U.S. patent application number 11/780059 was filed with the patent office on 2008-06-19 for optical probes and assays.
This patent application is currently assigned to Invitrogen Corporation. Invention is credited to Brian D. Hamman, Lewis R. Makings, Brian A. Pollok, Steven M. Rodems.
Application Number | 20080146460 11/780059 |
Document ID | / |
Family ID | 23185763 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080146460 |
Kind Code |
A1 |
Pollok; Brian A. ; et
al. |
June 19, 2008 |
OPTICAL PROBES AND ASSAYS
Abstract
This invention provides an optical probe useful as an optical
probe or sensor of post translational type modifications, such as
phosphorylation. The invention comprises a polypeptide moiety,
which contains a recognition motif for a post translational type
activity and a protease site, which is coupled to a probe moiety.
Modification of the polypeptide, by the post translational type
activity, results in a modulation of the rate at which a protease
cleaves the polypeptide which is sensed by a measurable change in
at least one optical property of the optical probe upon cleavage.
The present invention also includes a recombinant nucleic acid
molecule that encodes an optical probe and a vector and host cell
or library of cells that include the recombinant nucleic acid
molecule. The optical probe can be used in methods to determine
whether a sample, including a cell or a sample from an organism,
contains a post-translational type modification activity. Such
methods can also be used to determine whether a test chemical
modulates the activity of a modifying activity, and thus can be
used to identify therapeutic compositions. The identification of
such therapeutic compositions can be automated using a system that
includes an optical probe.
Inventors: |
Pollok; Brian A.; (San
Diego, CA) ; Hamman; Brian D.; (Poway, CA) ;
Rodems; Steven M.; (Poway, CA) ; Makings; Lewis
R.; (Encinitas, CA) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Invitrogen Corporation
Carlsbad
CA
|
Family ID: |
23185763 |
Appl. No.: |
11/780059 |
Filed: |
July 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10105735 |
Mar 22, 2002 |
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11780059 |
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09306542 |
May 5, 1999 |
6410255 |
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10105735 |
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Current U.S.
Class: |
506/18 ; 435/23;
506/39 |
Current CPC
Class: |
G01N 33/542 20130101;
G01N 2333/9121 20130101; C12Q 1/48 20130101; C12Q 1/42 20130101;
C12Q 1/37 20130101; C40B 60/12 20130101; G01N 2333/43595 20130101;
C40B 40/10 20130101; C40B 30/04 20130101; G01N 33/582 20130101;
G01N 33/6842 20130101; G01N 33/6803 20130101; A61P 43/00
20180101 |
Class at
Publication: |
506/18 ; 435/23;
506/39 |
International
Class: |
C40B 40/10 20060101
C40B040/10; C12Q 1/37 20060101 C12Q001/37; C40B 60/12 20060101
C40B060/12 |
Claims
1. An engineered optical probe for measuring a post-translational
type modification, activity comprising: a polypeptide comprising a
first fluorescent moiety attached to the N or C terminus of said
polypeptide, wherein said polypeptide comprises a recognition motif
for a post-translational modification within said polypeptide, and
a protease site located within said polypeptide; and wherein post
translational type modification of said recognition motif modulates
the rate of cleavage of said polypeptide by a protease with
specificity for said protease site and said polypeptide is a
non-naturally occurring polypeptide and said first fluorescent
moiety permits detection of a polypeptide fragment.
2. The engineered optical probe of claim 1, wherein said
polypeptide further comprises a second fluorescent moiety attached
to said polypeptide, wherein said recognition motif and said
protease site are located within said polypeptide between said
first fluorescent moiety and said second fluorescent moiety.
3. The engineered optical probe of claim 1, wherein said
polypeptide further comprises a luminescent moiety attached to said
polypeptide, wherein said recognition motif and said protease site
are located within said polypeptide between said first fluorescent
moiety and said luminescent moiety.
4. The engineered optical probe of claim 1, wherein said
polypeptide further comprises a quencher moiety attached to said
polypeptide, wherein said recognition motif and said protease site
are located within said polypeptide between said first fluorescent
moiety and said quencher moiety.
5. The engineered fluorescent protein of claim 1, wherein said
recognition motif is an engineered recognition motif.
6. The engineered fluorescent protein of claim 1, wherein said
protease site is an engineered protease site.
7. The engineered optical probe of claim 1, wherein said
polypeptide does not exceed 15 amino acids in length.
8. The engineered optical probe of claim 1, wherein said
polypeptide does not exceed 50 amino acids in length.
9. The engineered optical probe of claim 5, wherein said
polypeptide further comprises a quencher moiety, wherein said
engineered recognition motif and said protease site are located
within said polypeptide between said first fluorescent moiety and
said quencher moiety.
10. The engineered optical probe of claim 5, wherein said
polypeptide further comprises a second fluorescent moiety attached
to said polypeptide, wherein said engineered recognition motif and
said protease site are located within said polypeptide between said
first fluorescent moiety and said second fluorescent moiety.
11. The engineered optical probe of claim 5, wherein said
polypeptide further comprises a luminescent moiety attached to said
polypeptide, wherein said engineered recognition motif and said
protease site are located within said polypeptide between said
first fluorescent moiety and said luminescent moiety.
12. The engineered optical probe of claim 10, wherein said
post-translational type modification is phosphorylation or
dephosphorylation.
13. The engineered optical probe of claims 10, wherein said first
fluorescent moiety comprises a first fluorescent protein or homolog
thereof.
14. The engineered optical probe of claim 12, wherein said
phosphorylation or dephosphorylation is phosphorylation a tyrosine
kinase activity or dephosphorylation by a protein tyrosine
phosphatase activity.
15. The engineered optical probe of claim 12, wherein said
phosphorylation or said dephosphorylation is by a protein serine or
threonine kinase activity or dephosphorylation by protein serine or
threonine phosphatase activity.
16. The engineered optical probe of claim 12, wherein said
phosphorylation or said dephosphorylation occurs at the P'.sub.1
position with respect to said protease.
17. The engineered optical probe of claim 12, wherein said
phosphorylation or said dephosphorylation occurs at the P'.sub.2
position with respect to said protease.
18. The engineered optical probe of claim 13, wherein said
fluorescent protein is an Aequorea green fluorescent protein.
19. A library of engineered optical probes comprising at least 5
different members, wherein each member of said library of
engineered optical probes comprises, i) a polypeptide comprising a
recognition motif for a post-translational type modification within
said polypeptide, and a protease site located within said
polypeptide; ii) a first fluorescent moiety attached to the N or C
terminus of said polypeptide, wherein post translational type
modification of said recognition motif modulates the rate of
cleavage of said polypeptide by a protease with specificity for
said protease site, and wherein said recognition motif for said
post-translational type modification is a preferred substrate for a
defined sub-class or sub-family of post-translational enzyme
activities.
20. The library of engineered optical probes of claim 19, wherein
said library comprises at least 100 different members.
21. The library of engineered optical probes of claim 19, wherein
said engineered optical probe further comprises a second
fluorescent moiety attached to said polypeptide, wherein said
recognition motif and said protease site are located within said
polypeptide between said first fluorescent moiety and said second
fluorescent moiety.
22. The library of engineered optical probes of claim 19, wherein
said engineered optical probe further comprises a luminescent
moiety attached to said polypeptide, wherein said recognition motif
and said protease site are located within said polypeptide between
said first fluorescent moiety and said luminescent moiety.
23. The library of engineered optical probes of claim 19, wherein
said engineered optical probe further comprises a quencher moiety
attached to said polypeptide, wherein said recognition motif and
said protease site are located within said polypeptide between said
first fluorescent moiety and said quencher moiety.
24. The library of engineered optical probes of claim 19, wherein
said recognition motif is an engineered recognition motif.
25. The library of engineered optical probes of claim 19, wherein
said protease site is an engineered protease site.
26. The library of engineered optical probes of claim 19, wherein
said polypeptide does not exceed 15 amino acids in length.
27. The library of engineered optical probes of claim 19, wherein
said polypeptide does not exceed 50 amino acids in length.
28. The library of engineered optical probes of claim 19, wherein
said post-translational type modification is protein kinase
mediated phosphorylation.
29. The library of engineered optical probes of claim 19, wherein
said post-translational type modification is protein phosphatase
mediated dephosphorylation.
30. The library of engineered optical probes of claim 28, wherein
said protein kinase is a tyrosine kinase.
31. The library of engineered optical probes of claim 28, wherein
said protein kinase is a serine or threonine kinase.
32. The library of engineered optical probes of claim 29, wherein
said protein phosphatase is a tyrosine phosphatase activity.
33. The library of engineered optical probes of claim 29, wherein
said protein phosphatase is a serine or threonine protein
phosphatase.
34. The library of engineered optical probes of claim 19, wherein
said library comprises at least one preferred recognition motif for
a protein tyrosine kinase selected from the group consisting of PTK
group 1 and PTK group 2 of Table 1.
35. The library of engineered optical probes of claim 34, wherein
said library comprises at least one preferred recognition motif for
a protein tyrosine kinase selected from the group consisting of PTK
subgroup 1, PTK subgroup 2, PTK subgroup 3, PTK subgroup 4, PTK
subgroup 5, PTK subgroup 6, PTK subgroup 7, PTK subgroup 8 and PTK
subgroup 9 of Table 1.
36. The library of engineered optical probes of claim 34, wherein
said library comprises at least one preferred recognition motif for
a protein tyrosine kinase selected from the group consisting of PTK
subgroup 10, PTK subgroup 11, PTK subgroup 12, PTK subgroup 13, PTK
subgroup 14, PTK subgroup 15, PTK subgroup 16, PTK subgroup 17, PTK
subgroup 18, PTK subgroup 19, PTK subgroup 20, PTK subgroup 21, PTK
subgroup 22 and PTK subgroup 23 of Table 1.
37. The library of engineered optical probes of claim 31, wherein
said library comprises at least one preferred recognition motif for
a protein serine/threonine kinase selected from the group
consisting of the AGC group, the CaMK group, the CMGC group and the
OPK group of Table 1.
38. The library of engineered optical probes of claim 37, wherein
said library comprises at least one preferred recognition motif for
a protein serine/threonine kinase selected from the group
consisting of AGC subgroup 1, AGC subgroup 2, AGC subgroup 3, AGC
subgroup 4, AGC subgroup 5, AGC subgroup 6, AGC subgroup 7, AGC
subgroup 8 and AGC subgroup 9.
39. The library of engineered optical probes of claim 37, wherein
said library comprises at least one preferred recognition motif for
a protein serine/threonine kinase selected from the group
consisting of CaMK subgroup 1 and CaMK subgroup 2.
40. The library of engineered optical probes of claim 37, wherein
said library comprises at least one preferred recognition motif for
a protein serine/threonine kinase selected from the group
consisting of CMGC subgroup 1, CMGC subgroup 2, CMGC subgroup 3,
CMGC subgroup 4, CMGC subgroup and CMGC subgroup 6.
41. The library of engineered optical probes of claim 37, wherein
said library comprises at least one preferred recognition motif for
a protein kinase selected from the group consisting of OPK subgroup
1, OPK subgroup 2, OPK subgroup 3, OPK subgroup 4, OPK subgroup 5,
OPK subgroup 6, OPK subgroup 7, OPK subgroup 8, OPK subgroup 9, OPK
subgroup 10, OPK subgroup 11, OPK subgroup 12, OPK subgroup 13 and
OPK subgroup 14.
42. A system for spectroscopic measurements, comprising: at least
one reagent for an assay and a device, said device comprising a
container and a platform, wherein said container comprises a) an
optical probe, comprising: a first probe moiety attached to a
polypeptide, wherein said polypeptide comprises a recognition motif
for a post-translational modification activity, and a protease site
located within said polypeptide; and wherein post translational
type modification of said recognition motif within said polypeptide
by said post-translational modification activity modulates the rate
of a protease with specificity for said protease site to cleave
said polypeptide, b) a sample, c) a protease with specificity for
said protease site.
43. The system of claim 42, wherein said container comprises a
multiwell plate.
44. The system of claim 42, wherein said system further comprises a
detector for detecting at least one optical property of said
optical probe.
45. The system of claim 44, wherein said first probe moiety
comprises a first fluorescent moiety.
46. The system of claim 45, wherein said optical probe further
comprises a second fluorescent moiety attached to said polypeptide,
wherein said recognition motif and said protease site are located
within said polypeptide between said first fluorescent moiety and
said second fluorescent moiety.
47. The system of claim 45, wherein said optical probe further
comprises a luminescent moiety attached to said polypeptide,
wherein said recognition motif and said protease site are located
within said polypeptide between said first fluorescent moiety and
said luminescent moiety.
48. The system of claim 45, wherein said optical probe further
comprises, a quencher moiety attached to said polypeptide, wherein
said recognition motif and said protease site are located within
said polypeptide between said first fluorescent moiety and said
quencher moiety.
49. The system of claim 45, wherein said optical probe further
comprises a bead attached to said polypeptide, wherein said
recognition motif and said protease site are located within said
polypeptide between said first fluorescent moiety and said
bead.
50. The system of claim 44, wherein said optical property is
fluorescence emission.
51. The system of claim 44, wherein said optical property is
fluorescence anisotropy.
52. The system of claim 44, wherein said post-translational
modification activity is protein kinase mediated
phosphorylation.
53. The system of claim 44, wherein said post-translational
modification activity is protein phosphatase mediated
dephosphorylation.
54. The system of claim 52, wherein said protein kinase is a
tyrosine kinase.
55. The system of claim 52, wherein said protein kinase is a serine
or threonine kinase.
56. The system of claim 53, wherein said protein phosphatase is a
tyrosine phosphatase activity.
57. The system of claim 53, wherein said protein phosphatase is a
serine or threonine protein phosphatase.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application U.S. Ser.
No. 09/306,542, filed May 5, 1999, now allowed, the entire contents
of which are hereby incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
chemistry and biology. More particularly, the present invention
relates to optical probes for post translational type modification
activities, such as phosphorylation, and methods for their use.
INTRODUCTION
[0003] Systems and methods for rapidly identifying chemicals with
biological activity in samples, especially small liquid samples, is
of particular relevance to the agrochemical and pharmaceutical
fields. Various strategies are typically used to reduce processing
times and associated costs of screening large numbers of chemical
entities, including simplified assay design, automation, robotics
and miniaturization of sample size. The advent of high throughput
analysis and increasing use of miniaturized formats has led to the
development of high density plate formats. For example, containing
384, 864 and 3456 wells as described in U.S. patent application
Ser. No. 08/868,049 Entitled "Low Background Multi-Well Plates with
greater than 864 Wells for Fluorescence Measurements of Biological
and Biochemical Samples," filed Jul. 3, 1997, now pending. Even
higher density sample processing systems, for example using chips
that contain miniaturized microfluidic devices are being developed
(see, for example, R & D Magazine, November 1998, pages 38 to
43 entitled "Lab-on-a Chip: Biotech's next California Gold
Rush").
[0004] Higher density plates enable faster analysis and handling of
large sample or chemical libraries, such as in automated screening
systems, but place considerable constraints on the assays that can
be successfully employed within them. In particular, there is a
need to develop assays that are compatible with miniaturized
systems and which give accurate and reproducible assay results.
Central to this need is a requirement for high sensitivity assays
based on optical analysis, such as fluorescence or luminescence
that do not require wash steps (e.g. "addition only assays").
[0005] One of the largest and most important classes of
intracellular activities for which drugs may be particularly
valuable are those involved in post-translational modification
activities. These activities are typically directed to the
modification of proteins and nucleic acids within living cells to
effect changes in the biological activity and function of these
molecules. The major methods of protein or polypeptide,
post-translational modification include protein phosphorylation,
methylation, prenylation, glycosylation, ubiquitination sulfation
and proteolysis (see generally Cells. A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1998) review). Major methods of
nucleic acid modification include methylation, ADP-ribsoylation and
restriction digestion. A variety of environmental stimuli such as
the presence of growth factors, hormones, changes in the cell cycle
and toxins can transiently modulate the post-translational state of
many intracellular components. The rapid development of specific,
and effective inhibitors for a particular post-translational
activity requires the development of suitable assays that can
reliably and sensitively detect these activities in a high
throughput screening system.
[0006] In spite of their great potential importance however, there
are few existing methods of measuring such activities that are
homogenous, non radioactive and sensitive enough to accurately and
reproducible work in high throughput, or ultra high throughput
screening systems. Such assays, by reducing the time required to
identify and develop useful chemicals, can dramatically increase
the value of a new drug by enabling its patentability and
increasing it's period of exclusivity in the market.
[0007] Examples of such post-translational activities include,
amongst others, protein methylation and prenylation. Protein
prenylation involves the addition of isoprenoid moieties such as
farnesyl and geranylgeranyl to proteins, and is a major mechanism
of post-translational modification for many membrane-associated
proteins. (Clark, 1992 Protein isoprenylation and methylation at
carboxyl-terminal cysteine residues. Annu. Rev. Biochem. 61
355-386). In most cases, the amino acid derivatized with the
isoprenoid is a cysteine, or cysteines close to the
carboxyl-terminus of the protein. Present methods of measuring
protein prenylation and methylation typically involve labeling
cells with radioactive precursors such as [.sup.3H]-mevalonate or
[.sup.3H]-S-adenosylmethionine, isolation of the protein of
interest and measurement of radioactive incorporation. There is
thus a need for assays for these activities that are sensitive,
simple to use, non-radioactive and adaptable to high throughput
screening methods.
[0008] Another important example of post-translational modification
is protein glycosylation, which plays an extremely important role
in the function of a significant number of proteins (Varki, 1993,
Biological roles of oligosaccharides. Glycobiology 3 97-130).
Protein glycosylation, unlike most other types of
post-translational modification provides a wide diversity in the
oligosaccharides added to a protein because of the potential for
branching after the addition of the first sugar residue. Present
methods of measuring glycosylation typically involve determining
radioactive incorporation of a precursor oligosaccharide into a
protein, isolating the protein and then measuring specific
radioactive incorporation into a protein. There is thus a need for
fluorescence or luminescence based assays for these activities that
are adaptable to high throughput screening methods. It is one
objective of the present invention to provide optical probes and
methods of use that meet this need.
