment, the reporter compound is a
luminogenic compound that when bound to a peptide substrate is not
a substrate for a bioluminescent enzyme. Examples include
aminoluciferin or any other derivatives of luciferin. For example,
when aminoluciferin is enzymatically released from a peptide
substrate, it is available as a substrate for luciferase.
Luciferase is a biolumineseent enzyme that catalyzes the production
of light in the reaction between aminoluciferin and ATP. This
resulting light or luminescence produced is the detectable output
when such a luminogenic compound is used.
[0083] Preferably, the peptide substrate is added at micromolar
concentrations, such as at a concentration of at least 1 uM. Also
preferred is adding the peptide substrate at a concentration of 25
uM or less.
[0084] Activators can be added to the kinase reaction where
desired, e.g., where the kinase under investigation requires an
activator. It also may be desirable to add an activator to achieve
optimal kinase activity. Activators useful in the kinase reaction
include, but are not limited to, calcium, phospholipids and other
lipids, and phorbol 12-myristate 13-acetate (PMA) or similar
activators for Calcium-phospholipid-dependent protein kinase (PKC),
calcium and calmodulin for calmodulin-dependent protein kinase (CaM
K), cAMP for cAMP-dependent protein kinase (PKA) holoenzyme, cGMP
for cGMP-dependent protein kinase (PKG), DNA for DNA-PK. Activators
can be added at nanomolar or higher concentrations and at
micromolar or lower concentrations depending on the kinase under
investigation. A termination reagent can optionally be added to the
system in which the kinase reaction is occurring where an end point
is desired, e.g., for measuring and quantitating the activity of
protein kinase. The termination reagent usually is a metal
chelating reagent added at a concentration that is sufficient to
sequester the metal away from the kinase. In addition, any other
reagent that terminates the phosphorylation catalyzed by the kinase
can be used to terminate the phosphorylation reaction. For example,
EDTA, EGTA, and 1,10-phenanthroline are good chelators for
magnesium, calcium, and zinc, respectively. Other ion chelating
agents may be used. Additionally, kinases can be heat
inactivated.
[0085] The kinase reaction can also be performed using a
phosphopeptide as the phosphate donor and a nucleoside diphosphate
(NDP) as the phosphate acceptor, i.e., the reverse of the
previously described reaction. In this configuration, the kinase
reaction is performed in the same manner as is described above.
However, the output that is detected generally will be the inverse
of the output for kinase reactions where a phosphopeptide is the
phosphate donor. That is, where there is kinase activity in this
assay configuration, output will increase when dephosphorylation of
the phosphopeptide substrate and phosphorylation of the NDP
occur.
[0086] c. Peptidase Reaction:
[0087] A peptidase that is a hydrolase that acts on amide bonds of
the peptide substrate is added to the peptide substrate. Peptidases
that are particularly useful in the invention include those that
are free or substantially free of endopeptidase activity. In
addition, it is preferred to have a peptidase that cleaves a
non-phosphorylated peptide substrate at a first rate and cleaves a
phosphorylated peptide substrate at a second rate. For example, a
preferred peptidase shows relatively higher activity when cleaving
an amide bond that links an amino acid that has not been modified
by phosphorylation than it does cleaving an amide bond that links
an amino acid that has been modified by phosphorylation. This
difference in the ratio of fluorescence generated from the
non-phosphorylated peptides treated with protease compared to that
for the phosphorylated peptide treated with the same concentration
of protease can be used as in an indicator for the kinase, and
permits determination of whether a peptide substrate is
phosphorylated. A preferred peptidase is one that hydrolyzes
nonphosphorylated amino acids of a peptide substrate sequentially
and then dramatically slows hydrolysis when a phosphorylated amino
acid is reached. This slowing of hydrolysis results in the failure
of the reporter compound to be released in the majority of the
molecules of the phosphorylated amino acid. This results in
background fluorescence, or significantly lower fluorescence than
when a nonphosphorylated amino acid is present. The partial
hydrolysis of a phosphorylated amino acid is illustrated
schematically below. ##STR2##
[0088] In a preferred embodiment, the increase in activity of the
kinase enzyme is proportional to the decrease in detectable output
with increasing concentration of enzyme. Conversely, the activity
of the phosphatase enzyme is proportional to the increase in
output, e.g., fluorescence reading, when compared with the output,
e.g., fluorescence, recorded with increasing concentration of
phosphatase.
[0089] For a nonphosphorlyated peptide substrate, peptidase
activity has one rate. For a phosphorylated peptide substrate,
peptidase activity has a second rate. For example, for a given
enzyme/substrate pair and treatment with aminopeptidase M, the
output, e.g., fluorescent units, is higher for a nonphosphorylated
peptide substrate than it is for the phosphorylated peptide
substrate.
[0090] Preferably, the peptidase is an exopeptidase, which
hydrolyzes amino acids starting at a terminus of the peptide
substrate. In one preferred embodiment, the peptidase is an
aminopeptidase that cleaves a peptide from an amino terminus of a
peptide. Where an aminopeptidase is used, the peptide substrate has
its carboxy terminus linked to the reporter compound such that the
amino terminus of the peptide is free. The peptide substrate used
with an aminopeptidase can be represented as
NH.sub.2-peptide-CO-[reporter compound]-CO-peptide-NH.sub.2. Unless
otherwise indicated, when peptide substrates that are used with an
aminopeptidase are listed herein, it should be understood that the
peptide substrate has this configuration.