[0009] Protein kinases and phosphatases are generally recognized as
one of the more important general mechanisms of regulating protein
function. A recent review and analysis of diseases associated with
genetic defects in protein kinases
(www.nih.go.jp/mirror/pkr/pk_medicine.html) lists over 400 specific
disease states associated with these activities alone. Protein
kinases act on proteins via the addition of phosphate groups
(phosphorylation) primarily on the amino acids, tyrosine, serine or
threonine. Protein phosphatases in contrast, act to remove these
phosphate groups thereby reversing the effects of phosphorylation.
Changes in the phosphorylation state of proteins, can regulate the
enzymatic activity, protein localization and protein-protein
interactions of a particular protein within a cell. Such changes
can subsequently modulate virtually every aspect of cellular
metabolism, regulation, growth and differentiation. The overall
balance of kinase and phosphatase activities in a cell is a primary
determinant of the phosphorylation state of a protein at any one
time.
[0010] However, current methods of measuring protein kinases, have
many disadvantages, which prevents or hampers the ability to
rapidly screen for drugs using miniaturized automated formats of
many thousands of compounds.
[0011] For example, many current methods of measuring their
activity rely on the incorporation and measurement of .sup.32P into
the protein substrates of interest. In whole cells, this
necessitates the use of high levels of radioactivity to efficiently
label the cellular ATP pool and to ensure that the target protein
is efficiently labeled with radioactivity. After incubation with
test drugs, the cells must be lysed and the protein of interest
purified to determine its relative degree of phosphorylation. This
method requires large numbers of cells, long preincubation times,
careful manipulation, and washing steps to avoid artifactual
phosphorylation or dephosphorylation. Furthermore, this kinase
assay approach requires purification of the target protein, and
final radioactive incorporation into target proteins is usually
very low giving the assay poor sensitivity. In high throughput
screening operations, this approach requires large amounts of
radioactivity, which can be an environmental and health hazard.
[0012] Alternative kinase assay methods, such as those based on
phosphorylation-specific antibodies using ELISA-type approaches,
involve the difficulty of producing antibodies that distinguish
between phosphorylated and non-phosphorylated proteins.
[0013] Furthermore, most kinase measurements have the requirement
for cell lysis, multiple incubations, and washing stages are time
consuming, complex to automate, and potentially susceptible to
artifacts.
[0014] There is thus a need for assays for enzymes, such as those
involved in post-translational modification, that are sensitive,
simple to use, applicable to virtually any activity and adaptable
to high throughput screening methods. Preferably, such assays would
not utilize radioactive materials so that the assays would be safe
and not generate hazardous wastes. The present invention addresses
these needs, and provides additional benefits as well.
SUMMARY OF THE INVENTION
[0015] This invention provides a fluorescent or bioluminescent
substrate useful as an optical probe or sensor of post
translational type modifications, such as phosphorylation. In one
embodiment, the invention comprises a polypeptide moiety, which
contains a recognition motif for a post translational type activity
and a protease site, which is coupled to a probe moiety. Typically,
the presence of a modification at the recognition motif alters
protease activity at the protease site resulting in a modulation of
the cleavage rate of the protease. Cleavage is sensed by a
measurable change in at least one optical property of the optical
probe upon cleavage at the protease site, FIG. 1.
[0016] In one embodiment the probe is a fluorescent or luminescent
moiety.
[0017] In another embodiment, the invention further comprises a
fluorescent quencher coupled to the polypeptide that quenches
emission from the first probe moiety. In this embodiment, the first
probe moiety and the quencher moiety are coupled to the polypeptide
such that the recognition motif and the protease site are located
between them (FIG. 1). In this case, cleavage of the polypeptide by
a protease results in an alteration in the fluorescence emission of
the first probe moiety that may be used to determine
post-translational activity.
[0018] In another embodiment, the optical probe may further
comprise a second probe moiety coupled to the polypeptide that
participates in energy transfer with the first probe moiety. In
this embodiment, the first probe moiety and the second probe moiety
are coupled to the polypeptide such that recognition motif and the
protease site are located between them. In this case, cleavage of
the polypeptide by a protease results in an alteration in energy
transfer between the first probe moiety and the second probe moiety
that may be used to determine post-translational activity.
[0019] The invention also provides methods for using the optical
probes of the invention to determine whether a sample contains a
post-translational type modification activity such as protein
phosphorylation or dephosphorylation, methylation, prenylation or
glycosylation. The method consisting of; i), contacting the optical
probe with a sample, usually containing or suspected of containing
a post translational type activity; ii), contacting the sample and
optical probe with a protease, and iii), determining at least one
optical property of said optical probe, or product thereof.
[0020] In another embodiment, the invention provides methods for
using the optical probes of the invention to determine whether a
test chemical modulates the activity of a post-translational type
activity.
[0021] In another aspect, the invention provides a library of
optical probes, each with a unique peptide sequence for use in
selecting an optimal sequence specificity of a post-translational
type activity.
[0022] Another aspect of the present invention includes a compound
or therapeutic identified by at least one method of the present
invention. These methods can include monitoring the efficacy and/or
toxicology of said therapeutic in an in vitro or in vivo model. The
compound can be provided in therapeutically acceptable carrier and
can form a therapeutic composition.
[0023] A further aspect of the present invention includes various
systems for spectroscopic measurements. In one embodiment, the
system typically includes at least one reagent for an assay and a
device, said device comprising a container and a platform. The
container can include the optical sensor compounds of the present
invention, and additional reagents necessary for the
post-translational type activity. Addition of a sample to the
container, followed by the addition of a protease after a given
time results in a change in at least one fluorescent property of
the optical probes of the present invention that can be used to
determine the post-translational type activity of the sample.
[0024] In another embodiment the system can include a microfluidic
spectroscopic system comprising at least one fluid containing
structure with at least one electro-osmotic or electrophoretic
system to control fluid movement within that structure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings, which are incorporated in and
form part of the specification, merely illustrate embodiments of
the present invention. Together with the remainder of the
specification, they are meant to serve to explain certain
principles of the invention to those of skill in the art.
[0026] FIG. 1 shows a schematic representation of some different
embodiments of the present invention. In FIG. 1, a first probe
moiety, 1, is attached to a polypeptide, 5, that comprises a
post-translational modification recognition site for an activity, 6
(shown hatched in FIG. 1) and a protease site for a protease, 7,
(shown filled in FIG. 1). In one embodiment of the present
invention, the optical probe may be attached to a solid surface, 4,
such as a bead. In another embodiment, the optical probe may
further comprise a quencher, 2, that is separated from the first
probe moiety, 1, by the polypeptide, 5. In another embodiment, the
optical probe may comprise a second probe moiety, 3, that is again
separated from the first probe moiety 1, by the polypeptide, 5.
[0027] FIG. 2 shows fluorescence emission spectra of cleaved and
non-cleaved of certain optical probes of the present invention.
Dotted lines represent the spectra of the cleaved optical probe,
and dashed lines represent the non-cleaved probe.
[0028] FIG. 3 shows the dependency of 460/530 nm emission spectra
ratios of certain optical probes (+/- kinase treatment) of the
present invention upon incubation with increasing concentrations of
a protease (chymotrypsin). Open symbols represent control samples,
filled symbols represent phosphorylated samples, Squares represent
the Src-1 substrate, triangles represent the Src-2 substrate and
circles represent the Abl substrate.
[0029] FIG. 4 shows the comparison of fluorescent changes mediated
by certain optical probes of the present invention to
.sup.32P-incorporation for the detection of inhibitors of tyrosine
kinase activity. Triangles represent optical probe measurements,
squares represent .sup.32P-incorporation measurements.
[0030] FIG. 5 shows the detection of an inhibitor activity of
protein tyrosine phosphatase activity by orthovanadate, using
certain optical probes of the present invention.
[0031] FIG. 6 shows a mock high throughput screening validation to
verify that the present invention can be used to identify
inhibitors of serine/threonine kinase activity.
[0032] FIG. 7 shows the caspase-3 mediated cleavage of
phosphorylated (filled symbols) and non-phosphorylated (open
symbols) ERK kinase specific optical probes of the present
invention.
[0033] FIG. 8 shows the inhibition of ERK kinase activity by
roscovitine using certain optical probes of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention recognizes that optical probes can be
designed to act as optical sensors of post-translational activities
through the creation of engineered molecules. In the present
invention, post-translational modification of a polypeptide results
in the modulation of the rate and efficiency of cleavage of the
modified polypeptide compared to the non-modified peptide. The
attachment of at least one probe moiety to the peptide couples the
cleavage of the optical probe to a change in a fluorescence
property of the substrate that may be used to determine the amount
of post-translational activity in a sample, FIG. 1.
[0035] Abbreviations
[0036] t-Boc, tert-butyloxycarbonyl; Bzl, benzyl; CaMK, calmodulin
dependent kinase; CKI, casein kinase 1; PDGF, platelet derived
growth factor; Fmoc, fluorenylmethyloxycarbonyl; EGF, epidermal
growth factor; ELISA, enzyme-linked immuno absorbant assay; FGF,
fibroblast growth factor; HF, hydrogen fluoride; HOBT,
N-Hydroxybenzotriazole; PyBop,
Benzotriazole-1-yl-oxy-tris-pyyrolidino-phosphonium
hexafluorophosphate; TFA, trifluoroacteic acid.
DEFINITIONS
[0037] Generally, the nomenclature used herein and many of the
fluorescence, computer, detection, chemistry and laboratory
procedures described below are those well known and commonly
employed in the art. Standard techniques are usually used for
chemical synthesis, fluorescence, optics, molecular biology,
computer software and integration. Generally, chemical reactions,
cell assays and enzymatic reactions are performed according to the
manufacturer's specifications where appropriate. The techniques and
procedures are generally performed according to conventional
methods in the art and various general references. (Lakowicz, J. R.
Topics in Fluorescence Spectroscopy, (3 volumes) New York: Plenum
Press (1991), and Lakowicz, J. R. Emerging applications of
fluorescence spectroscopy to cellular imaging: lifetime imaging,
metal-ligand probes, multi-photon excitation and light quenching.
Scanning Microsc Suppl Vol. 10 (1996) pages 213-24, for
fluorescence techniques; Sambrook et al. Molecular Cloning: A
Laboratory Manual, 2.sup.nd ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., for molecular biology
methods; Cells: A Laboratory Manual, 1.sup.st edition (1998) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for cell
biology methods; Optics Guide 5 Melles Griot.RTM. Irvine Calif.,
and Optical Waveguide Theory, Snyder & Love published by
Chapman & Hall for general optical methods, which are
incorporated herein by reference which are provided throughout this
document).
[0038] As employed throughout the disclosure, the following terms,
unless otherwise indicated, shall be understood to have the
following meanings:
[0039] The term "acceptor" refers to a quencher that operates via
energy transfer. Acceptors may re-emit the transferred energy as
fluorescence and are "acceptor fluorescent moieties". Examples of
acceptors include coumarins and related fluorophores, xanthenes
such as fluoresceins, fluorescent proteins rhodols, and rhodamines,
resorufins, cyanines, difluoroboradiazaindacenes, and
phthalocyanines. Other chemical classes of acceptors generally do
not re-emit the transferred energy. Examples include indigos,
benzoquinones, anthraquinones, azo compounds, nitro compounds,
indoanilines, and di- and triphenylmethanes.
[0040] The term "bead" refers to a substantially spherical particle
such as a sphere or microsphere. Beads may be used within a wide
size range. Preferred beads are typically within the range of 0.01
to 100 .mu.m in diameter. Beads may be composed of any material and
may be substantially inert or comprise fluorescent, luminescence,
electro-luminescent, chemo-luminescent, magnetic or paramagnetic
probes. Such beads are commercially available from a variety of
sources including Molecular Probes, Sigma or Polysciences.
[0041] The terms "cleavage site" or "protease site" refers to the
bond cleaved by the protease (e.g. a scissile bond) and typically
the surrounding three amino acids of either side of the bond. The
letters "P.sub.1", "P.sub.2", "P.sub.3" etc, refer to the amino
acid positions, 1 amino acid, 2 amino acids and 3 amino acids
N-terminal to the scissile bond. The letters "P'.sub.1",
"P'.sub.2", "P'.sub.3", refer to the amino acids positions 1 amino
acid, 2 amino acids and 3 amino acids C-terminal to the scissile
bond, as shown below;
##STR00001##
[0042] The term "engineered recognition motif" refers to a
recognition motif that has been modified from the naturally
existing sequence by at least one amino acid substitution.
[0043] The term "engineered protease site" refers to a protease
site that has been modified from the naturally existing sequence by
at least one amino acid substitution.
[0044] The term "fluorescent moiety" refers to a moiety that can
absorb electromagnetic energy and is capable of at least partially
remitting some fraction of that energy as electromagnetic radiation
over some time period. Suitable fluorescent moieties include, but
are not limited to, coumarins and related dyes, xanthene dyes such
as fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes,
bimanes, acridines, isoindoles, dansyl dyes, aminophthalic
hydrazides such as luminol, and isoluminol derivatives,
aminophthalimides, aminonaphthalimides, aminobenzofurans,
aminoquinolines, dicyanohydroquinones, semiconductor fluorescent
nanocrystals, fluorescent proteins and fluorescent europium and
terbium complexes and related compounds.
[0045] The term "fluorescent property" refers to any one of the
following, the molar extinction coefficient at an appropriate
excitation wavelength, the fluorescent quantum efficiency, the
shape of the excitation or emission spectrum, the excitation
wavelength maximum, or the emission magnitude at any wavelength
during, or at one or more times after excitation of the fluorescent
moiety, the ratio of excitation amplitudes at two different
wavelengths, the ratio of emission amplitudes at two different
wavelengths, the excited state lifetime, the fluorescent anisotropy
or any other measurable property of a fluorescent moiety and the
like. Preferably fluorescent property refers to fluorescence
emission, or the fluorescence emission ratio at two or more
wavelengths.
[0046] The term "homolog" refers to two sequences or parts thereof,
that are greater than, or equal to 75% identical when optimally
aligned using the ALIGN program. Homology or sequence identity
refers to the following. Two amino acid sequences are homologous if
there is a partial or complete identity between their sequences.
For example, 85% homology means that 85% of the amino acids are
identical when the two sequences are aligned for maximum matching.
Gaps (in either of the two sequences being matched) are allowed in
maximizing matching; gap lengths of 5 or less are preferred with 2
or less being more preferred. Alternatively and preferably, two
protein sequences (or polypeptide sequences derived from them of at
least 30 amino acids in length) are homologous, as this term is
used herein, if they have an alignment score of more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O.,
in Atlas of Protein Sequence and Structure, 1972, volume 5,
National Biomedical Research Foundation, pp. 101-110, and
Supplement 2 to this volume, pp. 1-10.
[0047] The term "modulates" refers to either the enhancement or
inhibition (e.g. attenuation of the rate or efficiency) partially
or complete of an activity or process.
[0048] The term "modulator" refers to a chemical compound
(naturally occurring or non-naturally occurring), such as a
biological macromolecule (e.g., nucleic acid, protein, non-peptide,
or organic molecule), or an extract made from biological materials
such as bacteria, plants, fingi, or animal (particularly mammalian,
including human) cells or tissues. Modulators are evaluated for
potential activity as inhibitors or activators (directly or
indirectly) of a biological process or processes (e.g., agonist,
partial antagonist, partial agonist, inverse agonist, antagonist,
antineoplastic agents, cytotoxic agents, inhibitors of neoplastic
transformation or cell proliferation, cell proliferation-promoting
agents, and the like) by inclusion in screening assays described
herein. The activity of a modulator may be known, unknown or
partially known.
[0049] The term "non-naturally occurring" refers to the fact that
an object cannot be found in nature. For example, a polypeptide or
polynucleotide sequence that is present in an organism (including
viruses) that can be isolated from a source in nature and which has
not been intentionally modified by man in the laboratory is
naturally-occurring, while such a sequence that has been
intentionally modified by man is non-naturally occurring.
[0050] The term "optical property" refers to a physical property of
light, including the molar extinction coefficient at an appropriate
excitation wavelength, the fluorescent or luminescent quantum
efficiency, the shape of the excitation spectrum or emission
spectrum, the excitation wavelength maximum or emission wavelength
maximum, the ratio of excitation amplitudes at two different
wavelengths, the ratio of emission amplitudes at two different
wavelengths, the excited state lifetime, the fluorescent anisotropy
or any other measurable optical property of a compound, or any
product or emission derived from that compound, either
spontaneously or in response to electrical or chemical stimulation
or reaction.
[0051] The term "polypeptide" refers to a polymer in which the
monomers are amino acids and are joined together through amide
bonds, alternatively referred to as a peptide. Additionally,
unnatural amino acids, for example, beta-alanine, phenylglycine and
homoarginine are also meant to be included. Commonly encountered
amino acids, which are not gene-encoded, may also be used in the
present invention. For a general review see Spatola, A. F., in
Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,
B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983). All of
the amino acids used in the present invention may be either the D-
or L-isomer. The L-isomers are preferred. Chemically modified or
substituted amino acids including phosphorylated, sulfated,
methylated, or prenylated residues may also be used to create
polypeptides for specific applications.
[0052] The term "post-translational type modification" refers to
the enzymatic or non-enzymatic modification of an amino acid
residue (preferably enzymatic). Such covalent modifications include
phosphorylation, dephosphorylation glycosylation, methylation,
sulfation, ubiquitination, prenylation and ADP-ribsoylation.
Preferred post-translational type modifications include
phosphorylation and dephosphorylation.
[0053] The term post-translational includes non-covalent type
modifications including protein-protein interactions, and the
binding of allosteric, or other modulators or second messengers
such as calcium, or cAMP or inositol phosphates to the recognition
motif.
[0054] The term "probe moiety" refers to a chemical moiety useful
as a marker or indicator, or contrast agent for, absorption
spectroscopy, luminescence spectroscopy, fluorescence spectroscopy,
or magnetic detection.
[0055] The term "quencher" refers to a molecule or part of a
compound that is capable of reducing the emission from a probe
moiety. Such reduction includes reducing the light after the time
when a photon is normally emitted from a fluorescent moiety.