[0091] Aminopeptidases catalyze the release of an N-terminal amino
acid, X--|--Y from a peptide, amide, or arylamide, where X may be
most amino acids including Pro, although rates of hydrolysis vary.
When a terminal hydrophobic residue is followed by a prolyl
residue, the two may be released as an intact X-Pro dipeptide. For
a nonphosphorylated peptide substrate, the aminopeptidase
sequentially cleaves amino acids off the amino terminus of the
peptide substrate to free the reporter compound. In addition, a
dipeptidyl peptidase, which cleaves peptides from an amino terminus
of a peptide, can be used.
[0092] Preferred aminopeptidases include Aminopeptidase M (E.C.
3.4.11.2), and aminopeptidase II. Aminopeptidase M is a membrane
aminopeptidase. Other names for aminopeptidase M, include, but are
not limited to, membrane alanyl aminopeptidase, microsomal
aminopeptidase, aminopeptidase N, particle-bound aminopeptidase,
amino-oligopeptidase, alanine aminopeptidase, particle-bound
aminopeptidase, membrane aminopeptidase I, pseudo leucine
aminopeptidase, CD13, Cys-Gly dipeptidase, and peptidase E.
[0093] Aminopeptidase II is a peptidase isolated from Aspergillus
oryzae strain (ATCC20386), is a metalloenzyme, and is a
non-specific aminopeptidase (EC 3.4.11). Although the enzyme is
capable of cleaving almost any peptide bond, cleaving acidic, basic
neutral, hydrophobic or hydrophilic residues, the enzyme is less
active with proline when it is present as the penultimate
N-terminal amino acid. Any other peptidase that cleaves a
nonphosphorylated peptide substrate at a relatively different rate
than a phosphorylated peptide substrate and that is free or
substantially free of endopeptidase activity can be used for the
invention.
[0094] Notably, the peptidase can be added to the completed kinase
reaction without changing buffers, removing any component of the
kinase reaction, or any other step. Therefore, the screening assay
can be accomplished in a single tube or well. Preferably, at least
6.5 milliunits of the aminopeptidase is added. Also preferred is
adding 100 milliunits or less of the aminopeptidase. A unit of
aminopeptidase is defined as the amount of enzyme that will
hydrolyze 1 micromole of Leu-pNA per minute at 37.degree. C. and pH
7.0-7.5. Lower and higher amounts of peptidase can be used
depending on, e.g., peptide substrate concentration and reaction
time for peptide cleavage. The peptidase reaction can be performed
at any temperature at which the enzyme is active. Preferably, the
peptidase reaction is incubated at a temperature of at least
10.degree. C. Also preferred is a temperature of less than
40.degree. C. Preferably, the pcptidase reaction is carricd out for
5 seconds or more. Also preferred is carrying out the reaction for
180 minutes or less. Longer reaction times can be used depending,
e.g., on the enzyme and peptide substrate concentrations. Shorter
reaction times can be used, e.g., with lower peptide substrate
concentrations and higher units of aminopeptidase.
[0095] The peptidase can also be a carboxypeptidase, which cleaves
the carboxy-terminal amino acid from a peptide. The
carboxypeptidases that can be utilized include, but are not limited
to, carboxypeptidase A, which will remove any amino acid, and
carboxypeptidase B, which is specific for a terminal lysine or
arginine. Where a carboxypeptidase is used, the peptide substrate
has the amino-terminal of a peptide linked to the reporter compound
such that the carboxy terminus of the peptide is free. The peptide
substrate used with a carboxypeptidase can be represented as
COOH-peptide-NH-[reporter compound]-NH -peptide-COOH. Unless
otherwise indicated, when peptide substrates that are used with a
carboxypeptidase are listed herein, it should be understood that
the peptide substrate has this configuration.
[0096] If desired, a terminator of the peptidase is included.
Exemplary activators include, but are not limited to, actinonin,
bestatin, and amastatin. Inclusion of a peptidase terminator is
particularly useful where the detection or reading of output is
performed at a time later than the ending of the peptidase
reaction. Other reagents such as zinc chelators, e.g.,
1,10-phenanthroline, can also be used. Peptidase terminators can be
added in the micromolar or greater concentrations. Peptidase
terminators can also be added in the millimolar or lesser
concentrations.
[0097] d. Detecting Output
[0098] The output of the reporter compound used is detected after
the peptidase treatment of the peptide substrate. Where a
fluorogenic reporter compound is used, fluorescence can be used as
the output. Fluorometery can be used to detect fluorescence.
Fluorometers that are single-tube instruments or those that are
multi-well plate fluorescence readers can be used to detect
fluorescence. For example, the Fluorolog-2 spectroflurometer (SPEX
Industries, Inc., Edison, N.J.) equipped with quartz cuvettes can
be used for single tube assays. The Cytofluor.RTM. II multiwell
Fluorescence Plate Reader (PerSeptive Biosystems, Inc., Framingham,
Mass.) and the Fluoroscan Ascent CF (LabSystems OY, Helsinki,
Finland), both equipped with the appropriate filters, can be used
to detect fluorescence. The fluorescence units or readings can be
recorded. Where Rhodamine 110 is used as the reporter compound,
after peptidase treatment, the kinase reactions preferably are read
by exciting at 485 nm and reading emissions at 520 to 530 nm. Where
AMC is used, reactions preferably are read by exciting at 360 nm
and reading emissions at 420 nm.
[0099] Where a luminogenic reporter compound is used, luminescence
can be used as output. A luminometer apparatus or other suitable
apparatus (such as the Vector 1420 multiwell counter, Wallac Oy,
Perkin Elmer, Turku, Finland) can be used to detect the resulting
luminescence from the peptidase treatment.
[0100] Typical output from a kinase assay is illustrated in FIG. 1,
which shows higher fluorescence where less kinase is added and
lower fluorescence units where more kinase is added. The shape of
the titration curve can be explained by the kinase phosphorylating
the peptide substrate such that the ability of the aminopeptidase
to cleave the peptide substrate and release the reporter compound
is reduced as the concentration of the kinase present
increases.
[0101] In a preferred embodiment, relative output is determined by
comparing the output of a non-phosphorylated peptide substrate to
that of a phosphorylated peptide substrate where both peptides have
been treated with the same concentration of peptidase. For example,
the assay can use the change in relative fluorescence where a
fluorogenic compound is used or the change in relative luminescence
where a lumogenic compound is used. Relative change in the
detectable output of the reporter compound preferably is a ratio of
a test sample output to a control sample output. This ratio can be
expressed as the relative fluorescence units (RFU). For example,
output ratio can be calculated for a sample treated with a kinase
and a sample not treated with a kinase.
III. Methods for Assaying for Phosphatase Activity
[0102] Another preferred embodiment of the invention is an assay to
screen for phosphatase activity. In general, screening for
phosphatase activity is achieved similar to the screening for
kinase activity, with the major exception of using a substrate for
a phosphatase, typically a phosphopeptide substrate, instead of a
peptide substrate for a kinase. Other differences between the
kinase activity assay and the phosphatase activity assay are
described below and in the examples that follow.
[0103] In a preferred embodiment for detecting phosphatase activity
of a sample, the sample is contacted with a phosphopeptide
substrate and a phosphate acceptor. The peptide substrate includes
a reporter compound, a dephosphorylation site for a phosphatase,
and amino acids. A potential peptide substrate for protein
phosphatase must have a phosphoamino acid that can act as a
phosphate group donor. For example, a peptide substrate for a
serine/threonine phosphatase has a phosphorylated serine/threonine
and a peptide substrate for a tyrosine phosphatase has a
phosphorylated tyrosine. The phosphopeptide substrate is linked to
the reporter compound, as defined above.
[0104] In a preferred embodiment for detecting phosphatase activity
of a sample, the sample is contacted with a phosphopeptide
substrate and a phosphate acceptor. The peptide substrate includes
a reporter compound, a dephosphorylation site for a phosphatase,
and amino acids. A potential peptide substrate for protein
phosphatase must have a phosphoamino acid that can act as a
phosphate group donor. For example, a peptide substrate for a
serine/threonine phosphatase has a phosphorylated serine/threonine
and a peptide substrate for a tyrosine phosphatase has a
phosphorylated tyrosine. The utility of a potential peptide
substrate for the assay can be determined by incubating the
potential phosphopeptide substrate with the enzyme under conditions
where the enzyme is known to be active. The phosphopeptide
substrate is linked to the reporter compound, as defined above.
[0105] Although phosphatase substrate preferences are less
stringent than kinase substrate preferences, various protein
phosphatases indeed have known substrate preferences. (see, e.g.,
Eur. J. Biochem 219: 109-117 (1994)). For example, for
phosphatase-2B (PP-2B), which belongs to the family of
Ser/Thr-specific enzymes but also is active on phosphotyrosine
residues, is believed that higher-order structure is an important
determinant for its substrate specificity. However, a number of
shorter peptides are also appreciably dephosphorylated by PP-2B,
their efficiency as substrates depending on local structural
features. For instance, all the peptides that are appreciably
dephosphorylated by PP-2B contain basic residue(s) on the
amino-terminal side. A basic residue located at position -3
relative to the phosphorylated residue plays a particularly
relevant positive role in determining the dephosphorylation of
short phosphopeptides. Acidic residue(s) adjacent to the
carboxy-terminal side of the phosphoamino acid are conversely
powerful negative determinants, preventing the dephosphorylatjon of
otherwise suitable peptide substrates. However, PP-2B displays an
only moderate preference for phosphothreonyl peptides, which are
conversely strikingly preferred over their phosphoseryl
counterparts by the other classes of Ser/Thr-specific protein
phosphatases. Moreover PP-2B does not perceive as a strong negative
determinant the motif Ser/Thr-Pro in peptides where this motif
prevents dephosphorylation by the other classes of Ser/Thr protein
phosphatases. Whenever tested on phosphotyrosyl peptides, PP-2B
exhibits a specificity that is strikingly different from that of
T-cell protein tyrosine phosphatase, a bonafide protein tyrosine
phosphatase. In particular, while the latter enzyme is especially
active toward a number of phosphopeptides reproducing the
phosphoacceptor sites of src products and of PP-2B whose
amino-terminal moieties are predominantly acidic, the artificial
substrate phospho-angiotensin II, bearing an arginine residue at
position -2, is far preferred by PP-2B over all phosphotyrosyl
peptides of similar size. Collectively taken, these results show
that the specificity of PP-2B, rather than resting on a given
consensus sequence, is determined by a variety of primary and
higher-order structural features conferring to it an overall
selectivity that is different from those of any other known protein
phosphatase.