Quenching may occur by any of several mechanisms, including
fluorescence resonance energy transfer, photoinduced electron
transfer, paramagnetic enhancement of intersystem crossing, Dexter
exchange coupling, and excitation coupling, such as the formation
of dark complexes. Preferred quenchers include those that operate
by fluorescence resonance energy transfer.
[0056] The term "recognition motif" refers to all or part of a
polypeptide sequence recognized by a post-translational
modification activity to enable a polypeptide to become modified by
that post-translational modification activity. Typically, the
affinity of a protein, e.g. enzyme, for the recognition motif is
about 1 mM (apparent K.sub.d), preferably a greater affinity of
about 10 .mu.M or less, more preferably, 1 .mu.M or most preferably
has an apparent K.sub.d of about 0.1 .mu.M. The term is not meant
to be limited to optimal or preferred recognition motifs, but
encompasses all sequences that can specifically confer substrate
recognition to a peptide. Preferably the recognition motif is a
phosphorylated recognition motif (e.g. includes a phosphate group),
or other post-translationally modified residues. Typically the
recognition motif will, at least partially, comprise a protease
site. The protease site may be located at any location within
recognition motif.
[0057] The term "test chemical" refers to a chemical to be tested
by one or more screening method(s) of the invention as a putative
modulator. A test chemical can be any chemical, such as an
inorganic chemical, an organic chemical, a protein, a peptide, a
carbohydrate, a lipid, or a combination thereof. Usually, various
predetermined concentrations of test chemicals are used for
screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar.
Test chemical controls can include the measurement of a signal in
the absence of the test compound or comparison to a compound known
to modulate the target.
Introduction
[0058] The present invention recognizes for the first time that
optical probes can be designed to measure a range of
post-translational type activities. The advantages of the present
invention include compositions that can be used in methods,
particularly methods for high throughput and miniaturized screening
systems for drug discovery and profiling. Theoretical probes
provide for assays, that typically exhibit a large dynamic range,
increased sensitivity and allow ratiometric readouts for the
detection of post-translational type activities.
[0059] As a non-limiting introduction to the breadth of the
invention, the invention includes several general and useful
aspects, including:
[0060] 1) A polypeptide moiety, which contains a recognition motif
for a post translational type activity and a protease site, which
is coupled to a first fluorescent moiety. Typically, the presence
of a modification at the recognition motif alters protease activity
at the protease site resulting in a modulation of the cleavage rate
of the protease. Cleavage is sensed by a measurable change in at
least one optical property of the optical probe upon cleavage at
the protease site. In different embodiments, the invention may
further comprise a second optical probe, such as fluorescent
quencher or second fluorescent moiety or luminescent moiety.
Typically the second optical probe is coupled to the polypeptide
such that recognition motif and the protease site are located
between them.
[0061] 2) Methods for using the optical probes of (1) to determine
whether a sample contains a post-translational type modification
activity such as protein phosphorylation or dephosphorylation.
[0062] 3) Methods for using the optical probes of (1) to determine
whether a test chemical modulates the activity of a
post-translational type activity.
[0063] 4) Libraries of optical probes, each with a unique peptide
sequence for use in selecting an optimal sequence specificity of a
post-translational type activity.
[0064] 5) A compound or therapeutic identified by at least one
method of the present invention.
[0065] 6) Systems for spectroscopic measurements using the optical
probes (1) and methods above.
[0066] 7) Microfluidic spectroscopic systems for using the optical
probe (1) comprising at least one fluid containing structure with
at least one electro-osmotic or electrophoretic system to control
fluid movement within that structure.
[0067] These aspects of the invention and others described herein,
can be achieved by using the methods and compositions of matter
described herein. To gain a full appreciation of the scope of the
invention, it will be further recognized that various aspects of
the invention can be combined to make desirable embodiments of the
invention. Such combinations result in particularly useful and
robust embodiments of the invention.
Designing Peptide Sequences for Use in the Optical Probes of the
Present Invention.
[0068] Generally peptide sequences for measuring a
post-translational type activity encompass a post-translational
recognition motif that contains a residue that, when modified,
modulates the rate of cleavage of the substrate by a protease as
compared to the unmodified form. Typically, such peptides contain a
single scissile bond (bond that is cleaved within the substrate)
for a specific protease and exhibit reasonable solubility (e.g. 0.1
mg/ml or greater) in aqueous solution. The design and size of
peptide sequences for specific optical probes, and the choice of a
particular protease, is dependent upon the application for which
the optical probe is to be used. For example, for resonance energy
transfer type applications, the peptide separating the fluorescent
or luminescent moieties will typically be in the range of 5 to 50
amino acids in length, preferably 10 to 25 amino acids in length,
or more preferably 10 to 15 amino acids in length. For polarization
based applications the peptide may be significantly larger, up to
and including entire protein domains, for example 50 to 100 amino
acids in length. Smaller peptides, in the range of 5 to 50 amino
acids may also be used. Typically the protease site may be located
at any position either completely or partially within the
recognition motif. The recognition motif and protease site may be
located at any position within the peptide with respect to the
optical probe moiety. The section below describes the design of
suitable peptide substrates for use in the present invention.
Subsequent sections describe the selection and coupling of suitable
fluorescent moieties for use in the invention. The following
representative examples are offered by way of illustration, not by
way of limitation.
A Design of Peptides for Measuring Protein Phosphorylation
[0069] In general protein kinases act on proteins via the addition
of phosphate groups (phosphorylation) primarily on the amino acids,
tyrosine, serine or threonine through a free hydroxyl group. The
protein kinases that enzymatically catalyze these reactions may be
classified into a number of distinct families based on shared
structural and functional properties. Typically, kinases within a
family have a similar overall topology, have similar modes of
regulation and have similar substrate specificity's (see, Table 1,
and may be used with the invention, as well as those recognition
motifs developed in the future). For example, members of the AGC
(protein kinase A, G or C) families of kinases typically prefer
phosphorylation recognition motifs with basic amino acids (R or K),
those in the CMGC group typically prefer proline containing motifs,
etc. In Table 1, blank cells in the "Substrate Preference" column
indicate that the complete information for every member of a
particular class was not available, or indicates that the family
was too small to define a clear substrate preference, or indicates
that no clearly defined substrate preference yet exists.
[0070] Within sub-families, particular members have specific
preferences for amino acids at specific positions within the
substrate. These preferences have been extensively characterized
for a number of kinases as described herein. Additional methods for
identifying the substrate specificities and binding recognition
motifs of new kinases are known in the art and may be used with the
present invention. Such methods enable the substrate specificity of
virtually any kinase known now, or discovered in the future, to be
rapidly identified, for example see U.S. Pat. No. 5,532,167 by
Cantley et al., issued Jul. 2, 1996, and PCT application WO
98/54577 by Lai et al., filed May 28, 1998.
TABLE-US-00001 TABLE 1 MAIN GROUPS SUB-GROUPS DESCRIPTION SUBSTRATE
PREFERENCE AGC GROUP Group 1 Cyclic nucleotide regulated Arg/Lys
Directed protein kinase family Group 2 Diacylglycerol- Arg/Lys
Directed activated/phospholipid-dependent protein kinase family
Group 3 Related to protein kinase A and Arg/Lys Directed protein
kinase C Group 4 Kinases that phosphorylate G Negative charge
Directed protein-coupled receptors Group 5 Budding yeast
AGC-related Not available protein kinases Group 6 Kinases that
phosphorylate Arg/Lys Directed ribosomal protein S6 family Group 7
Budding yeast DBF2/20 family Group 8 Flowering plant PVPK1 protein
kinase homologue family Group 9 Other AGC related kinase Various
families CAMK GROUP CaMK Group 1 Kinases regulated by Ca.sup.2+/CaM
Arg/Lys Directed and close relatives CaMK Group 2 CaMK group II
Arg/Lys Directed CaMK Other Other CaMK related kinase Various
families CMGC GROUP CMGC Group 1 Cyclic-dependent kinases (CDKs)
Ser/Pro Directed and close relatives family CMGC Group 2 ERK (MAP)
kinase family Ser/Pro Directed CMGC Group 3 Glycogen synthase
kinase 3 Ser/Pro Directed family CMGC Group 4 Casein kinase II
family Negative charge directed CMGC Group 5 C1k family CMGC Group
6 CMGC Group other Various PTK GROUP 1 Non-membrane spanning
protein tyrosine kinases PTK Group 1 Src family IY directed PTK
Group 2 Tec/Atk family PTK Group 3 Csk family IYM directed PTK
Group 4 Fes (Fps) family IYE directed PTK Group 5 Abl family IYA
directed PTK Group 6 Syk/ZAP70 family YE directed PTK Group 7
Tyk2/Jak 1 family PTK Group 8 Ack family PTK Group 9 Focal adhesion
kinase (Fak) family PTK GROUP 2 Membrane spanning protein tyrosine
kinases PTK Group 10 Epidermal growth factor receptor EEEYF
directed family (EGF) PTK Group 11 Eph/Elk/Eck receptor PTK Group
12 Axl family PTK Group 13 Tie/Tek family PTK Group 14 PDGF family
EEEYV directed PTK Group 15 FGF family EXYXF directed PTK Group 16
Insulin receptor family YMMM directed PTK Group 17 LTK/ALK family
PTK Group 18 Ros/Sevenless family PTK Group 19 Trk/Ror family PTK
Group 20 DDR/TKT family PTK Group 21 Hepatocyte growth factor
receptor family PTK Group 22 Nematode Kin 15/16 family PTK Group 23
Other membrane spanning kinases Various OPK GROUP Other protein
Kinases (not falling in major groups) OPK Group 1 Polo family OPK
Group 2 MEK/STE7 family OPK Group 3 PAK/STE20 family OPK Group 4
MEKK/STE11 family OPK Group 5 NimA family OPK Group 6 Wee1/mik1
family OPK Group 7 Kinases involved in transcriptional control
family OPK Group 8 Raf family OPK Group 9 Activin/TGFb receptor
family OPK Group 10 Flowering plant putative receptor kinases and
close relatives OPK Group 11 PSK/PTK "mixed lineage" leucine zipper
domain family OPK Group 12 Casein kinase 1 family OPK Group 13 PKN
prokaryotic protein kinase family OPK Group 14 Other protein kinase
families Various (each with no close relatives)
[0071] Eukaryotic protein phosphatases are structurally and
functionally diverse enzymes that are represented by three distinct
gene families. Two of these, dephosphorylate phosphoserine and
phosphothreonine residues, whereas the protein tyrosine
phosphatases (PTPs) dephosphorylate phosphotyrosine amino acids. A
subfamily of the PTPs, the dual specificity phosphatases,
dephosphorylates all three phosphoamino acids. Within each family,
the catalytic domains are highly conserved, with functional
diversity endowed by regulatory domains and subunits.
[0072] The protein serine or threonine phosphatases type 1 and 2A
account for as much as 95% of the phosphatase activity in cell
extracts (Brautigan and Shriner, Methods. Enzymol. 159: 339-346
(1988). These enzymes have broad substrate specificities and may be
regulated in vivo through targeting of the enzymes to discrete
sub-cellular localizations.
[0073] The total number of protein tyrosine phosphatases encoded in
the mammalian genome has been estimated at between 500 and
approximately 2000. These estimates are imprecise due to the large
number of sequence database entries that are different splice forms
or duplicates of the same PTP sequence.
[0074] i) Tyrosine Phosphorylation or Dephosphorylation
[0075] Optical probes for detecting tyrosine kinase activity
according to the present invention are designed by incorporating
the desired phosphorylation motif into a peptide, and by ensuring
that the only aromatic residue (Tyr, Trp or Phe) in the substrate
is the tyrosine that is phosphorylated. It may also be preferable
in certain cases to eliminate or reduce the number of negatively
charged amino acids in the P'.sub.1, P'.sub.2 or P'.sub.3
positions. If this is the case, then phosphorylation of the
tyrosine residue by the tyrosine directed protein kinase activity
modulates the rate of optical probe hydrolysis by chymotrypsin
compared to the non-phosphorylated optical probe. The present
inventors have recognized that elimination of negatively charged
residues in the optical probe C-terminal to the scissile bond
improves the efficiency of cleavage of non-phosphorylated optical
probe, on occasion significantly increasing the utility of the
optical probes for measuring kinase or phosphatase activities. This
approach can be used to create specific optical probes for
virtually all known tyrosine kinase activities by routine
optimization of the reaction conditions as described herein.
Specific illustrative examples for different tyrosine kinase
classes are shown in Table 2, below for use with chymotrypsin.
TABLE-US-00002 TABLE 2 Optimal recogni- tion motif for Optical
probe Kinase the kinase specific motif c-FGR MEEIYGIFF .sup.(2)
MEEIYGILS SEQ. ID. NO. 1 Lyn DEEIYEELE .sup.(2) DEEIYESLE SEQ. ID.
NO: 2 Src-1 GEEEIYGEFEK .sup.(1) GEEEIYGEIEK SEQ. ID. NO: 3 C-Abl
AXVIYAAPF .sup.(1) AEAIYAAPL SEQ. ID. NO: 4 CSK XEPIYMFFF .sup.(2)
EPIYMLSL SEQ. ID. NO: 5 Insulin receptor XEEEYMMMF .sup.(1)
EEEYMMMM SEQ. ID. NO: 6 PDGF receptor EEEEYVFIX .sup.(1) EEEEYVVIX
SEQ. ID. NO: 7 EGF receptor EEEEYFELV .sup.(1) EEEEYVLLV SEQ. ID.
NO: 8 FGF receptor AEEEYFFLF .sup.(1) AEEEYFVLM SEQ. ID. NO: 9
[0076] In Table 2, bold residues indicate those considered to be
significant in kinase recognition, and italicized residues are
those that can be substituted to enable effective modulation of the
proteolytic sensitivity of the optical probe towards chymotrypsin
upon phosphorylation. The tyrosine that is phosphorylated is
underlined, and the indicated references are (1) Songyang, et al.,
Current Biology 4:973-983, 1994, and (2) Ruzzene, et al., Eur. J.
Biochem. 246: 433-439.
[0077] Optical probes for detecting protein tyro sine phosphatase
activity according to the present invention are designed by
incorporating the desired phosphorylation motif into a peptide, for
example like those in (Table 2), or other such motifs developed now
or in the future, and either enzymatically or chemically
phosphorylating the appropriate amino acid. Dephosphorylation of
the tyrosine residue in such optical probes by a tyrosine directed
protein phosphatase activity modulates the rate of optical probe
hydrolysis by chymotrypsin compared to the phosphorylated optical
probe.
[0078] ii) Serine/Threonine Phosphorylation or
Dephosphorylation
[0079] To develop optical probes for measuring serine or threonine
kinase activities, peptides are designed to incorporate a single
aromatic amino acid (Tyr, Trp or Phe) that is typically located
within about three amino acids of a serine or threonine residue,
which is phosphorylated by an appropriate serine or threonine
specific kinase. It is also preferable in certain cases (depending
on the protease selected) to eliminate or reduce the number of
negatively charged amino acids (e.g. Asp or Glu residues) in the
P'.sub.1, P'.sub.2 or P'.sub.3 positions to ensure that the effect
of phosphorylation of the serine or threonine residue provides a
large modulation in proteolytic sensitivity of the optical probe
upon phosphorylation. Examples of such sequences are provided in
Table 3, below, for use with chymotrypsin.
TABLE-US-00003 TABLE 3 Optical probe Optical probe Kinase Optimal
Motif with p-Ser in P'.sub.1 with p-Ser in P'.sub.2 Protein kinase
A RRRR IIFI .sup.(1) RRRFSIIII RRFR III SEQ. ID. NO: 10 SEQ. ID.
No: 11 Protein kinase C RRRKFSFRRK.sup.(5) RRRKFSLRRKA SEQ. ID. NO:
CaMK I LRRRLSDSNL .sup.(6) LRRRFSASNL SEQ. ID. NO: 13 CaMK II
KRQQSFDLF .sup.(2) KRQFSIDLK KRFQ IDLK SEQ. ID. NO: 14 SEQ. ID. NO:
15 Casein kinase I F TG IIFF .sup.(2) GDQ Y DK GDQ YL DK SEQ. ID.
NO: 16 SEQ. ID. NO: 17 Casein kinase II EDEESEDEE .sup.(2)
EDEFSEDEE EDFESEDEE SEQ. ID. NO: 18 SEQ. ID. NO: 19 CycA/cdk2 HHHR
PRKR .sup.(1) HHHFSPRKR HHFR PRKR SEQ. ID. NO: 20 SEQ. ID. NO: 21
CycB/cdc2 HHHKSPRRR .sup.(1) HHHFSPRRR HHFKSPRRR SEQ. ID. NO: 22
SEQ. ID. NO: 23 ERK RVDEPDSPGEK.sup.(4) RVDEPFSPGEK SEQ. ID. NO: 24
Glycogen Synthase PRPASVPP (6) PRPFSVPP SEQ. ID. NO: 25 SLKI RRFG
LRRF .sup.(1) RRRFSLRRI RRFG LRRI SEQ. ID. NO: 26 SEQ. ID. NO: 27
SRPK2 RRRH RRRR .sup.(3) RRRF RRRR RRFH RRRR SEQ. ID. NO: 28 SEQ.
ID. NO: 29
[0080] In Table 3, bold residues indicate those considered to be
significant in kinase recognition, and italicized residues are
those that can be substituted to enable effective modulation of the
proteolytic sensitivity of the optical probe towards chymotrypsin
upon phosphorylation. The serine that is phosphorylated is
underlined, and the indicated references are (1) Songyang, et al.,
Current Biology 4: 973-983, (1994), (2) Songyang, et al., Mol.
Cell. Biol. 16: 6486-6493, (1996), (3) Wang, et al., J. Cell Biol.
140: 737-750, (1998), and (4) Gonzalez et al., J. Biol. Chem., 266:
22159-22163, (1991). (5) Nishikawa et al., J. Biol. Chem. 272:
952-960 (1997), (6) Kemp and Pearson Meth. Enzymology 200: 121-155
(1991).
[0081] Optical probes for detecting protein serine or threonine
phosphatase activity according to the present invention are
designed by incorporating the desired phosphorylation motif into a
peptide, for example like those in (Table 3), or other such motifs
developed now or in the future, and either enzymatically or
chemically phosphorylating the appropriate amino acid.
Dephosphorylation of the serine or threonine residue in such an
optical probe by a serine or threonine directed protein phosphatase
activity modulates the rate of optical probe hydrolysis by
chymotrypsin compared to the phosphorylated optical probe.
B Design of Peptides for Measuring Protein Prenylation.
[0082] Protein prenylation typically occurs through the addition of
isoprenyl groups to cysteine residues located near the C-terminus
of proteins. Typically the linkage of the isoprenyl moiety to
cysteine occurs through the formation of a thioether with the
cysteine sulfhydral. After the creation of the thioether
intermediate, the modified protein may undergo proteolytic
processing and cleavage to produce a product in which the cysteine
is the C-terminal amino acid. A number of different isoprenylation
activities have been identified that recognize distinct recognition
motifs, as shown in Table 4.
TABLE-US-00004 TABLE 4 Examples of proteins Description of Activity
Recognition motif modified Farnesyl transferase.sup.(1) . . . CAAX
Lamins, p21.sup.ras Type 1 geranylgeranyl . . . CAAL Smgp21B,
G-protein transferase.sup.(1) .gamma.-subunit Type 2 geranylgeranyl
. . . CXC or XCC Rab 3A transferase.sup.(2)
[0083] (1) Clarke, S. (1992) Protein isoprenylation and methylation
at carboxyl-terminal cysteine residues. Ann. Rev. Biochem. 61:
355-386, (2) Kawata et al., Post-translationally processed
structure of the human platelet protein smg p21B: Evidence for
geranylgeranylation and carboxyl methylation of the C-terminal
cysteine. Proc. Natl. Acad. Sci. 87: 8960-8964 (1997). Optical
probes for detecting protein prenylation activity according to the
present invention are designed by incorporating the desired
prenylation motif into a peptide, for example like those in (Table
4), or other such motifs developed now or in the future, usually
within three amino acids of the C-terminus. Additional amino acids
may be incorporated N-terminal to the cysteine reside that is
modified to enable subsequent coupling of a first probe moiety,
provided that they do not introduce additional prenylation sites.
Under these circumstances, prenylation of the optical probe results
in an increase in the rate of cleavage of the substrate upon
exposure to an isoprenylated protein-specific endoprotease. Such a
protease activity results in the cleavage of the substrate between
the modified amino acid and the adjacent amino acid to liberate an
intact tripeptide, and a new substrate with a C-terminal modified
cysteine residue that results in a measurable change in at least
one fluorescent property of the optical probe. Such a change can be
used to measure protein prenylation activity as described in the
section entitled "Assays using optical probes".
C Design of Peptides for Measuring Protein Glycosylation
[0084] In general, oligosaccharides may be either N-linked or
O-linked to a protein or peptide. In the case of N-linked
oligosaccharides, an N-acetylglucosamine residue is typically
coupled to an asparagine residue. In the case of O-linked
oligosaccharides, N-acetylgalactosamine is typically coupled to a
serine or threonine residue. Optical probes for detecting N-linked
protein glycosylation activity according to the present invention
are designed by incorporating the desired glycosylation motif into
a peptide, for example like those in (Table 5), or other such
motifs developed now or in the future.
TABLE-US-00005 TABLE 5 Glycosylation Activity Consensus Sequence
motif N-glycosylation.sup.(1) NXT or NXS O-Glycosylation.sup.(2)
X.sub.1TPX.sub.2P in preferred sequences X.sub.1 = uncharged, and
X.sub.2 = small amino acids .sup.(1)Gooley et al., (1991) Biochem.
Biophys. Res. Comm. 178: (3) 1194-201; and .sup.(2)Yoshida et al.,
(1997) J. Biol. Chem. 272: (27) 16884-8.
[0085] To provide the required modulation of proteolytic
sensitivity of the substrate upon glycosylation the peptide should
contain only one asparagine residue and no other basic amino acids
such as lysine, arginine, histidine or glutamine residues. Under
these conditions, the rate of cleavage of the substrate by trypsin
is modulated by N-linked glycosylation of the asparagine residue in
the substrate, which can be coupled to an optical readout using the
methods described herein for example in the section entitled
"Assays using optical probes".
Choice of Protease
[0086] Generally proteases for use in the present invention
typically have the following characteristics: They are commonly
available at high purity, are substantially stable, and recognize a
substrate recognition motif that comprises at least one position in
which the presence, or absence, of a post-translationally modified
residue modulates the activity of the protease towards that
substrate.
[0087] Preferred substrates possess well defined protease sites,
and exhibit a significant modulation e.g. at least 2 fold, or more
preferably at least 5 fold modulation of activity towards a
post-translationally modified residue compared to a non-modified
residue.
A Choice of Protease for Measuring Protein Phosphorylation
[0088] Proteases that may be used to measure peptide
phosphorylation or dephosphorylation include those that recognize a
substrate recognition motif that comprises at least one position in
which the presence or absence of a phosphorylated residue modulates
the activity of the protease towards that substrate. For example
like those in (Table 6), or other such proteases developed now or
in the future.
TABLE-US-00006 TABLE 6 Peptide bond Primary Name EC number Type
cleaved Specificity Caspase 3 Cysteine DXXD-P'.sub.1 P.sub.1 = Asp,
P'.sub.1 = neutral preferred Cathepsin G EC 3.4.21.20 Serine
P.sub.1-P'.sub.1 P.sub.1 = aromatic preferred, W, Y, F Chymotrypsin
EC 3.4.21.1 Serine P.sub.1-P'.sub.1 P.sub.1 = aromatic preferred,
W, Y, F Elastase EC 3.4.21.36 Serine P.sub.1-P'.sub.1 P.sub.1 =
uncharged, non aromatic, e.g. A, V, L, I, G, S, T P'.sub.1 =
non-specific Endoproteinase Unknown P.sub.1-Asp P'.sub.1 = Asp or
P'.sub.1 = Cysteic acid Asp-N P.sub.1 = non-specific Endoproteinase
EC 3.4.21.9 Serine Glu-P'.sub.1 P.sub.1 = Glu or Asp Glu-N P'.sub.1
= non-specific Streptomyces EC 3.4.21.82 Serine Glu-P'.sub.1
P.sub.1 = Glu or Asp griseus P'.sub.1 = non-specific GluSGP
Staphylococcus EC 3.4.21.19 Serine Glu-P'.sub.1 P.sub.1 = Glu or
Asp aureus V8 P'.sub.1 = non-specific
[0089] The flexibility in choice of phosphorylated amino acid
(tyrosine, serine or threonine) combined with the flexibility in
choice of the protease enables virtually any protein kinase or
phosphatase activity to be measured using the present invention. It
should be further noted that the above examples are illustrative of
peptides that could be used to develop optical probes as described
herein. Many other alternative substrates for a specific
post-translational modification are possible by virtue of the
inherent flexibility in the approach.
[0090] A contemplated version of the method is to use inducible
controlling nucleotide sequences to produce a sudden increase in
the expression of the protease within a cell, for the development
of a cell based assay. An appropriate optical property would
typically be monitored at one or more time intervals after the
onset of increased expression of the protease.
B Choice of Protease for Measuring Protein Prenylation.
[0091] In the case of protein prenylation, proteases that exhibit
modulated rates of cleavage of prenylated compared to
non-prenylated substrates are preferred. For example, the yeast a
factor maturation enzyme Ste24p Tam et al., (1998) Dual roles for
Ste24p in yeast .alpha.factor maturation; NH2-terminal proteolysis
and COOH-terminal CAAX processing. J. Biol. Chem. 142(3) 635-49;
and the isoprenylated protein endoprotease; Ma et al., (1992)
Substrate specificity of the isoprenylated protein endoprotease.
Biochemistry 31 (47) 11772-7, or other such proteases developed now
or in the future.
C Choice of Protease for Measuring Protein Glycosylation
[0092] Preferred proteases for use in the present invention to
measure N-linked glycosylation include enzymes that primarily
recognize basic amino acids that can be modified by either
enzymatic or non-enzymatic glycosylation reactions to create
modified substrates with modulated rates of cleavage compared to
non-modified substrates. For example, bovine trypsin, porcine
trypsin and pineapple bromelian or other such proteases developed
now or in the future. (see, Casey and Lang (1976) Tryptic
hydrolysis at asparagine residues in globulin chains. Biochim.
Biophys. Acta 434: 184-8; Loh and Gainer, (1980) Evidence that
glycosylation of pro-opiocortin and A CTH influences their
proteolysis by trypsin and blood proteases. Mol. Cell. Endocrinol.
(1) 35-44; Gil et al., (1991), Effect of non-enzymatic
glycosylation on reactivity in proteolysis. Acta Cient Venez 42:
(1) 16-23.)
Choice of Probe Moieties
[0093] The choice of the probe moiety is governed by a number of
factors including, the type of measurements being made, the
availability of specific instrumentation and the ease of coupling
of the probe moiety to the peptide. Additionally, other factors
that are specific to a particular application are also relevant and
include, the effect of labeling on the solubility of the peptide,
kinetics of the optical probe with respect to the
post-translational activity or protease, and the required detection
sensitivity of the assay. Fortunately numerous probe moieties are
commercially available or can be readily made so that availability
of probe moieties to meet a desired situation is not limiting.
[0094] For fluorescent probes, preferred fluorophores typically
exhibit good quantum yields, lifetimes, and extinction
coefficients, are resistant to collisional quenching and bleaching,
and should preferably be easily conjugated to the ligand.
Particularly desirable, are fluorophores that show absorbance and
emission in the red and near infrared range, which are useful in
whole animal studies, because of reduced scattering background
fluorescence, and greater transmission through tissues. Examples of
such moieties include cyanines, oxazines, thiazines, porphyrins,
phthalocyanines, fluorescent infrared-emitting polynuclear aromatic
hydrocarbons such as violanthrones, fluorescent proteins, near IR
squaraine dyes. (For example as shown in Dyes and Pigments, 17
19-27 (1991), U.S. Pat. No. 5,631,169 to Lakowicz et al., issued
May 20, 1997, and organo-metallic complexes such as the ruthenium
and lanthanide complexes of U.S. Pat. Nos. 4,745,076 and 4,670,572,
the disclosures of which are incorporated herein by reference). The
lanthanide complexes have the advantage of not being quenched by
oxygen, and the long lifetimes may allow easy suppression of the
autofluorescence of biological samples. Specific materials include
fluoroscein isothicyanate (especially
fluorescein-5-isothiocyanate), dichlorotriazinylaminofluorescein,
tetramethylrhodamine-5 (and -6)-isothiocyanate,
1,3-bis-(2-dialkylamino-5-thienyl)-substituted squarines, and the
succinimidyl esters of: 5 (and 6) carboxyfluoroscein; 5 (and
6)-carboxytetramethylrhodamine; and
7-amino-4-methylcoumarin-3-acetic acid. Semiconductor fluorescent
nanocrystals are available with a range of emission spectra, are
highly fluorescent and are also preferred, (see Bruchez et al.,
Science 281: 2013-2016).
[0095] Preferred luminescent probes include chemi-luminescent,
electro-luminescent and bioluminescent compounds. Preferred
bioluminescent compounds include bioluminescent proteins such as
firefly, bacterial or click beetle luciferases, aequorins and other
photoproteins, for example as described in U.S. Pat. Nos.
5,221,623, issued Jun. 22, 1989 to Thompson et al., and 5,683,888
issued Nov. 4, 1997 to Campbell, 5,674,713 issued Sep. 7, 1997 to
DeLuca et al., 5,650,289 issued Jul. 22, 1997 to Wood and U.S. Pat.
No. 5,843,746 issued Dec. 1, 1998 to Tatsumi et al. Preferred
electro-luminescent probes include ruthenium complexes, as for
example described in U.S. Pat. No. 5,597,910 issued to Jan. 28,
1997 to Gudibande. Preferred chemi-luminescent substrates include
those based on 1,2-dioxetanes, as for example described in U.S.
Pat. Nos. 4,372,745 issued Feb. 8, 1983 to Mandle et al., 5,656,207
issued Aug. 12, 1997 to Woodhead et al., and 5,800,999 issued Sep.
1, 1998 issued to Bronstein et al.
[0096] Preferred probes for use as NMR contrast agents include
chelates of paramagnetic, ferromagnetic or diamagnetic metal ions
complexed to lipophilic complexes as described in U.S. Pat. Nos.
5,628,982, issued May 13, 1997 to Lauffer et al. and U.S. Pat. No.
5,242,681, issued Sep. 7, 1993 to Elgavish et al., and fluorine-18-
and 19 containing compounds J. Nucl. Med. 39 1884-91 (1998).
[0097] In some applications it may be desirable to derivatize the
compounds above to render them more hydrophobic and permeable
through cell membranes. The derivatizing groups should undergo
hydrolysis inside cells to regenerate the compounds thus trapping
them within cells. For this purpose, it is preferred that any
phenolic hydroxyls or free amines in the dye structures are
acylated with C.sub.1-C.sub.4 acyl groups (e.g. formyl, acetyl,
n-butryl) or converted to various esters and carbonates, as
described in Bundgaard, H., Design of Prodrugs, Elsevier Science
Publishers (1985), Chapter 1, page 3 et seq., Further modification
of the fluorescent moieties may also be accomplished, as required
as described in U.S. Pat. No. 5,741,657 issued Apr. 21, 1998 to
Tsien et al.
[0098] The probe may be attached to the polypeptide by a linker
that provides a spacer between the probe and the peptide thereby
preventing sterric interference of the probe on the interaction
between the recognition motif and the post-translational-type
activity. Preferred spacers are substantially stable under cellular
conditions and easily coupled to the peptide and probe. Preferred
examples include flexible aliphatic linkers such as .gamma.-amino
n-butyric acid (GABA), diaminopentane, and aminohexanoyl as well as
rigid aromatic linkers. Such linkers are known in the art and
described for example in the Handbook of Fluorescent Probes and
Research Chemicals, by Richard Haugland, published by Molecular
Probes.
[0099] Additionally non-covalent methods of attachment may also be
used to label the peptide moiety. For example, the peptide may be
designed to encompass a specific binding site for a fluorescent
moiety as described in the pending U.S. patent applications,
identified by Ser. No. 08/955,050, filed Oct. 21, 1997, entitled
Methods of using synthetic molecules and target sequences; Ser. No.
08/955,859, filed Oct. 21, 1997, entitled Synthetic molecules that
specifically react with target sequences, and Ser. No. 08/955,206,
filed Oct. 21, 1997, entitled Target sequences for synthetic
molecules. Labeling may then be achieved by incubation of the
peptide with the membrane permeate fluorescent binding partner,
which has the advantages of enabling the expression of peptides
within intact living cells, and the subsequent labeling of these
peptides in situ to create optical probes within intact living
cells.
Fluorescent Proteins
[0100] For some cell based applications, preferred fluorescent
moieties include endogenously fluorescent proteins, functional
engineered fluorescent proteins, and homologs thereof. Because the
entire fluorophore and peptide can be expressed within intact
living cells without the addition of other co-factors or
fluorophores, such optical probes provide the ability to monitor
post-translational activities within defined cell populations,
tissues or an entire transgenic organism. For example by the use of
inducible controlling nucleotide sequences to produce a sudden
increase in the expression of the optical probe and suitable
protease. Endogenously fluorescent proteins have been isolated and
cloned from a number of marine species including the sea pansies
Renilla reniformis, R. kollikeri and R. mullerei and from the sea
pens Ptilosarcus, Stylatula and Acanthoptilum, as well as from the
Pacific Northwest jellyfish, Aequorea victoria; Szent-Gyorgyi et
al. (SPIE conference 1999), D. C. Prasher et al., Gene, 111:229-233
(1992). These proteins are capable of forming a highly fluorescent,
intrinsic chromophore through the cyclization and oxidation of
internal amino acids within the protein that can be spectrally
resolved from weakly fluorescent amino acids such as tryptophan and
tyrosine.
[0101] Additionally fluorescent proteins have also been observed in
other organisms, although in most cases these require the addition
of some exogenous factor to enable fluorescence development. For
example, the cloning and expression of yellow fluorescent protein
from Vibrio fischeri strain Y-1 has been described by T. O. Baldwin
et al., Biochemistry (1990) 29:5509-15. This protein requires
flavins as fluorescent co-factors. The cloning of
Peridinin-chlorophyll .alpha. binding protein from the
dinoflagellate Symbiodinium sp. was described by B. J. Morris et
al., Plant Molecular Biology, (1994) 24:673:77. One useful aspect
of this protein is that it fluoresces in red. The cloning of
phycobiliproteins from marine cyanobacteria such as Synechococcus,
e.g., phycoerythrin and phycocyanin, is described in S. M. Wilbanks
et al., J. Biol. Chem. (1993) 268:1226-35. These proteins require
phycobilins as fluorescent co-factors, whose insertion into the
proteins involves auxiliary enzymes. The proteins fluoresce at
yellow to red wavelengths.
[0102] A variety of mutants of the GFP from Aequorea victoria have
been created that have distinct spectral properties, improved
brightness and enhanced expression and folding in mammalian cells
compared to the native GFP, Table 7, (Green Fluorescent Proteins,
Chapter 2, pages 19 to 47, edited Sullivan and Kay, Academic Press,
U.S. Pat. Nos. 5,625,048 to Tsien et al., issued Apr. 29, 1997;
5,777,079 to Tsien et al., issued Jul. 7, 1998; and U.S. Pat. No.
5,804,387 to Cormack et al., issued Sep. 8, 1998). In many cases
these functional engineered fluorescent proteins have superior
spectral properties to wild-type Aequorea GFP and are preferred for
use in the optical probes of the invention.
TABLE-US-00007 TABLE 7 Quantum Yield (.PHI.) & Relative
Sensitivity To Common Molar Excitation & Fluorescence Low pH
Mutations Name Extinction (.epsilon.) Emission Max At 37.degree. C.