[0106] Preferred peptide substrates for the phosphatase assays
include a peptide substrate that includes a reporter compound and
at least one amino acid and that is useful in a phosphatase
reaction, a reporter compound and at least two amino acids and that
is useful in a phosphatase reaction, and a reporter compound and at
least four amino acids and that is useful in a phosphatase
reaction. Those peptide substrates that are useful in a phosphatase
reaction are those that can be dephosphorylated by a phosphatase of
interest. Preferred peptide substrates include Y(PO.sub.3) (SEQ.
ID. NO:33)-Reporter Compound-Y(PO.sub.3), where reporter compound
is any reporter compound; Y(PO.sub.3)--R-110-Y(PO.sub.3); and
AAY(PO.sub.3)AXAA (SEQ. ID. NO:34)-R-110-AAXAY(PO.sub.3)AA, where X
is any amino acid. Other preferred peptide substrates are listed in
the Examples.
[0107] Added to the phosphatase reaction is a peptidase that
cleaves a non-phosphorylated peptide substrate at a faster rate
than it cleaves a phosphorylated peptide substrate, as is described
in detail above. This permits assessment of dephosphorylation of
the phosphopeptide substrate. The detectable output of the reporter
compound can be detected, as is described in detail above.
Representative output results of a phosphatase activity assay are
shown, for example, in FIG. 6.
[0108] The protein phosphatase activity assay can be represented
schematically with the following equations. ##STR3##
[0109] In the above equations, RC is the reporter compound,
PO.sub.3 is a phosphate group, and M is a metal or a divalent
cation.
[0110] The de-phosphorylation of a phosphopeptide substrate and
hydrolysis thereof is illustrated schematically below. ##STR4##
[0111] In a preferred embodiment for a serine/threonine
phosphatase, the phosphatase reaction includes a phosphopeptide
substrate, buffer, such as Tris-HCl, pH 7.5, bovine serum albumin
(BSA), a metal or divalent cation, such as MgCl.sub.2 or
MnCl.sub.2. In addition, as is known in the art, other activators,
such as calmodulin, may be added to achieve optimal enzymatic
activity. Reactions can be incubated at any temperature at which
the phosphatase is active. Preferably, the reactions are incubated
at room temperature for 30 minutes.
[0112] Where desired, phosphatase reactions can be terminated, such
as by adding okadaic acid, EDTA and/or EGTA. After termination
(where used), peptidase is added, and the reaction is incubated at
25.degree. C., preferably for at least 60 minutes. Where the
reporter compound is a fluorogenic compound, such as Rhodamine 110,
fluorescence can be read at an excitation of 480 nm and an emission
at 520 nm.
IV. Methods for Screening for Alterations in or to Kinase
Activity
[0113] A further embodiment of the invention is an assay to screen
for alterations in or to a kinase reaction. Alterations include,
but are not limited to, activations or inhibitions of a kinase
reaction. For this, a test substance that is a potential activator
or inhibitor of a kinase is added to the assay along with the
kinase. An assay typically includes a buffer, a cation, NTP,
peptide substrate, and 0.05 units or greater of the kinase of
interest.
[0114] The potential inhibitor or activator is added to the
reaction to determine whether a compound inhibits or stimulates the
phosphorylation reaction. In addition, a peptidase is added to the
reaction as detailed above. The potential inhibitor or activator
can produce a change in the detectable output from the reporter
compound. For example, where a potential inhibitor is included in
the assay, typically an increase in the detectable output from the
reporter compound indicates inhibition of the kinase. This increase
would be due to inhibition of the kinase, leading to reduced
phosphorylation of the peptide substrate. With fewer amino acids of
the peptide substrate phosphorylated, the peptidase can cleave more
molecules of the peptide substrates to liberate more reporter
compound than a non-inhibited kinase reaction. Conversely, where a
potential enhancer is included in the assay, a decrease in output
from the reporter compound when compared to a control reaction
without the potential enhancer indicates the enhancement of the
kinase.
[0115] In a preferred embodiment, output from a test sample
contacted with a test substance is compared to output of a control
sample that has not been contacted with the test substance.
Preferably, a ratio is calculated from these detected outputs. The
ratio is a measure of the phosphorylation (or lack thereof) of the
reporter compound by the kinase.
V. Methods for Screening for Alterations in or to Phosphatase
Activity
[0116] An additional embodiment of the invention is an assay to
screen for alterations in or to a phosphatase reaction. Alterations
include, but are not limited to, activations or inhibitions of a
phosphatase reaction. For this, a test substance that is a
potential inhibitor of a phosphatase is added to the assay along
with the phosphatase. An assay typically includes a buffer, a
cation, a phosphopeptide substrate, and 0.1 units or greater of the
phosphatase of interest.
[0117] The potential inhibitor or activator is added to the
reaction to determine whether a compound inhibits or stimulates the
dephosphorylation reaction. In addition, a peptidase is added to
the reaction as detailed above. The potential inhibitor or
activator can produce a change in the detectable output from the
reporter compound. For example, where a potential inhibitor is
included in the assay, typically a decrease in the detectable
output from the reporter compound indicates inhibition of the
phosphatase. This decrease would be due to inhibition of the
phosphatase, leading to decreased dephosphorylation of the peptide
substrate. With more amino acids of the peptide substrate remaining
phosphorylated, the peptidase can cleave fewer molecules of the
peptide substrate to liberate less reporter compound to a
non-inhibited phosphatase reaction. Conversely, where a potential
enhancer is included in the assay, an increase in output from the
reporter compound when compared to a control reaction without the
potential enhancer indicates the enhancement of the
phosphatase.
[0118] In a preferred embodiment, output from a test sample
contacted with a test substance is compared to output of a control
sample that has not been contacted with the test substance as is
described above.
VI. Kits
[0119] The invention also relates to kits for carrying out the
methods described above. In a preferred embodiment, the kit
includes a substrate that includes a reporter compound, a buffer
that supports enzymatic activity of the transferase, at least one
of a phosphate donor and a phosphate acceptor, and a peptidase
compatible with the substrate. The transferase under investigation
may be included in the kit, or may be provided by the user. The
transferase can be a kinase, a phosphatase, or another transferase
under investigation. Where the transferase is a kinase, the
substrate preferably is a peptide substrate that acts as a
phosphate group acceptor, and the phosphate donor preferably is an
NTP that the kinase is capable of using. Where the transferase is a
phosphatase, the substrate preferably is a phosphopeptide substrate
that acts as a phosphate group donor. Other components, such as
activators of the transferase under investigation, a terminator for
the transferase, a terminator for the peptidase, and the like, all
of which has been described previously, can also be included. In a
preferred embodiment, the kit for screening for transferase
activity also optionally includes a transferase that can be used
for a control reaction. A kit including a transferase can also be
used to determine whether a test substance alters the activity of
the transferase. For example, the kit can be used to determine
whether a test substance enhances or inhibits the transferase under
study.
[0120] In one preferred embodiment, the substrate is a kinase
substrate. In another preferred embodiment, the substrate is a
phosphatase substrate. Preferably, the peptidase of the kit is an
aminopeptidase, although it can be another peptidase, such as a
carboxypeptidase. Preferred aminopeptidases include, but are not
limited to Aminopeptidase M and Aminopeptidase II. A preferred
reporter compound for the substrate is a fluorogenic or luminogenic
compound, as described above.
EXAMPLES
[0121] The following Examples are provided for illustrative
purposes only. The Examples are included herein solely to aid in a
more complete understanding of the presently described invention.
The Examples do not limit the scope of the invention described or
claimed herein in any fashion.
Example 1
Detection of Ser/Thr Kinase using R110-Modified Peptide Substrate
and Aminopeptidase M
[0122] PKA Assay With LRRASLG-(R110)-GLSARRL. The kinase activity
of the catalytic subunit of cAMP-dependent protein kinase (PKA)
from Promega Corp., Madison, Wis. was tested in triplicate in a
96-well plate using the following reaction components: 40 mM
Tris-HCl, pH 7.5, 20 mM MgCl.sub.2, 0.1 mg/ml bovine serum albumin
(BSA), 50 .mu.M ATP, and 5 .mu.M of LRRASLG (SEQ. ID.
NO:1)-R110-GLSARRL, a bis-rhodamine peptide kinase substrate also
known as "bis-kemptide." The final reaction volume was 50 uL. The
amount of PKA added to each reaction was titrated in 2-fold unit
increments, in a range from 0.001 unit to 1 unit. Control reactions
with 0 units were also run. All kinase reactions were incubated at
room temperature for 20 minutes.
[0123] The kinase reactions were terminated by adding a
termination/detection reagent (25 ul) containing 100 mM EDTA and 25
mU aminopeptidase M (Calbiochem, San Diego, Calif.).
[0124] Terminated reactions were incubated at room temperature for
30 minutes, and aminopeptidase activity was then terminated by the
addition of a final concentration of 2.5 uM actinonin/well.
Enzymatic activity of the kinase was measured by taking a reading
of the fluorescence, at the time of addition of actinonin and 3 hrs
later to test the stability of the signal, with an excitation at
480 nm and fluorescence emission at 520-530 nm.
[0125] As shown in FIG. 1, there is a corresponding decrease in
fluorescence output with increasing concentration or units of
enzyme in the reaction. In addition, it was also determined that
greater units of the catalytic subunit of PKA yielded lower
fluorcscent output. This can be explained by phosphorylation of the
peptide substrate, which decreases the rate of cleavage by
Aminopeptidase M to result in decreased release of Rhodamine 110.
Moreover, the signal was very stable over time as shown by almost
identical profile obtained 3 hrs after termination of
aminopeptidase activity.