% max F at pH 6 S65T type S65T, S72A, N149K, Emerald .PHI. = 0.68
487 100 91 M153T, I167T .epsilon. = 57,500 509 F64L, S65T, V163A
.PHI. = 0.58 488 54 43 .epsilon. = 42,000 511 F64L, S65T (EGFP)
EGFP .PHI. = 0.60 488 20 57 .epsilon. = 55,900 507 S65T .PHI. =
0.64 489 12 56 .epsilon. = 52,000 511 Y66H type F64L, Y66H, Y145F,
P4-3E .PHI. = 0.27 384 100 N.D. V163A .epsilon. = 22,000 448 F64L,
Y66H, Y145F .PHI. = 0.26 383 82 57 .epsilon. = 26,300 447 Y66H,
Y145F P4-3 .PHI. = 0.3 382 51 64 .epsilon. = 22,300 446 Y66H BFP
.PHI. = 0.24 384 15 59 .epsilon. = 21,000 448 Y66W type S65A, Y66W,
S72A W1C .PHI. = 0.39 435 100 82 N146I, M153T, .epsilon. = 21,200
495 V163A F64L, S65T, Y66W, W1B .PHI. = 0.4 434 452 80 71 N146I,
M153T, .epsilon. = 32,500 476 (505) V163A Y66W, N146I, hW7 .PHI. =
0.42 434 452 61 88 M153T, V163A .epsilon. = 23,900 476 (505) Y66W
436 N.D. N.D. 485 T203Y type S65G, S72A, K79R, Topaz .PHI. = 0.60
514 100 14 T203Y .epsilon. = 94,500 527 S65G, V68L, S72A 10C .PHI.
= 0.61 514 58 21 T203Y .epsilon. = 83,400 527 S65G, V68L, Q69K
h10C+ .PHI. = 0.71 516 50 54 S72A, T203Y .epsilon. = 62,000 529
S65G, S72A, .PHI. = 0.78 508 12 30 T203H .epsilon. = 48,500 518
S65G, S72A .PHI. = 0.70 512 6 28 T203F .epsilon. = 65,500 522 T203I
type T203I, S72A, Sapphire .PHI. = 0.64 395 100 90 Y145F .epsilon.
= 29,000 511 T203I H9 .PHI. = 0.6 395 13 80 T202F .epsilon. =
20,000 511
Cell Based Assays
[0103] Recombinant production of optical probes within living cells
involves expressing nucleic acids having sequences that encode the
fluorescent protein and substrate peptide as a fusion protein. In
one embodiment described below, the optical probe comprises a first
fluorescent protein, a peptide containing a post-translational
modification recognition motif and a protease site, and a second
fluorescent protein fused together as a single polypeptide chain.
Nucleic acids encoding fluorescent proteins can be obtained by
methods known in the art. For example, a nucleic acid encoding the
protein can be isolated by polymerase chain reaction of cDNA from a
suitable organism using primers based on the DNA sequence of the
fluorescent protein. PCR methods are described in, for example,
U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor
Symp. Quant. Biol. 51:263; and Erlich, ed., PCR Technology,
(Stockton Press, NY, 1989).
[0104] Suitable clones expressing the optical probes of the
invention may then be identified, isolated and characterized by
fluorescence activated cell sorting (FACS) typically enabling the
analysis of a few thousand cells per second.
[0105] The construction of expression vectors and the expression of
genes in transfected cells involve the use of molecular cloning
techniques also well known in the art. Sambrook et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., (1989) and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (most recent Supplement). Nucleic acids used to
transfect cells with sequences coding for expression of the
polypeptide of interest generally will be in the form of an
expression vector including expression control sequences
operatively linked to a nucleotide sequence coding for expression
of the polypeptide comprising the optical probe.
Methods of Measurement
[0106] Methods that are preferred with the present invention
include, fluorescence spectroscopy, luminescence spectroscopy,
absorption spectroscopy and magnetic detection
[0107] Fluorescent methods that are preferred with the present
invention include, continuous or time resolved fluorescence
spectroscopy, fluorescence correlation spectroscopy, fluorescence
polarization spectroscopy, and resonance energy based fluorescence
spectroscopy. Methods of performing such assays on fluorescent
materials are well known in the art and are described in, e.g.,
Lakowicz, J. R., Topics in Fluorescence Spectroscopy, volumes 1 to
3, New York: Plenum Press (1991); Herman, B., Resonance energy
transfer microscopy, in: Fluorescence Microscopy of Living Cells in
Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D.
L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.
219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Park:
Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361. The
selection and use of specific fluorophores or quenchers for
particular applications is known in the art, for example see,
Berlman, I. B. Energy transfer parameters of aromatic compounds,
Academic Press, New York and London (1973), that contains tables of
spectral overlap integrals for the selection of resonance energy
transfer partners. Additional information sources include the
Molecular Probes Catalog, 1999; and Tsien et al., 1990 Handbook of
Biological Confocal Microscopy pp. 169-178.
Assays Using Optical Probes
[0108] Methods for determining whether a sample has an activity
typically involve contacting the sample with an optical probe,
incubating the mixture under conditions to enable post
translational modification of the substrate, and then adding a
protease. Finally the degree of post-translational type activity in
the sample is detected by determining at least one optical property
of the optical probe or product thereof. In some cases, the optical
probe and the protease may be added to a sample at the same time.
Alternatively in the case where the sample contains cells, the
method would typically involve stimulation of the cells, and then
either lyzing the cells in the presence of the substrate, or in the
case where the substrate is expressed within the cells, lyzing the
cells in the presence of a protease to measure substrate
modification. The method used to determine the degree of
post-translational type activity is dependent on the assay format
used.
[0109] In one aspect, the method may be based on the difference in
fluorescence anisotropy of the optical probe before and after
cleavage with a protease. In this case the optical probe typically
comprises a polypeptide moiety, which contains a recognition motif
for a post translational type activity and a protease site, which
is coupled to a fluorescent moiety (FIG. 1). Modification of the
polypeptide, by the post translational type activity, results in a
modulation of the rate at which a protease cleaves the polypeptide
which is sensed by a measurable change in fluorescence polarization
of the optical probe upon cleavage.
[0110] Polarization measurements are based on the relative
rotational movement of the fluorophore compared to the excited
state life-time of that fluorophore. For globular molecules in
dilute solution, the relationship between polarization (p) and the
degree of rotational movement can be readily derived (see Weber,
Polarization of the fluorescence of solutions, in Fluorescence and
Phosphorescence Analysis, Don Hercules (ed.), Interscience
Publishers New York. Chapter 8, pages 217-240 (1966)). Rotational
movement can be related to the rotational diffusion constant of the
molecule, and hence to the molecular volume. In practice there is a
close correlation between the molecular size and relative
polarization of emitted light from a fluorophore. As a consequence,
a significant change in fluorescence polarization can occur when
the optical probes of the present invention are acted upon by a
protease. Polarization based measurements are relatively easy to
set up, and can be obtained over a wide concentration, temperature,
and ionic strength range.
[0111] In one embodiment of this method, fluorescence anisotropy
measurements may be enhanced by attaching one end of the peptide to
a solid matrix, or a bead. In either case, cleavage of the optical
probe results in a larger drop in fluorescence polarization because
of the increased rotational flexibility of the optical probe once
separated from the solid matrix or bead.
[0112] In another aspect, the present invention takes advantage of
resonance energy transfer either between two fluorescent moieties
(FRET), or a bioluminescent moiety and fluorescent moiety
(bioluminescent resonance energy transfer, BRET), or a fluorescent
moiety and a quencher (resonance energy transfer, RET) to provide a
fluorescent readout.
[0113] In FRET applications, the optical probe typically comprises
a first fluorescent moiety and a second fluorescent moiety coupled
to the polypeptide such that the recognition motif and the protease
site are located between them (FIG. 1). In this case, cleavage of
the polypeptide by a protease results in an alteration in energy
transfer between the first fluorescent moiety and the second
fluorescent moiety that may be used to determine post-translational
activity. In this case, the fluorescent moieties are typically
chosen such that the excitation spectrum of one of the moieties
(the acceptor fluorescent moiety) overlaps with the emission
spectrum of the donor fluorescent moiety. The donor fluorescent
moiety is excited by light of appropriate intensity within the
donor fluorescent moiety's excitation spectrum and under conditions
in which direct excitation of the acceptor fluorophore is
minimized. The donor fluorescent moiety then transfers the absorbed
energy by non radiative means to the acceptor, which subsequently
re-emits some of the absorbed energy as fluorescence emission, at a
characteristic wavelength. FRET can be manifested as a reduction in
the intensity of the fluorescent signal from the donor, reduction
in the lifetime of its excited state, and an increase in emission
of fluorescence from the acceptor fluorescent moiety. When the
peptide substrate that connects the donor fluorescent moiety and
acceptor fluorescent moiety is cleaved, the donor fluorescent
moiety and the acceptor fluorescent moiety physically separate, and
FRET is diminished or eliminated. Under these circumstances,
fluorescence emission from the donor increases and fluorescence
emission from the acceptor decreases.
[0114] The efficiency of FRET is dependent on the separation
distance and the orientation of the donor fluorescent moiety and
acceptor fluorescent moiety, the fluorescent quantum yield of the
donor moiety and the energetic overlap with the acceptor moiety.
Forster derived the relationship:
E=(F.sup.0-F)/F.sup.0=R.sub.0.sup.6/(R.sup.6+R.sub.0.sup.6)
where E is the efficiency of FRET, F and F.sup.0 are the
fluorescence intensities of the donor in the presence and absence
of the acceptor, respectively, and R is the distance between the
donor and the acceptor. R.sub.0, the distance at which the energy
transfer efficiency is 50%, of maximum is given (in .ANG.) by
R.sub.0=9.79.times.10.sup.3(K.sup.2QJn.sup.-4).sup.1/6
where K.sup.2 is an orientation factor having an average value
close to 0.67 for freely mobile donors and acceptors, Q is the
quantum yield of the unquenched fluorescent donor, n is the
refractive index of the intervening medium, and J is the overlap
integral, which expresses in quantitative terms the degree of
spectral overlap,
J=.intg..sup.oo.sub.0.epsilon..lamda.F.sub..lamda..lamda..sup.4d.lamda./-
.intg..sup.oo.sub.0F.sub..lamda.d.lamda.
where .epsilon..sub..lamda. is the molar absorptivity of the
acceptor in M.sup.-1 cm.sup.-1 and F.sub..lamda. is the donor
fluorescence at wavelength .lamda. measured in cm. Forster, T.
(1948) Ann. Physik 2: 55-75. The characteristic distance R.sub.0 at
which FRET is 50% efficient depends on the quantum yield of the
donor, the extinction coefficient of the acceptor, the overlap
between the donor's emission spectrum and the acceptor's excitation
spectrum and the orientation factor between the two
fluorophores.
[0115] Preferably, changes in the degree of FRET are determined as
a function of the change in the ratio of the amount of fluorescence
from the donor and acceptor moieties, a process referred to as
"ratioing." By calculating the ratio, the assay is insensitive to
fluctuations in substrate concentration, photobleaching and
excitation intensity making the assay more robust. This is of
particular importance in automated screening applications where the
quality of the data produced is important for its subsequent
analysis and interpretation.
[0116] A contemplated variation of the above assay is to either
introduce, or express, the optical probe into living eukaryotic or
prokaryotic cells to enable the measurement of intracellular
post-translational activities.
[0117] In one aspect, the method would involve an optical probe
comprising a first fluorescent protein, a peptide containing a
post-translational modification recognition motif and a protease
site, and a second fluorescent protein fused together as a single
polypeptide chain. In this case the first fluorescent protein and
the second fluorescent protein would be selected to enable FRET to
occur as described above. A preferred pair of functional engineered
fluorescent proteins for example being, Topaz (S65G, S72A, K79R,
T203Y) and W1B (F64L, S65T, Y66W, N146I, M153T, V163A) (Table
7).
[0118] In another aspect the method would involve an optical probe
comprising a peptide containing one or more binding sites for a
fluorescent moiety, a post-translational modification recognition
motif and a protease site. For example, the binding site could
comprises a sequence that recognizes a fluorescent moiety as
described in the pending U.S. patent applications, identified by
Ser. No. 08/955,050, filed Oct. 21, 1997, entitled Methods of using
synthetic molecules and target sequences; Ser. No. 08/955,859,
filed Oct. 21, 1997, entitled Synthetic molecules that specifically
react with target sequences, and Ser. No. 08/955,206, filed Oct.
21, 1997, entitled Target sequences for synthetic molecules. In
this case, expression of the peptide comprising the
post-translational recognition motif, protease site and binding
site could be accomplished using genetic means as described above.
The addition of a membrane permeate fluorescent moiety capable of
binding to the binding site would enable the creation, in situ of
an optical probe.
[0119] In both cases, a contemplated version of the method is to
use inducible controlling nucleotide sequences to produce a sudden
increase in the expression of either the optical probe or the
post-translational activity being assayed, e.g., by inducing
expression of the construct. A suitable protease could be expressed
within the cell, or induced, or introduced using a membrane
translocating sequence U.S. Pat. No. 5,807,746, issued Sep. 15 1998
to Lin et al. The efficiency of FRET is typically monitored at one
or more time intervals after the onset of increased expression of
the protease.
[0120] In another aspect the method would involve the introduction
of an optical probe of the present invention into the cell through
the use of a membrane translocating sequence, as described
herein.
[0121] In BRET applications, the optical probe typically comprises
a luminescent moiety and a fluorescent moiety coupled to the
polypeptide such that the recognition motif and the protease site
are located between them (FIG. 1). In this case, cleavage of the
polypeptide by a protease results in an alteration in energy
transfer between the luminescent moiety and the fluorescent moiety
that may be used to determine post-translational type activities.
In this case, the luminescent and fluorescent moieties are
typically chosen such that the emission spectrum of the luminescent
moiety overlaps with the excitation spectrum of the fluorescent
moiety. Because the luminescent moiety provides light through a
chemi-luminescent, electro-luminescent or bioluminescent reaction,
there is no requirement for direct light excitation to create the
excited state in the luminescent moiety. Instead appropriate
substrates, or voltage must be provided to the luminescent moiety,
to create an excited state within the luminescent moiety. In the
case of bioluminescent proteins, such substrates are, generically
referred to as luciferins (for example see U.S. Pat. No. 5,650,289
issued Jul. 22, 1997 to Wood). If BRET occurs, the energy from the
excited state of the luminescent moiety is transferred to the
fluorescent moiety by non radiative means, rather than being
emitted as light from the luminescent moiety. Because the
luminescent and fluorescent moieties emit light at characteristic
wavelengths, the emission ratio of the two can provide a
ratiometric readout as described for FRET based applications. BRET
can be manifested as a reduction in the intensity of the
fluorescent signal from the luminescent moiety, reduction in the
lifetime of its excited state, and an increase in emission of
fluorescence from the fluorescent moiety. When the peptide
substrate that connects the luminescent moiety and fluorescent
moiety is cleaved, the luminescent moiety and the fluorescent
moiety physically separate, and BRET is diminished or eliminated.
Under these circumstances light emission from the luminescent
moiety increases and fluorescence emission from the fluorescent
moiety decreases. The efficiency of BRET is dependent on the same
separation and orientation factors as described above for FRET.
[0122] In RET applications, the optical probe typically comprises a
first fluorescent moiety and a quencher moiety coupled to the
polypeptide such that the recognition motif and the protease site
are located between them (FIG. 1). In this case, cleavage of the
polypeptide by a protease results in an alteration in energy
transfer between the first fluorescent moiety and the quencher
moiety that may be used to determine post-translational activity.
In this case, the fluorescent moiety and the quencher moiety are
typically chosen such that the absorption spectrum of one of the
quencher (the acceptor moiety) overlaps with the emission spectrum
of the donor fluorescent moiety. The donor fluorescent moiety is
excited by light of appropriate intensity within the donor
fluorescent moiety's excitation spectrum. The donor fluorescent
moiety then transfers the absorbed energy by non radiative means to
the quencher, which in this case does not re-emit any of the
absorbed energy as light. RET can be manifested as a reduction in
the intensity of the fluorescent signal from the donor or a
reduction in the lifetime of its excited state. When the peptide
substrate that connects the donor fluorescent moiety and quencher
moiety is cleaved, the donor fluorescent moiety and the quencher
moiety physically separate, and RET is diminished or eliminated.
Under these circumstances fluorescence emission from the
fluorescent moiety increases.
[0123] The post-translational modification assays of the present
invention can be used in drug screening assays to identify
compounds that alter a post translational type activity. In one
embodiment, the assay is performed on a sample in vitro (e.g. in a
sample isolated from a cell, or cell lysate or purified enzyme)
containing the activity. A sample containing a known amount of
activity is mixed with an optical probe of the invention and with a
test chemical. The amount of the activity in the sample is then
determined after addition of a protease as described herein, for
example, by determining at least one optical property of the probe.
Then the optical property of the sample in the presence of the test
chemical is compared with the optical property of the sample in the
absence of the test compound. A difference indicates that the test
compound alters the activity.
[0124] In another embodiment, the ability of a test chemical to
alter a post-translational type activity, in a cell based assay may
be determined. In these assays, cells transfected with an
expression vector encoding an optical probe of the invention, as
described above, are exposed to different amounts of the test
chemical, and the effect on FRET or fluorescence polarization in
each cell can be determined after induction or introduction of a
suitable protease. Typically, as with any method of the present
invention, the difference in FRET or polarization of treated cells
is compared to that of untreated controls.
[0125] Additionally libraries of optical probes can be created by
producing peptides containing a diverse population of amino acid
sequences. Such libraries are useful for the identification and
characterization of novel post-translational type activities that
have unknown or poorly defined substrate specificities.
[0126] As used herein, a "library" refers to a collection
containing at least 5 different members, preferably at least 100
different members and more preferably at least 200 different
members. The amino acid sequences for the peptide will typically be
in the range or 10 to 20 amino acids in length and may be
completely random or biased towards a particular sequence based on
a particular structural motif, for example based on a known
substrate for a particular post-translational activity. In some
instances the library will created genetically and the individual
members expressed in bacterial or a mammalian cells. Suitable
clones expressing the optical probes of the invention may then be
identified, isolated and characterized by fluorescence activated
cell sorting (FACS) typically enabling the analysis of a few
thousand cells per second.