[0126] Other protein kinase sources (e.g., Calzyme Laboratories
(San Luis Obispo, Calif.)) were also tested and gave essentially
similar results.
Example 2
Inhibition of Ser/Thr Protein Kinases
[0127] PKA Assay With Inhibitors. The effects of various inhibitors
of PKA kinase were tested. A known and specific inhibitor of PKA
(PKI-"protein kinase inhibitor"), a general and nonspecific
inhibitor of PKA (staurosporin
((9S-(9.alpha.,10.beta.,11.beta.,13.alpha.)-2,3,10,11,12,13-Hexahydro-10--
methoxy-9-methyl-11-(methylamino)-epoxy-1H,9H-diindolo[1,2,3-gh:3',2',1'-1-
m]pyrrolo{3,4-j]][1,7]benzodiazonin-1-one))), a poor inhibitor of
PKA (H7) (1-(5-isoquinolinesulfonyl)-2-methylpiperzine), and a
compound that does not inhibit PKA (U0126)
(1,4-diamino-2,3-dicyano-1,4-bis-(2-aminophenylthio)butadiene) were
tested for their effect on the kinase activity of PKA. Kinase
reactions and aminopeptidase reactions were run under conditions
similar to those described in Example 1 except that inhibitors were
included at increasing concentrations and 0.5 units of PKA was
used. A control was included which did not contain any
inhibitor.
[0128] Fluorescence was detected with an excitation of 480 nm and
fluorescence emission at 520-530 nm. As shown in FIG. 2, increasing
the concentration of PKI and staurosporin resulted in an increase
in fluorescence output indicating inhibition of PKA enzyme
activity. The compounds H7 and U0126 were without effect since
fluorescence output did not change. It is also apparent the PKI is
more a potent inhibitor than staurosporin as lower concentrations
of the former was capable of inhibiting 50% of enzyme activity
(IC.sub.50) than the latter (FIG. 2).
[0129] Kinase reactions were also run with two additional peptide
substrates: Peptide substrate bis-SPK-2 (KKALRRASLKG (SEQ. ID.
NO:2)-R110-GKLSARRLAKK) and bis-SPK-4 (KKALRKASVRG (SEQ. ID.
NO:3)-R110-GRVSAKRLAKK) under similar conditions as those listed
above. This test demonstrated that the peptide substrate bis-SPK-2
is a better substrate for PKA than the peptide substrate bis-SPK-4.
In addition, a monoamide peptide substrate was compared to a
bisamide peptide substrate, and similar profiles were obtained,
except that the background was higher for the mono substituted
Rhodamine 110 compared to the bis-substituted Rhodamine 110
derivative. This property of Rhodamine 110 derivatives is well
known in the art (Results not shown).
[0130] The reactions were carried out in single tube, 96-well, and
384-well formats, and both white and black plates were used. Black
plates were preferred due to their lower reflectivity resulting in
lower equipment associated-background. Other serine/threonine
protein kinases, such as PKG, PKC, AKT, were also tested using
substrate SPK-2, and the change in output fluorescence in each
assay was inversely proportional to the amount of enzyme in the
reaction (Results not shown).
Example 3
Detection of Ser/Thr Kinase Activity using AMC-Modified Peptide
Substrate and Aminopeptidase M
[0131] cAMP-dependent Protein Kinase (PKA) assay with LRRASLG-AMC.
Other fluorogenic reporters, such as 7-amino-4-methylcoumarin
(AMC), were tested for their suitability for use with PKA. AMC was
linked to peptide LRRASLG in an amide bond via a free amino group,
producing the peptide substrate kemptide-AMC. This substrate was
used under assay conditions that were identical to those used with
the Rhodamine 110-modified peptide substrate, except that the
substrate was added at a concentration of 40 uM. Reactions were
incubated at room temperature for 30 minutes and Aminopeptidase M
was added at a final concentration of 50 mU/well and incubated at
room temperature for 60 minutes. Reactions were carried out in the
presence and absence of 50 uM ATP to show that the
phosphotransferase activity of PKA requires ATP. Fluorescent data
were obtained at sixty minutes, without addition of actonin.
Fluorescence was detected with an excitation of 360 nm and a
fluorescence emission at 420 nm.
[0132] The results in FIG. 3 show that in the absence of ATP, there
is no change in the fluorescence output with increasing enzyme
concentrations. In the presence of ATP, the fluorescence output
decreased in proportion to the increase in amount of enzyme. These
data show that any fluorogenic reporter compound can be used in the
invention.
Example 4
Detection of Tyrosine Kinase using R110-Modified Peptide Substrate
and Aminopeptidase M
[0133] Tyrosine Kinase Assays. The kinase activity of tyrosine
kinases was demonstrated using peptide substrates containing
tyrosine as the phosphorylatable amino acid residue. The kinase
activity of several enzymes of the Src family of protein tyrosine
kinases, such as Fyn, Lyn A, Lyk, Src, Src N1, and for the kinase
activity of a growth factor receptor tyrosine kinases (insulin
receptor) were tested.