[0127] Alternatively, the peptides may be chemically synthesized
and individual members attached to a solid matrix and arranged
within a two dimensional array.
[0128] Typically, the library will contain variable peptides in
which only a few, e.g., one to ten, amino acid positions are
varied, but in which the probability of substitution is very high.
Typically each member of the optical probe library will contain a
single defined protease site, and a variable post-translational
type recognition motif, such that randomized sequences comply with
the design considerations for the particular post-translational
type activity (described above). In one embodiment the array
includes systematically substituted amino acids attached to a
substrate, as described in U.S. Pat. No. 5,770,456, issued Jun. 23,
1998 to Holmes.
[0129] Screening of the library to identify optimal substrates may
be achieved by incubating the array with a sample containing the
post-translational activity, adding an appropriate protease, and
then detecting at least one optical property from each member of
the library. Those library members that are more efficiently
modified by the post-translational type activity may then be
identified by the degree to which the optical property of each
library member is altered after exposure to the post-translational
activity.
[0130] Alternatively libraries of known recognition motifs may be
created in order to create an activity profile of
post-translational activities in a sample. In this case, screening
of the library is used to characterize the relative
post-translational activities within by incubating the array with a
sample containing the post-translational activities, adding an
appropriate protease, and then detecting at least one optical
property from each member of the library. Those library members
that are more efficiently modified after exposure to the sample may
then be identified by the degree to which the optical property of
each library member is altered after exposure to the sample to
determine the post-translational activities present within the
sample.
A System for Spectroscopic Measurements
[0131] The optical probes of the present invention can be used with
various systems for spectroscopic measurement. In one embodiment,
the system comprises: a reagent for an assay, and a device
comprising at least one plate or container, preferably a multi-well
platform, and a second platform to hold said plate or container for
detecting a signal from a sample. The system can further comprise a
detector, such as a detector appropriate for detecting a signal
from a sample or a plate on in a container as such detectors are
known in the art or are later developed. The system can comprise
multiple plates or containers or multi-well platforms. In this
context, a reagent for an assay includes any reagent useful to
perform biochemical or biological in vitro or in vivo testing
procedures, such as, for example, buffers, co-factors, proteins
such as enzymes or proteases, carbohydrates, lipids, nucleic acids,
active fragments thereof, organic solvents such as DMSO, chemicals,
analytes, therapeutics, compositions, cells, antibodies, ligands,
and the like. In this context, an active fragment is a portion of a
reagent that has substantially the activity of the parent reagent.
The choice of optical probe depends on the type of assay to be
performed. For example, FRET based assays would typically comprise
an optical probe with two fluorophores. Fluorescent polarization
based assays would typically be completed with optical probes
comprising one fluorescent moiety (FIG. 1).
[0132] The optical probes of the present invention are suited for
use with systems and methods that utilize automated and
integratable workstations for identifying modulators, and chemicals
having useful activity. Such systems are described generally in the
art (see, U.S. Pat. Nos. 4,000,976 to Kramer et al. (issued Jan. 4,
1977), 5,104,621 to Pfost et al. (issued Apr. 14, 1992), 5,125,748
to Bjornson et al. (issued Jun. 30, 1992), 5,139,744 to Kowalski
(issued Aug. 18, 1992), 5,206,568 Bjornson et al. (issued Apr. 27,
1993), 5,350,564 to Mazza et al. (Sep. 27, 1994), 5,589,351 to
Harootunian (issued Dec. 31, 1996), and PCT Application Nos: WO
93/20612 to Baxter Deutschland GMBH (published Oct. 14, 1993), WO
96/05488 to McNeil et al. (published Feb. 22, 1996), WO 93/13423 to
Agong et al. (published Jul. 8, 1993) and PCT/US98/09526 to Stylli
et al., filed May 14, 1998.
[0133] Typically, such a system includes: A) a storage and
retrieval module comprising storage locations for storing a
plurality of chemicals in solution in addressable chemical wells, a
chemical well retriever and having programmable selection and
retrieval of the addressable chemical wells and having a storage
capacity for at least 100,000 addressable wells, B) a sample
distribution module comprising a liquid handler to aspirate or
dispense solutions from selected addressable chemical wells, the
chemical distribution module having programmable selection of, and
aspiration from, the selected addressable chemical wells and
programmable dispensation into selected addressable sample wells
(including dispensation into arrays of addressable wells with
different densities of addressable wells per centimeter squared) or
at locations, preferably pre-selected, on a plate, C) a sample
transporter to transport the selected addressable chemical wells to
the sample distribution module and optionally having programmable
control of transport of the selected addressable chemical wells or
locations on a plate (including adaptive routing and parallel
processing), D) a reaction module comprising either a reagent
dispenser to dispense reagents into the selected addressable sample
wells or locations on a plate or a fluorescent detector to detect
chemical reactions in the selected addressable sample wells or
locations on a plate, and a data processing and integration
module.
[0134] The storage and retrieval module, the sample distribution
module, and the reaction module are integrated and programmably
controlled by the data processing and integration module. The
storage and retrieval module, the sample distribution module, the
sample transporter, the reaction module and the data processing and
integration module are operably linked to facilitate rapid
processing of the addressable sample wells or locations on a plate.
Typically, devices of the invention can process at least 100,000
addressable wells or locations on a plate in 24 hours. This type of
system is described in the PCT application WO/98/52047 by Stylli et
al., entitled "Systems and method for rapidly identifying useful
chemicals in liquid samples."
[0135] If desired, each separate module is integrated and
programmably controlled to facilitate the rapid processing of
liquid samples, as well as being operably linked to facilitate the
rapid processing of liquid samples. In one embodiment the invention
provides for a reaction module that is a fluorescence detector to
monitor fluorescence. The fluorescence detector is integrated to
other workstations with the data processing and integration module
and operably linked with the sample transporter. Preferably, the
fluorescence detector is of the type described herein and can be
used for epi-fluorescence. Other fluorescence detectors that are
compatible with the data processing and integration module and the
sample transporter, if operable linkage to the sample transporter
is desired, can be used as known in the art or developed in the
future. For some embodiments of the invention, particularly for
plates with 96, 192, 384 and 864 wells per plate, detectors are
available for integration into the system. Such detectors are
described in U.S. Pat. No. 5,589,351 (Harootunian), U.S. Pat. No.
5,355,215 (Schroeder), U.S. patent application (Ser. No. pending),
entitled "Detector and Screening Device for Ion Channels" filed
Jul. 17, 1998, and PCT patent application WO 93/13423 (Akong).
Alternatively, an entire plate may be "read" using an imager, such
as a Molecular Dynamics Fluor-Imager 595 (Sunnyvale, Calif.).
Multi-well platforms having greater than 864 wells, including 3,456
wells, can also be used in the present invention (see, for example,
the PCT Application PCT/US98/11061, filed Jun. 2, 1998. These
higher density well plates require miniaturized assay volumes that
necessitate the use of highly sensitivity assays that do not
require washing. The present invention provides such assays as
described herein.
[0136] In another embodiment, the system comprises a microvolume
liquid handling system that uses electrokinetic forces to control
the movement of fluids through channels of the system, for example
as described in U.S. Pat. No. 5,800,690 issued Sep. 1, 1998 to Chow
et al., European patent application EP 0 810 438 A2 filed May 5,
1997, by Pelc et al. and PCT application WO 98/00231 filed 24 Jun.
1997 by Parce et al. These systems use "chip" based analysis
systems to provide massively parallel miniaturized analysis. Such
systems are preferred systems of spectroscopic measurements in some
instances that require miniaturized analysis.
[0137] In another embodiment, the system may comprise a two
dimensional array of optical probes dispersed on a substratum, for
example as described in U.S. Pat. Nos. 4,216,245 issued Aug. 5,
1980 to Johnson, 5,721,435 issued Feb. 24, 1998 to Troll, and
5,601,980 issued Feb. 11, 1997 issued to Gordon et al. Such a
system provides the ability to rapidly profile large numbers of
optical probes and or large numbers of samples in a simple,
miniaturized high throughput format.
A Method for Identifying a Chemical, Modulator or a Therapeutic
[0138] The optical probes of the present invention can also be used
for testing a therapeutic for useful therapeutic activity or
toxicological activity. A therapeutic is identified by contacting a
test chemical suspected of having a modulating activity of a
biological process or target with a biological process or target on
a plate or in a container, such as at least one well of a
multi-well platform, that also comprises an optical probe. The test
chemical can be part of a library of test chemicals that is
screened for activity, such as biological activity. The library can
have individual members that are tested individually or in
combination, or the library can be a combination of individual
members. Such libraries can have at least two members, preferably
greater than about 100 members or greater than about 1,000 members,
more preferably greater than about 10,000 members, and most
preferably greater than about 100,000 or 1,000,000 members. After
appropriate incubation of the sample with the optical probe, a
protease is added and at least one optical property (such as FRET
or polarization) of the sample is determined and compared to a
non-treated control. If the sample having the test chemical
exhibits increased or decreased FRET or polarization relative to
that of the control or background levels, then a candidate
modulator has been identified
[0139] The candidate modulator can be further characterized and
monitored for structure, potency, toxicology, and pharmacology
using well-known methods. The structure of a candidate modulator
identified by the invention can be determined or confirmed by
methods known in the art, such as mass spectroscopy. For putative
modulators stored for extended periods of time, the structure,
activity, and potency of the putative modulator can be
confirmed.
[0140] Depending on the system used to identify a candidate
modulator, the candidate modulator will have putative
pharmacological activity. For example, if the candidate modulator
is found to inhibit a protein tyrosine phosphatase involved, for
example in T-cell proliferation in vitro, then the candidate
modulator would have presumptive pharmacological properties as an
immunosuppressant or anti-inflammatory (see, Suthanthiran et al.,
Am. J. Kidney Disease, 28:159-172 (1996)). Such nexuses are known
in the art for several disease states, and more are expected to be
discovered over time. Based on such nexuses, appropriate
confirmatory in vitro and in vivo models of pharmacological
activity, as well as toxicology, can be selected. The optical
probes, and methods of use described herein, enable rapid
pharmacological profiling to assess selectivity and specificity,
and toxicity. This data can subsequently be used to develop new
candidates with improved characteristics.
Bioavailability and Toxicology of Candidate Modulators
[0141] Once identified, candidate modulators can be evaluated for
bioavailability and toxicological effects using known methods (see,
Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk
Assessment, Hemisphere Publishing Corp., Washington (1985); U.S.
Pat. No. 5,196,313 to Culbreth (issued Mar. 23, 1993) and U.S. Pat.
No. 5,567,952 to Benet (issued Oct. 22, 1996). For example,
toxicology of a candidate modulator can be established by
determining in vitro toxicity towards a cell line, such as a
mammalian i.e. human, cell line. Candidate modulators can be
treated with, for example, tissue extracts, such as preparations of
liver, such as microsomal preparations, to determine increased or
decreased toxicological properties of the chemical after being
metabolized by a whole organism. The results of these types of
studies are often predictive of toxicological properties of
chemicals in animals, such as mammals, including humans.
[0142] The toxicological activity can be measured using reporter
genes that are activated during toxicological activity or by cell
lysis (see WO 98/13353, published Apr. 2, 1998). Preferred reporter
genes produce a fluorescent or luminescent translational product
(such as, for example, a Green Fluorescent Protein (see, for
example, U.S. Pat. No. 5,625,048 to Tsien et al., issued Apr. 29,
1998; U.S. Pat. No. 5,777,079 to Tsien et al., issued Jul. 7, 1998;
WO 96/23810 to Tsien, published Aug. 8, 1996; WO 97/28261,
published Aug. 7, 1997; PCT/US97/12410, filed Jul. 16, 1997;
PCT/US97/14595, filed Aug. 15, 1997)) or a translational product
that can produce a fluorescent or luminescent product (such as, for
example, beta-lactamase (see, for example, U.S. Pat. No. 5,741,657
to Tsien, issued Apr. 21, 1998, and WO 96/30540, published Oct. 3,
1996)), such as an enzymatic degradation product. Cell lysis can be
detected in the present invention as a reduction in a fluorescence
signal from at least one photon-producing agent within a cell in
the presence of at least one photon reducing agent. Such
toxicological determinations can be made using prokaryotic or
eukaryotic cells, optionally using toxicological profiling, such as
described in PCT/US94/00583, filed Jan. 21, 1994 (WO 94/17208),
German Patent No 69406772.5-08, issued Nov. 25, 1997; EPC 0680517,
issued Nov. 12, 1994; U.S. Pat. No. 5,589,337, issued Dec. 31,
1996; EPO 651825, issued Jan. 14, 1998; and U.S. Pat. No.
5,585,232, issued Dec. 17, 1996).
[0143] Alternatively, or in addition to these in vitro studies, the
bioavailability and toxicological properties of a candidate
modulator in an animal model, such as mice, rats, rabbits, or
monkeys, can be determined using established methods (see, Lu,
supra (1985); and Creasey, Drug Disposition in Humans, The Basis of
Clinical Pharmacology, Oxford University Press, Oxford (1979),
Osweiler, Toxicology, Williams and Wilkins, Baltimore, Md. (1995),
Yang, Toxicology of Chemical Mixtures; Case Studies, Mechanisms,
and Novel Approaches, Academic Press, Inc., San Diego, Calif.
(1994), Burrell et al., Toxicology of the Immune System; A Human
Approach, Van Nostrand Reinhold, Co. (1997), Niesink et al.,
Toxicology; Principles and Applications, CRC Press, Boca Raton,
Fla. (1996)). Depending on the toxicity, target organ, tissue,
locus, and presumptive mechanism of the candidate modulator, the
skilled artisan would not be burdened to determine appropriate
doses, LD50 values, routes of administration, and regimes that
would be appropriate to determine the toxicological properties of
the candidate modulator. In addition to animal models, human
clinical trials can be performed following established procedures,
such as those set forth by the United States Food and Drug
Administration (USFDA) or equivalents of other governments. These
toxicity studies provide the basis for determining the therapeutic
utility of a candidate modulator in vivo.
Efficacy of Candidate Modulators
[0144] Efficacy of a candidate modulator can be established using
several art-recognized methods, such as in vitro methods, animal
models, or human clinical trials (see, Creasey, supra (1979)).
Recognized in vitro models exist for several diseases or
conditions. For example, the ability of a chemical to extend the
life-span of HIV-infected cells in vitro is recognized as an
acceptable model to identify chemicals expected to be efficacious
to treat HIV infection or AIDS (see, Daluge et al., Antimicro.
Agents Chemother. 41:1082-1093 (1995)). Furthermore, the ability of
cyclosporin A (CsA) to prevent proliferation of T-cells in vitro
has been established as an acceptable model to identify chemicals
expected to be efficacious as immunosuppressants (see, Suthanthiran
et al., supra, (1996)). For nearly every class of therapeutic,
disease, or condition, an acceptable in vitro or animal model is
available. Such models exist, for example, for gastro-intestinal
disorders, cancers, cardiology, neurobiology, and immunology. In
addition, these in vitro methods can use tissue extracts, such as
preparations of liver, such as microsomal preparations, to provide
a reliable indication of the effects of metabolism on the candidate
modulator. Similarly, acceptable animal models may be used to
establish efficacy of chemicals to treat various diseases or
conditions. For example, the rabbit knee is an accepted model for
testing chemicals for efficacy in treating arthritis (see, Shaw and
Lacy, J. Bone Joint Surg. (Br) 55:197-205 (1973)). Hydrocortisone,
which is approved for use in humans to treat arthritis, is
efficacious in this model which confirms the validity of this model
(see, McDonough, Phys. Ther. 62:835-839 (1982)). When choosing an
appropriate model to determine efficacy of a candidate modulator,
the skilled artisan can be guided by the state of the art to choose
an appropriate model, dose, and route of administration, regime,
and endpoint and as such would not be unduly burdened.
[0145] In addition to animal models, human clinical trials can be
used to determine the efficacy of a candidate modulator in humans.
The USFDA, or equivalent governmental agencies, have established
procedures for such studies (see, www.fda.gov).
Selectivity of Candidate Modulators
[0146] The in vitro and in vivo methods described above also
establish the selectivity of a candidate modulator. It is
recognized that chemicals can modulate a wide variety of biological
processes or be selective. Panels of enzymes or panels of cells
based on the present invention, or a combination of both, can be
used to determine the specificity of the candidate modulator.
Selectivity is evident, for example, in the field of chemotherapy,
where the selectivity of a chemical to be toxic towards cancerous
cells, but not towards non-cancerous cells, is obviously desirable.
Selective modulators are preferable because they have fewer side
effects in the clinical setting. The selectivity of a candidate
modulator can be established in vitro by testing the toxicity and
effect of a candidate modulator on a plurality of cell lines that
exhibit a variety of cellular pathways and sensitivities. The data
obtained from these in vitro toxicity studies can be extended into
in vivo animal model studies, including human clinical trials, to
determine toxicity, efficacy, and selectivity of the candidate
modulator suing art-recognized methods.
[0147] For example arrays of kinase or phosphatase optical probes
may be used to rapidly profile the selectivity of a test chemical
with respect to its ability to inhibit related kinases or
phosphatases. Such arrays may be located within a microtiter plate,
or as a printed array, for example as disclosed in U.S. Pat. Nos.
4,216,245 issued Aug. 5, 1980 to Johnson, 5,721,435 issued Feb. 24,
1998 to Troll, and 5,601,980 issued Feb. 11, 1997 issued to Gordon
et al. Such a system provides the ability to rapidly profile large
numbers of kinases or phosphatases in the presence or absence of a
test chemical in order to profile in a simple, miniaturized high
throughput format the selectivity of a candidate modulator.
An Identified Chemical, Modulator, or Therapeutic and
Compositions
[0148] The invention includes compositions, such as novel
chemicals, and therapeutics identified by at least one method of
the present invention as having activity by the operation of
methods, systems or components described herein. Novel chemicals,
as used herein, do not include chemicals already publicly known in
the art as of the filing date of this application. Typically, a
chemical would be identified as having activity from using the
invention and then its structure revealed from a proprietary
database of chemical structures or determined using analytical
techniques such as mass spectroscopy.
[0149] One embodiment of the invention is a chemical with useful
activity, comprising a chemical identified by the method described
above. Such compositions include small organic molecules, nucleic
acids, peptides and other molecules readily synthesized by
techniques available in the art and developed in the future. For
example, the following combinatorial compounds are suitable for
screening: peptoids (PCT Publication No. WO 91/19735, 26 Dec.
1991), encoded peptides (PCT Publication No. WO 93/20242, 14 Oct.
1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan.
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomeres such
as hydantoins, benzodiazepines and dipeptides (Hobbs DeWitt, S. et
al., Proc. Nat. Acad. Sci. USA 90: 6909-6913 (1993)), vinylogous
polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568
(1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose
scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc. 114:
9217-9218 (1992)), analogous organic syntheses of small compound
libraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)),
oligocarbamates (Cho, C. Y. et al., Science 261: 1303 (1993)),
and/or peptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem.
59: 658 (1994)). See, generally, Gordon, E. M. et al., J. Med.
Chem. 37: 1385 (1994). The contents of all of the aforementioned
publications are incorporated herein by reference.
[0150] The present invention also encompasses the identified
compositions in a pharmaceutical composition comprising a
pharmaceutically acceptable carrier prepared for storage and
subsequent administration, which have a pharmaceutically effective
amount of the products disclosed above in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,
stabilizers, dyes and even flavoring agents may be provided in the
pharmaceutical composition. For example, sodium benzoate, acsorbic
acid and esters of p-hydroxybenzoic acid may be added as
preservatives. In addition, antioxidants and suspending agents may
be used.
[0151] The compositions of the present invention may be formulated
and used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions,
suspensions for injectable administration; and the like.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Suitable
excipients are, for example, water, saline, dextrose, mannitol,
lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. If desired, absorption enhancing preparations
(e.g., liposomes), may be utilized.
[0152] The pharmaceutically effective amount of the composition
required as a dose will depend on the route of administration, the
type of animal being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize. In practicing the
methods of the invention, the products or compositions can be used
alone or in combination with one another, or in combination with
other therapeutic or diagnostic agents. These products can be
utilized in vivo, ordinarily in a mammal, preferably in a human, or
in vitro. In employing them in vivo, the products or compositions
can be administered to the mammal in a variety of ways, including
parenterally, intravenously, subcutaneously, intramuscularly,
colonically, rectally, nasally or intraperitoneally, employing a
variety of dosage forms. Such methods may also be applied to
testing chemical activity in vivo.
[0153] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed, and
the specific use for which these compounds are employed. The
determination of effective dosage levels, that is the dosage levels
necessary to achieve the desired result, can be accomplished by one
skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0154] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage for the products of the present
invention can range broadly depending upon the desired affects and
the therapeutic indication. Typically, dosages may be between about
10 mg/kg and 100 mg/kg body weight, and preferably between about
100 .mu.g/kg and 10 mg/kg body weight. Administration is preferably
oral on a daily basis.
[0155] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See e.g., Fingl et al., in The Pharmacological Basis of
Therapeutics, 1975). It should be noted that the attending
physician would know how to and when to terminate, interrupt, or
adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administrated
dose in the management of the disorder of interest will vary with
the severity of the condition to be treated and to the route of
administration. The severity of the condition may, for example, be
evaluated, in part, by standard prognostic evaluation methods.
Further, the dose and perhaps dose frequency, will also vary
according to the age, body weight, and response of the individual
patient. A program comparable to that discussed above may be used
in veterinary medicine.
[0156] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,
Easton, Pa. (1990). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0157] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated.
[0158] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external micro-environment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0159] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the active
ingredients, these pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The
preparations formulated for oral administration may be in the form
of tablets, dragees, capsules, or solutions. The pharmaceutical
compositions of the present invention may be manufactured in a
manner that is itself known, for example, by means of conventional
mixing, dissolving, granulating, dragee-making, levitating,
emulsifying, encapsulating, entrapping, or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared
as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides,
or liposomes. Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents that
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0160] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses. For this purpose,
concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses. Such formulations can be made using methods known
in the art (see, for example, U.S. Pat. Nos. 5,733,888 (injectable
compositions); 5,726,181 (poorly water soluble compounds);
5,707,641 (therapeutically active proteins or peptides); 5,667,809
(lipophilic agents); 5,576,012 (solubilizing polymeric agents);
5,707,615 (anti-viral formulations); 5,683,676 (particulate
medicaments); 5,654,286 (topical formulations); 5,688,529 (oral
suspensions); 5,445,829 (extended release formulations); 5,653,987
(liquid formulations); 5,641,515 (controlled release formulations)
and 5,601,845 (spheroid formulations).
EXAMPLES
[0161] The following examples are offered by way of illustration,
not by way of limitation.
Example 1
Measurement of Tyrosine Kinase Activity Using Optical Probes
[0162] Peptides were prepared by traditional solid-phase synthesis
see, Merrifield, J. Amer. Chem. Soc., 85:2149-2154 (1963); Fields,
G. B., et al., Principles and practice of solid-phase peptide
synthesis, pages 77-183 in Synthetic Peptides: A Users Guide,
Grant, G. R., ed., W. H. Freeman and Co. New York, (1992), in
conjunction with the "tea-bag" methodology using Boc/benzyl based
chemistry. See, Houghten et al., Proc. Natl. Acad. Sci. USA
82:513-5135 (1985). Peptides were assembled on
methylbenzhydrylamine resin (MBHA resin) using traditional
Boc/Benzyl based chemistry. A minor modification to the protocol
(in the case of the abl-specific substrate (AEAIYAAPL, SEQ. I.D.
No. 4) was the use of a base sensitive protecting group (Fmoc) for
the side chain of the C-terminal lysine residue. Bags, made of a
polypropylene mesh material were filled with MBHA resin. The bags
("tea-bags") were placed in a Nalgene.TM. bottle with
dichloromethane (DCM), enough to cover the bags, and shaken 5 min
to allow the swelling of the resin. The DCM solution was then
discarded and the actual synthesis was carried out. (All subsequent
steps involved the addition of enough solvent to cover all the bags
and vigorous shaking to ensure adequate solvent transfer).
[0163] The bags were washed 3 times, first with 5%
diisopropylethylamine (DIEA) in DCM (neutralization step) for 2
minutes, and then twice with 100% DCM (each for one minute) to
remove excess base. After neutralization, the bags were sorted and
placed into a Nalgene.TM. bottle containing a solution of the amino
acid of interest in DCM, an equal amount of diisopropylcarbodiimide
(DIC) in DCM was added to activate the coupling reaction. A 5-fold
excess of amino acid and DIC was used for all of the couplings. The
bottle was shaken for one hour to ensure completion of the
reaction. The reaction mixture was discarded and the packets were
washed in DMF twice for 1 minute to remove excess amino acids and
by-products like diisopropylurea. Two final washes with DCM were
performed to remove any excess DMF. The N-.alpha.-t-Boc was removed
by acidolysis using a solution of 55% TFA in DCM for 30 minutes
leaving the TFA salt of the .alpha.-amino group. The bags were
washed successively with DCM (1.times.1 minute), isopropanol
(2.times.1 minute) and DCM (1.times.1 minute). The synthesis was
completed by repeating the same procedure while substituting for
the corresponding amino acid at the coupling step.
[0164] After removal of the N-.alpha.-t-Boc from the
.gamma.-Amino-n-butyric acid (GABA), the bags were washed 3 times,
2 minutes each, with 5% DIEA in DCM, then with DCM (3.times.2
minutes). The bags were sorted, placed in a Nalgene.TM. bottle
containing a solution of fluorescein isothiocyanate (FITC) in
DCM/DMF (80/20) and shaken for 2 minutes (2-fold excess). Neat DIEA
was then added to the FITC solution. The bottle was shaken for 3
hours to ensure completion of the reaction. The reaction mixture
was discarded and the bags were washed in DCM (4.times.2 minutes)
and DMF (1.times.2 minutes).
[0165] The Fmoc group on the side chain of the C-terminal lysine
residue was removed using a solution of 20% piperidine in DMF for
25 minutes. The bags were washed successively with DMF (2.times.2
minutes), DCM (1.times.2 minutes) and DMF (1.times.2 minutes). Bags
were then placed in a Nalgene.TM. bottle containing a solution of
7-hydroxycoumarin-3-carboxylic acid (1.5-fold excess) in DMF and
shaken for 2 minutes. A solution of PyBop/HOBt in DMF was added to
the bottle and the mixture was shaken for 2 minutes. Neat DIEA was
then added and the mixture was shaken for 2 hours. The reaction
mixture was discarded, the packets were washed with DMF (3.times.2
minutes) and DCM (3.times.2 minutes), and placed in a desiccator
and dried under vacuum in preparation for cleavage.
[0166] All peptides were side chain deprotected and cleaved from
the resin at 0.degree. C. with liquid HF in presence of anisole as
a carbocation scavenger. The reaction was allowed to proceed for 60
minutes. Liquid HF was then removed using a strong flow of N.sub.2
for 90 minutes followed by the use of aspirator vacuum for 60
minutes while maintaining the temperature at 0.degree. C. The
reaction vessels were removed from the apparatus and the residual
anisole was removed with two ethylether washes. The peptides were
extracted with two 30 ml 10% AcOH washes. For each peptide, the
extraction solutions were pooled and lyophilized. The crude
peptides were weighed and stored under nitrogen to await
purification.
Automated Peptide Synthesis.
[0167] Alternatively, fluorescent peptide substrates were made
using an automated peptide synthesizer (ABI 432A, Applied
Biosystems, Foster City, Calif.) using Fmoc/t-Boc chemistry. See
Fields, G. B., et al., Principles and practice of solid-phase
peptide synthesis, pages 77-183 in Synthetic Peptides: A Users
Guide, Grant, G. R., ed., W. H. Freeman and Co. New York, (1992).
Briefly, after the automated peptide synthesis of the desired
peptide (containing an unprotected N-terminal GABA and a Fmoc
protected C-terminal lysine) was complete, the synthesis column was
removed from the ABI432A synthesizer. The column containing the
peptide attached to the resin was manually flushed with DMF to
swell the resin. A two ml solution of 100 .mu.M FITC (5 fold
excess) in 10% DIEA/DMF was then slowly injected through the
synthesis column over a period of 2 hours using a syringe pump. The
synthesis column was washed with (5.times.10 ml) DMF and THF
(3.times.5 ml) and finally dried with a stream of dry nitrogen. The
dried resin was suspended in 1 ml of trifluoroacetic (TFA)
containing 50 .mu.l of ethanedithiol and 50 .mu.l of thioanisole,
this mixture was stirred under nitrogen for 4 hours. The peptide
was precipitated from the TFA solution by the addition of 20 ml of
ether. The solid was further washed with ether (3.times.20 ml) to
remove the thiol scavengers. The precipitated peptide (still mixed
with the cleaved resin) was dried under vacuum. Finally, the
C-terminal lysine of the peptide (in the case of the abl-specific
peptide (AEAIYAAPL, SEQ. ID. NO: 4) was labeled with the
N-hydroxysuccimidyl ester of 7-hydroxycoumarin-3-carboxylic acid
(NHS-coumarin ester). This was accomplished by incubating a 5-fold
excess of the NHS-coumarin ester with the peptide overnight at room
temperature in a solution of DMF containing 10% DIEA. After removal
of the solvent the peptide was purified as described below.
[0168] A fluorescein/rhodamine fluorescent substrate was produced
using an identical procedure to that described above with the
exception that an amine reactive rhodamine fluorophore (Lissamine
rhodamine B sulfonyl chloride) was used to label the C-terminal
lysine. Previous attempts to synthesize peptides with a C-terminal
lysine labeled with rhodamine while the peptides were still
attached to the resin were not successful. The method described
above (labeling with rhodamine after cleavage from the resin)
avoids the problematic tendency of rhodamine labels to bind
irreversibly to the resins. Reaction of amine reactive rhodamine
derivatives while the peptides are still attached to the resin
apparently precludes them from reacting with the C-terminal
lysine.
[0169] The crude peptides were purified by reversed-phase
high-performance liquid chromatography on a C.sub.18 column using
established methods. The mobile phase solvents were 0.1% TFA in
water (Solvent A) and 0.1% TFA in Acetonitrile (Solvent B). The
fractions containing the purified material were pooled and
lyophilized and the purified peptides were characterized by
analytical reverse phase-HPLC and by mass spectral analysis.
Peptide concentrations were determined by absorbance spectroscopy,
using coumarin and fluorescein extinction coefficients of 35,000
and 75,000 M.sup.-1 cm.sup.-1, respectively. Peptides were stable
at 4.degree. C. for at least one month and indefinitely at
-20.degree. C.
Preparation of Phosphorylated Optical Probes.
[0170] To prepare a sample of phosphorylated optical probe, the
peptides were incubated with excess tyrosine kinase activity for a
sufficient time to ensure complete phosphorylation of the peptide.
Typically for v-Abl kinase reactions, the reaction buffer consisted
of: 0.1.times. phosphate buffered saline (PBS), 5 mM MgCl.sub.2,
200 .mu.M ATP and not more than 10% of the total reaction volume of
the tyrosine kinase enzyme. Reaction volumes were typically 20
.mu.L, but were also performed at 10 .mu.L and 100 .mu.L.
Recombinant v-Abl kinase was typically purchased from Calbiochem.
Kinase reactions were quenched by the addition of 20 mM EDTA, pH 8.
The degree of phosphorylation of the peptide was monitored over
time by removing samples of the reaction mixture and analyzing them
by reverse-phase high-performance liquid chromatography.
[0171] Alternatively, phosphorylated optical probes could be
prepared directly during the peptide synthesis by simply using the
O-benzyl protected phosphate derivative of the desired hydroxyl
containing amino acid. For example,
N.alpha.-Fmoc-O-benzyl-L-phosphotyrosine is commercially available
and is compatible with standard Fmoc solid phase peptide synthesis.
See White, P. et al. in "Innovations & Perspectives in Solid
Phase Synthesis and Combinatorial Libraries, 4th International
Symposium", R. Epton (Ed.), Mayflower Scientific Ltd. Birmingham,
(1966), pp 557. Thus, phosphorylated optical probes could be
readily produced using protocols similar to those described above
for automated peptide synthesis using Fmoc chemistry and purified
as described below.
[0172] For example, using a Dionex HPLC apparatus and a C18
reverse-phase column by running a gradient elution profile
consisting of either 5 to 80% acetonitrile/0.1% trifluoroacetic
acid (.about.pH 3) or 5 to 80% acetonitrile/0.1% triethylamine
(.about.pH 7.5) over 25 minutes. Alternatively, the degree of
phosphorylation was determined by mass spectroscopy. Using both
methods, the degree of peptide phosphorylation was typically
greater than 95% after incubation with the kinase. Negative control
peptides were incubated under identical conditions to those for the
phosphorylated peptides, but were incubated in the absence of
ATP.
Fluorescence Changes Upon Cleavage
[0173] To initially test cleavage of the optical probes,
fluorescence emission measurements were made in a cuvette, using a
steady-state fluorimeter (SPEX). In the case of
fluorescein/coumarin labeled peptides, emission spectra between 420
and 600 nm were obtained by excitation at 405 nm, (where coumarin
absorbs maximally and there is little direct excitation of
fluorescein). Typically the concentration of the optical probes was
100 nM, and the total reaction volume was 700 .mu.L.
[0174] FIG. 2 shows that cleavage of the non-phosphorylated optical
probes by chymotrypsin results in a large increase in fluorescence
emission at around 460 nm, and smaller decrease in emission at 530
nm that is caused by the loss of fluorescence resonance energy
transfer (FRET) between the donor (coumarin) and acceptor
(fluorescein). By comparison, the phosphorylated optical probe, is
not degraded by a chymotrypsin, and exhibits almost no change in
emission characteristics at either wavelength upon incubation with
the protease. The substantial 30-fold difference in emission ratios
of phosphorylated (non-cleaved) substrate and non-phosphorylated
(cleaved) substrate provides the basis for one aspect of the
present invention. It should be further noted that since the
emission spectra varies independently at two distinct wavelengths,
it is possible to calculate an emission ratio, which has several
significant advantages compared to single wavelength measurements.
These include greater sensitivity and reproducibility in screening
applications because the ratio is largely independent (within
certain limits) of the absolute light intensity and optical probe
concentration.
[0175] To confirm that the emission ratio is directly related to
the degree of optical probe phosphorylation, mixtures of
phosphorylated and non-phosphorylated peptides were mixed in
defined amounts and diluted to 100 .mu.L with 0.1.times.PBS and
then added to a 96-well multiwell plate. Emission ratios (460/530)
were acquired with a Cytofluor plate reader (Perspective
Biosystems) using a 395 nm excitation filter [full-width
half-maximum (FWHM) of 25 nm] a 460 nm emission filter (FWHM=40 nm)
and a 530 nm emission filter (FWHM=50 nm). Measurements were made
before, and 1 minute after, addition of 0.04 nMol bovine
alpha-chymotrypsin (Calbiochem, 230832, 1,018 USP units/mg), and
the 460/530 emissions ratios calculated. The results, shown in
Table 8, demonstrate a direct relationship between the degree of
optical probe phosphorylation and the 460/530 emission ratio.
TABLE-US-00008 TABLE 8 % of Phosphorylated 460/530 Emission Peptide
Best Fit Ratio Actual Data 0 0.904 0.904 10 2.118 2.086 20 3.331
3.256 30 4.545 4.193 40 5.758 5.440 50 6.972 6.498 60 8.186 7.919
70 9.399 9.332 80 10.613 10.598 90 11.826 11.865 100 13.040
13.044
Optimization of Protease Concentration
[0176] To determine the relative proteolytic sensitivity of the
phosphorylated and non-phosphorylated optical probe (AEAIYAAPL,
SEQ. ID. NO: 4), samples of both were incubated with various
concentrations of chymotrypsin, in 0.1.times.PBS. Fluorescence
measurements were made on a 96-well plate reader as described
previously. In FIG. 3, the open symbols represent the control,
non-phosphorylated optical probe. In this case, cleavage of the
optical probe, as indicated by the 460/530 emission ratio, is
already significant at 10 nM chymotrypsin and reaches a maximum
value of around 12, in the presence of 100 nM protease under these
conditions. By comparison, the phosphorylated optical probe (filled
symbols) does not begin to exhibit a comparable change in emission
ratio until exposed to a 1000-fold higher concentration of protease
(10 .mu.M). These results demonstrate that maximal differences in
emission ratio between phosphorylated and non-phosphorylated
optical probe can occur, in this case, at protease concentrations
between 0.1 to 1 .mu.M chymotrypsin. Under these conditions,
virtually all of the non-phosphorylated optical probe has been
cleaved whereas virtually all of the phosphorylated optical probe
is still intact. Optimal protease incubation conditions for other
specific optical probes can be determined using similar procedures
and protocols.