[0134] Conditions for the tyrosine kinase reactions include
Tris-HCl, pH 7.5, 0.1 mg/ml BSA, 20 mM MgCl.sub.2, 1 mM MnCl.sub.2,
0.2 mM EGTA, 100 uM sodium vanadate, 8 mM beta glycerophosphate, 2
uM bis-PTK-5 (YIYGAFKRRG (SEQ. ID. NO:4)-R110-GRRKFAGYIY), in a
volume of 50 ul/well. Enzyme titrations for the tyrosine kinase
lck, were run with 2-fold increments of enzyme from 0.07 mU to 40
mU, as well as a control containing no enzyme. Reactions were
carried out with and without 100 uM ATP at room temperature for 30
minutes. Samples were run in Dynex.RTM. Microfluor(.RTM. 2, black,
96-well plates (Dynex Technologies, Inc., Chantilly, Va.).
[0135] Kinase reactions were terminated by the addition of 25 ul of
100 mM EDTA. Aminopeptidase M (50 mU) was added and incubated at
25.degree. C. for 90 minutes. Fluorescence was then read using an
excitation at 480 nm and emission at 520-530 nm, as in Example 1.
Results shown in FIG. 4 indicate that where 100 um ATP was included
the decrease in fluorescence output was proportional to the
increase in amount of enzyme in the reaction, and little or no
change was observed in the absence of ATP.
[0136] In another experiment, it was shown that a nonspecific
inhibitor of the tyrosine kinase lck (staurosporin), but not a
compound that does not inhibit the kinase (U0126), can reverse the
change in fluorescence confirming that the change in fluorescence
is attributable to the enzyme activity of lck (FIG. 5).
Example 5
Detection of Ser/Thr Phosphatase Activity using R110-Modified
Peptide Substrate and Aminopeptidase M
[0137] PP2A activity with STP-R-110. The phosphatase activity of
phosphatase 2A (PP2A) was carried out in 50 ul volume containing 5
uM of phosphopeptide substrate bis-STP-R110 (RRAT(PO.sub.3)VA (SEQ.
ID. NO:5)-R110-AV(PO.sub.3)TARR), 40 mM Tris-HCl, pH 7.5, and 0.1
mg/ml BSA. Phosphatase reactions were initiated by adding the
enzyme serine/threonine phosphatase PP2A from Promega (Madison,
Wisconsin). The amount of PP2A added to each reaction was titrated
in 1/2 increments, in a range from 0.0075 ng (0.015 munits) to 7.5
nanograms (15 munits), plus control reactions containing no enzyme.
Phosphatase reactions were carried out for 10 minutes at room
temperature in 96-well plates.
[0138] Phosphatase reactions were terminated with 25 ul of 2 uM
okadaic acid ((9,10-Deepithio-9,10-didehydroacanthifolicin) (sodium
salt)), a known inhibitor of PP2A. After termination, 25 mU/well of
Aminopeptidase M was added in 40 mM Tris buffer, pH 7.5, 0.1 mg/ml
BSA. The aminopeptidase reaction was incubated at room temperature,
25.degree. C., for 90 minutes. Fluorescence was read as in Example
1. The results in FIG. 6 show that PP2A dephosphorylated the
substrate efficiently, and fluorescence increased proportionally to
the amount of enzyme in the reaction.
[0139] The specificity of the phosphatase activity using this
substrate in the assay system was also validated using a specific
inhibitor of PP2A (okadaic acid) and with PP1 inhibitor-2, which is
known to inhibit PP1 but not PP2A, and staurosporin, which also
does not inhibit PP2A. The same assay protocol described herein was
used the presence of 2 nanograms of PP2A (4 munits) and 5 uM of the
phosphopeptide substrate STP5. Inhibitors were included in the
reactions at concentrations ranging from zero inhibitor up to 100
nM. It is clear from FIG. 7 that dephosphorylation of the substrate
by PP2A was only inhibited in the presence of increasing
concentrations of okadaic acid and not in the presence of PP1
inhibitor-2 or staurosporin, confirming the specificity of assay
for PP2A. The concentration of okadaic acid required to inhibit 50%
of PP2A activity (IC.sub.50) was less than 1 nM, which agrees with
known values for okadaic acid inhibition of PP2A.
[0140] Phosphatase PP2A was also tested in a 384-well format. The
reproducibility in the 384 plates was found to be excellent.
[0141] Enzyme activity of PP1, PP2B, and PP2C were also tested
using the same substrate but with appropriate known cofactors added
for each enzyme to obtain optimal enzyme activity in the assay. The
results obtained show excellent proportionality between the
fluorescence output and dephosphorylation of the substrate. The
amount of enzyme in the reaction and the activity of each enzyme
was dependent on the presence of the corresponding activator.
Furthermore, the addition of specific inhibitors abolished the
phosphatase activity of the corresponding enzyme.
Example 6
Detection of Phosphatase Activity using Rhodamine 110-Modified
Peptide Substrate and Aminopeptidase II
[0142] PP2A activity with STP-R110. Phosphatase activity of PP2A
was tested using the same conditions as described in Example 5,
above, except that PP2A was tested at concentrations ranging from
0.000001 mU to 0.01 mU, plus a control reaction containing no
enzyme. Reactions were carried out at room temperature in 96-well
plates for 10 minutes and were terminated with okadaic acid
(9,10-Deepithio-9,10-didehydroacanthifolicin). In place of
Aminopeptidase M, 25 mU of Aminopeptidase II was added and the
reaction incubated for 90 minutes at room temperature. Fluorescence
output was read as described earlier. FIG. 8 shows that an increase
in phosphatase activity resulted in an increase in fluorescence.
These data show that any aminopeptidase can be used to in the
invention.
Example 7
Detection of Tyrosine Phosphatase Activity using R110-Modified
Peptide Substrate and Aminopeptidase M
[0143] CD45 and PTP-1B assays with PTK5-R110. Phosphatase reactions
were carried out using either CD45, which is a recombinant human
receptor protein tyrosine phosphatase, or PTP-1B, which is a
soluble tyrosine phosphatase. Reactions were carried out in 50 ul
volume containing 1 uM of phosphopeptide substrate bis-PTK5p-R110
(YIY(PO.sub.3)GAFKRRG (SEQ. ID. NO:6)-R110-GRRKFAG(PO.sub.3)YIY),
40 mM Tris-HCl, pH 7.5, 0.1 mg/ml BSA. Reactions were carried out
in the presence of increasing concentrations of phosphatase (0-2
units of CD45, or 0-0.025 units of PTP-1B) for 10 minutes at room
temperature in 96-well plates.
[0144] Phosphatase reactions were terminated with 25 ul of a
solution containing 40 mM Tris-HCl, pH 7.5, 0.1 mg/ml BSA, 300 uM
Na.sub.3VO.sub.4, and 1 mU/ul of Aminopeptidase M. Reactions were
incubated for additional 90 minutes at room temperature.
Fluorescence was read as earlier described for Example 1. The
results in FIG. 9 show that the increase in the fluorescence output
is proportional to the amount of phosphatase added per reaction.
The assay was also very sensitive to low concentrations of
phosphatase. A similar profile was obtained with other tyrosine
phosphatases, including YOP 51.
Example 8
Detection of Tyrosine Phosphatase Activity using R110-Modified
Peptide Substrate and Aminopeptidase M
[0145] PTP 1B activity with PTK5-R110. The effect of various
inhibitors were tested on the dephosphorylation of bis-PTK5p-R110
by the enzyme tyrosine phosphatase PTP-1B. Sodium vanadate,
(Na.sub.3VO.sub.4, a specific inhibitor of PTP-1B) and staurosporin
(a known inhibitor of PKA but not of PTP-1B) were tested.
Inhibitors were included in the reactions at concentrations ranging
from zero inhibitor up to 50 uM. Phosphatase reactions were
initiated by adding PTB-1B, which was added to each reaction at 25
mU/well. Control reactions containing no enzyme were also run.
Phosphatase reactions were carried out generally as described in
Example 7 for PTP-1B.
[0146] Phosphatase reactions were terminated with 25ul of a
solution of Na.sub.3VO.sub.4 and 25 mU/well of Aminopeptidase M.
Reactions were incubated at room temperature for 60 minutes.
Fluorescence was read as earlier described. The results in FIG. 10
show that dephosphorylation of the substrate by PTB-1B was only
inhibited in the presence of increasing concentrations of
Na.sub.3VO.sub.4 and not in the presence of staurosporin.
Increasing concentrations of Na.sub.3VO.sub.4 resulted in a
decreased fluorescence output indicating inhibition of PTP 1B
enzyme activity, while staurosporin had no effect since
fluorescence output was unchanged with increasing amounts of
staurosporin.
Example 9
Detection of Ser/Thr Kinase using Luciferin Modified Peptide
Substrate and Aminopeptidase M
[0147] PKA Assay with LRRASLG-(Luciferin). Protein kinase assay is
carried out at room temperature in a 50 ul volume in a 96-well
plate, with the peptide substrate LRRASLG-Luciferin at 50 uM, and
varying enzyme concentrations (0.001 to 1 unit) of protein kinase A
in reaction buffer as described in Example 1. Reactions are
terminated after 20 minutes by heat inactivation at 70.degree. C.
for 5 minutes. The reaction mixture is cooled off to room
temperature, and 25 ul of detection buffer containing 50 mU/ul of
Aminopeptidase M in 40 mM Tris HCl, pH 7.5 and 0.1 mg/ml BSA. The
reaction is kept at room temperature for additional 60 minutes
before optional termination by addition of actinonin at a final
concentration of 2.5 uM. Luciferase (Promega Corporation) at 100
ug/ml in a 25 ul of steady glow buffer (Promega Corp.) is added,
and then luminescence is read at 30 minutes in an Orion plate
luminometer, Berthold Detection Systems (Pforzheim, Germany).
Expression of enzyme activity is expected to be similar to that
described for fluorescently labeled substrates shown in Example 1,
i.e., a decrease in luminescence output in response to increase in
enzyme concentration or activity.
[0148] It is understood that the various preferred embodiments are
shown and described above to illustrate different possible features
of the invention and the varying ways in which these features may
be combined. Apart from combining the different features of the
above embodiments in varying ways, other modifications are also
considered to be within the scope of the invention. The invention
is not intended to be limited to the preferred embodiments
described above, but rather is intended to be limited only by the
claims set out below. Thus, the invention encompasses all alternate
embodiments that fall literally or equivalently within the scope of
these claims.
* * * * *