Example 2
Validation of Optical Probes for Screening for Protein Tyrosine
Kinase Inhibitors
[0177] To validate the invention in a high throughput screening
format, optical probe-based assays were carried out in a 96-well
plate reader. The results demonstrated highly reproducible and
accurate results with the present invention. As shown in Table 9,
the calculation of emission ratios significantly reduces the
standard deviation and C.V. values compared to intensity
measurements at either 460 or 530 nm. The reduction of errors is an
important consideration in the design and analysis of screening
systems, and particularly automated high throughput and ultra-high
through screening systems.
TABLE-US-00009 TABLE 9 Emission Emission 460 nm 530 nm Ratio Mean
1290 1922 0.67 Standard 3.7 4.8 0.01 Deviation C.V. 2.9% 2.5%
1.5%
[0178] Analysis of the kinetics of phosphorylation of the optical
probe revealed values for the apparent Km for the substrate of 40
.mu.M, and an apparent Km for ATP of 8 .mu.M. The turnover of the
optical probe by v-Abl was 9.5 sec.sup.-1, in agreement with
published values for purified tyrosine kinases and optimal peptide
substrates. For example, Seethala and Menzel., Anal Biochem. 253:
210-218 (1997); Songyang et al., Nature 373: 536-539 (1995).
Comparison to Other Methods of Screening.
[0179] To determine how the optical probe-based kinase assay
compared to other screening methods, such as the direct measurement
of .sup.32P-incorporation into a peptide, a direct comparison of
the two methods was completed. Samples of fluorescent substrate (2
.mu.M) were phosphorylated with v-Abl kinase as described above in
either the presence, or absence, of .gamma.-labeled .sup.32P-ATP
(0.5 .mu.M or 10 .mu.Ci per 20 .mu.l reaction.) In the case of the
.sup.32P-incorporation experiments, radioactive incorporation was
determined by the binding of the optical probe to P81 filters as
described previously (e.g., Seethala and Menzel, Anal. Biochem.,
253:210-218, 1997). Abl peptide binding to P81 filters proved to be
able to reliably capture a constant fraction of phosphorylated
peptide. Radioactive incorporation into the optical probe was
monitored using a TopCount liquid scintillation counter (Packard).
Incubation of the optical probe with chymotrypsin (100 nM) and
measurement of fluorescence emission ratios were as described in
Example 1. Results from a typical experiment are shown in Table 10,
and demonstrate that both methods of measuring tyrosine kinase
activity give similar results and reliably indicate the level of
substrate phosphorylation.
TABLE-US-00010 TABLE 10 Concentration % Phosphorylation %
Phosphorylation as of Enzyme as Determined by Determined by
.sup.32P- (ng/assay) Fluorescence Incorporation 0 0.0 0.0 0.5 3.1
4.6 1.5 9.0 12.3 4.0 36.8 42.1 10.0 69.2 65.6 22.0 96.0 95.5 44.0
100.0 100.0
Characterization of a Protein Tyrosine Kinase Inhibitor in a
Screening Assay
[0180] To demonstrate that the present invention can effectively
identify inhibitors of tyrosine kinase activity, a direct
comparison was completed to compare the effect of an inhibitor of
tyrosine phosphorylation on either fluorescence changes after
incubation with chymotrypsin, or .sup.32P-incorporation (FIG. 4).
The results demonstrated almost identical dose dependencies and
inhibition curves for the inhibitor using either method of
measuring tyrosine kinase activity. These experiments therefore
demonstrate that the present invention provides for a sensitive and
convenient system of measuring phosphorylation, and that the
results obtained with the assay system are directly comparable to
those obtained with by measuring .sup.32P-incorporation.
Example 3
Measurement of Other Tyrosine Kinase Activities Using Optical
Probes
Measurement of Src Kinase Activity
[0181] To measure Src kinase activity, two optical probes Src-1
(GEEEIYGEIEK, SEQ. ID. NO: 3) and Src-2 (GEEEIYGVIEK, SEQ. ID. NO:
29) were developed. In the case of the Src-1 kinase substrate, and
as shown in Table 4, a second aromatic amino acid was changed to
isoleucine in the optical probe. In the second substrate, Src-2,
the negatively charged amino acid (Glu=E) in the P'.sub.2 position
with respect to the protease site, was changed to valine (Val=V) to
enable more efficient cleavage of the non-phosphorylated optical
probe by chymotrypsin. Src kinase (Upstate Biotechnology) reaction
conditions were the same as described in Example 1, except 25 mM
glycerol phosphate and 1 mM DTT were also added. Incubation of the
optical probe with chymotrypsin (100 nM) and measurement of
fluorescence emission ratios were as described in Example 1. The
apparent K.sub.ms for the two substrates, (determined by
fluorescence measurements after protease incubation) with respect
to the Src kinase were 11 .mu.M and 19 .mu.M respectively. Other
optical probes, designed as described herein, can be generated to
create specific optical probes for a range of tyrosine kinase
activities that can subsequently be optimized using the methods
described herein.
Example 4
Measurement of Protein Tyrosine Phosphatase Activities Using
Optical Probes
[0182] To demonstrate that the present invention could also be used
to determine protein tyrosine phosphatase activities, experiments
were completed using phosphorylated optical probes incubated with
protein tyrosine phosphatases. Optical probes were first
phosphorylated to completion as described above, and samples of
phosphorylated optical probe, at a final concentration of 500 nM,
were incubated with various concentrations of protein tyrosine
phosphatase-B (PTP-B) agarose (Upstate Biotechnology) for 20
minutes at 30.degree. C. in 0.1.times.PBS. At the required time
interval, PTP-B-agarose was removed by a brief microfuge spin prior
to transfer to 96-well Cytofluor plates for fluorescence
measurements, after addition of chymotrypsin (100 nM), as described
in Example 1.
[0183] Results from a typical experiment are shown in Table 11. In
this experiment, the relative rates of dephosphorylation of the
phosphorylated Src-2-specific substrate (SEQ. ID. NO: 29) and an
abl-specific (SEQ. I.D. No. 4) substrate optical probes by the
protein tyrosine phosphatase PTP-B were compared.
TABLE-US-00011 TABLE 11 Fluorescence Fluorescence emission emission
ratio of Log concentration of ratio of Src-2 (SEQ. ID. abl (SEQ.
ID. NO: 4) protein tyrosine NO: 29) peptide after peptide after
protease phosphatase [PTP-B] protease treatment treatment -7.7 17.7
n.d. -8.7 17.6 17.7 -9.7 15.8 17.8 -10.7 5.40 16.6 -11.7 1.99 8.60
-12.7 1.75 5.70 -13.7 1.62 5.30 -14.7 1.60 5.30
[0184] In the case of the tyrosine phosphatase PTP-B, the Src-2
substrate is more readily de-phosphorylated than the abl substrate.
Analysis of the enzyme kinetics, by virtue of a Michaelis-Menten
plot, demonstrates that the apparent K.sub.m for the Src-2 optical
probe is 1.3 .mu.M and the k.sub.cat is 79 sec.sup.-1. The
K.sub.cat/K.sub.m for this substrate is nearly 10.sup.8 M.sup.-1
sec.sup.-1, indicating extremely efficient recognition of the
phosphotyrosine containing optical probe. These experiments
therefore demonstrate that the present invention provides for a
sensitive and convenient system of measuring protein tyrosine
phosphatase activity, and that the results obtained with the assay
system are directly comparable to those obtained with other methods
of measuring dephosphorylation. The relative broad substrate
specificity of phosphotyrosine phosphatases (see, for example,
Barford, et al. (1995) Nature Struct. Biol. 2: 1043-1053), suggest
that the approach will be useful for measuring a wide range of
protein tyrosine kinase activities.
Example 5
Validation of Optical Probes for Screening for Protein Tyrosine
Phosphatase Inhibitors
[0185] To demonstrate that the present invention can be used to
identify and characterize protein tyrosine phosphatase inhibitors,
experiments were carried in the absence, or presence of various
concentrations of orthovanadate, a well characterized competitive
inhibitor of tyrosine specific phosphatase activities.
Ortho-vanadate competitively inhibited tyrosine phosphatase
activity with an apparent IC.sub.50 of 420 nM (FIG. 5) using the
optical probes of the invention. This value is consistent with
literature values of orthovanadate inhibition of PTP-B obtained by
measuring .sup.32P-labeling. This result demonstrates that the
present invention can be used for the development of sensitive and
selective screening assays for the identification and
characterization of protein phosphatase activities.
Example 6
Measurement of Serine/Threonine Phosphorylation Using Optical
Probes
Measurement of Protein Kinase A Activity
[0186] To demonstrate that optical probe could be developed to
measure serine or threonine kinases, peptides was designed that
could be effectively recognized and phosphorylated by protein
kinase A. In this case, the substrate was designed with a single
aromatic amino acid (F) that was located immediately N-terminal to
the phosphorylation site for protein kinase A, underlined in SEQ.
ID. NO: 12, below, (the P'.sub.1 position with respect to the
protease cleavage site of chymotrypsin). This results in a
modulation of the rate of optical probe cleavage by chymotrypsin
after phosphorylation.
[0187] The peptide (RRRKFSLRRKA, SEQ. ID. NO: 12) was labeled with
fluorescein isothiocyanate at the N-terminus and
7-hydroxycoumarin-3-carboxamide at the C-terminus as described
above. To determine the relative proteolytic activity of the
phosphorylated and non-phosphorylated optical probes, samples of
both were prepared. To do this, 10 microM of the substrate, in a
total volume of 10 .mu.L, was phosphorylated to completion by
incubation with excess protein kinase A for one hour at 30.degree.
C. in a buffer consisting of 50 mM TRIS-Cl, pH 7.5, 10 mM
MgCl.sub.2, and 200 .mu.M ATP. Mock kinase reactions with no ATP
were used to create non-phosphorylated, control samples. In both
cases, the samples were diluted 10 fold with buffer containing 50
mM HEPES, pH 7.5, 10 mM CaCl.sub.2 and 0.01% Brij-35, and incubated
with 0.8 nM chymotrypsin. Fluorescence emission ratios were
monitored for one hour and are shown in Table 12.
TABLE-US-00012 TABLE 12 Fold Time of Cleavage Non-phosphorylated
Phosphorylated Difference in (minutes) Optical probe Optical probe
Ratios 0 0.34 0.01 0.33 0.01 1.0 10 3.15 0.06 0.54 0.01 5.8 20 6.71
0.29 0.78 0.01 8.6 30 9.86 0.43 1.02 0.02 9.7 40 12.00 0.4 1.27
0.03 9.5 50 13.34 0.40 1.53 0.03 8.7 60 14.02 0.27 1.78 0.03
7.9
The maximum fold difference in 460/530 ratio was about 9.7 after 30
minutes of treatment with chymotrypsin. This ratio change therefore
provides a robust and sensitive measure of protein phosphorylation
that by virtue of its high signal to noise ratio is well suited for
high throughput screening applications.
Example 7
Validation of Optical Probes for Screening for Serine/Threonine
Kinase Inhibitors
[0188] To demonstrate that the present invention could be used to
identify and characterize serine/threonine kinase inhibitors,
experiments were carried out with a number of previously
characterized inhibitors of serine threonine kinase activity. In
the case of the ATP-competitive inhibitors staurosporin and H-89,
inhibitor at a final concentration of 10 .mu.M was preincubated
with protein kinase A and the fluorescent substrate (SEQ. ID. NO:
28) in 50 mM TRIS-Cl, pH 7.5, 10 mM MgCl.sub.2, and the reactions
were initiated by the addition of ATP (10 .mu.M) For the substrate
competitive inhibitor PKI, inhibitor (2.8 .mu.M) was pre-incubated
with enzyme before the addition of optical probe and ATP (100
.mu.M) in the buffer described above. After one hour incubation at
30.degree. C., chymotrypsin to a final concentration of 0.8 nM was
added and the 460/530 ratio was determined after one hour, as
described above (Example 6). The results showed almost complete
inhibition of protein kinase A activity at the concentrations of
inhibitor tested (Table 13A and 13B).
TABLE-US-00013 TABLE 13A Kinase + the inhibitor Kinase + Negative
Control Positive Control Staurosporin the inhibitor (No Active
Kinase) (Active Kinase) (10 .mu.M) H-89 (10 .mu.M) 460/530 460/530
460/530 460/530 Emission Ratio Emission Ratio Emission Ratio
Emission Ratio 14.02 0.27 1.78 0.03 13.92 0.07 13.29 0.14
TABLE-US-00014 TABLE 13B Negative Control Positive Control Kinase +
the inhibitor (No Active Kinase) (Active Kinase) PKI (2.8 .mu.M)
460/530 460/530 460/530 Emission Ratio Emission Ratio Emission
Ratio 11.67 0.48 2.11 0.19 11.71 0.15
Validation of the Use of Optical Probes for High Throughput
Screening
[0189] To demonstrate that the present invention could reproducibly
detect inhibitors in a high throughput type-screening assay, a
screen was performed in a 96-well plate. The experiment was set up
with randomly spiked wells containing a known protein kinase A
inhibitor, staurosporin, under conditions where approximately 20%
of the substrate was converted to phosphorylated product. The
96-well plate was set up with appropriate no-ATP and no-inhibitor
controls. Fifteen wells were chosen at random and received 5 .mu.L
of 360 nM staurosporin (in 3% DMSO). The final concentration of
staurosporin in the five spiked wells was 60 nM, equal to the
IC.sub.80 determined empirically. All other wells (including the no
inhibitor wells) received 5 .mu.L of 3% DMSO. All wells (except
blanks) then received 10 .mu.L of kinase reaction mix which
included the optical probe (final concentration in the kinase
reaction was 3.3 .mu.M), buffer, and protein kinase A. After a 5
min pre-incubation, kinase reactions were started by the addition
of 15 .mu.L of 20 .mu.M ATP, and incubated for 15 minutes at
30.degree. C. Final kinase reaction concentrations were: 3.3 .mu.M
fluorescent substrate (SEQ. ID. NO: 28), 60 nM staurosporin, 0.004
units protein kinase A, 10 .mu.M, ATP, 10 mM MgCl.sub.2. Kinase
reactions were terminated by the addition of 30 .mu.L of a buffer
containing 50 mM HEPES, pH 7.4, 0.01% Brij-35, and 20 mM EDTA.
Initial 460/530 ratios were obtained, and then the chymotrypsin
reaction was started by the addition of 40 .mu.L of a buffer
containing 50 mM HEPES, pH 7.4, 0.01% Brij-35, and 2 nM
chymotrypsin (50 ng/ml). Final chymotrypsin concentration in the
reaction was 0.8 nM (20 ng/ml). 460/530 emission ratios were
obtained after 30 minutes. In this assay format, all wells spiked
with staurosporin were correctly assigned as positive hits for
kinase inhibition (FIG. 6; filled in dots). Furthermore, in those
wells, kinase activity was inhibited by about 80% when compared to
the no ATP (negative) controls. The assay was highly reproducible,
exhibiting a low coefficient of variance (Table 14).
TABLE-US-00015 TABLE 14 Samples Coefficient of Variance No ATP (n =
7) 1.6% Control Kinase (n = 7) 2.6% Kinase + Inhibitor (n = 15)
1.8% Kinase, no Inhibitor (n = 65) 2.6% Coefficient of Variance (in
%) = 100 .times. (Standard Deviation/Mean) N = number of wells
Example 8
Measurement of Other Serine Threonine Kinase Activities
Measurement of Casein Kinase 1 Activity
[0190] To measure casein kinase 1 activity, an optical probe was
designed as described above. In this case, the kinase substrate was
designed so that the point of phosphorylation was located at the
P'.sub.2 position (underlined in SEQ. I.D. NO: 17 below) with
respect to the scissile bond cleaved by chymotrypsin, which enables
the creation of a recognition motif suitable for casein kinase 1. A
substrate peptide (GDQDYLSLDK, SEQ. ID. NO: 17) was synthesized and
labeled with fluorescein isothiocyanate at the N-terminus and
7-hydroxycoumarin-3-carboxamide at the C-terminus as described in
Example 1.
[0191] Samples of phosphorylated and non-phosphorylated optical
probes were prepared and tested as described in Example 1. In this
case, complete phosphorylation of the optical probe (1 .mu.M) was
obtained after room temperature incubation for 15 to 30 minutes in
the presence of 500 Units of casein kinase 1 (New England BioLabs)
in 50 mM Tris-Cl, pH 7.5, 10 mM MgCl.sub.2, 5 mM DTT, and 200 .mu.M
ATP.
[0192] To determine the relative proteolytic sensitivity of
phosphorylated and non-phosphorylated optical probes, samples of
both were incubated with various concentrations of chymotrypsin and
the cleavage monitored by measuring the 460/530 emission ratio as
described in Example 1. The results, shown in Table 15, demonstrate
that the non-phosphorylated optical probe is significantly more
susceptible to proteolytic cleavage at low concentrations of
chymotrypsin than the phosphorylated optical probe.
TABLE-US-00016 TABLE 15 Negative Control Positive Control
Concentration of (No Active Kinase) (Active Kinase Fold
Chymotrypsin 460/530 Only) 460/530 difference in (.mu.M) Emission
Ratio Emission Ratio ratios 0.04 3.0 1.2 2.5 0.1 6.6 1.4 4.7 0.2
10.9 1.7 6.4 0.3 12.3 1.9 6.5 0.4 14.3 2.3 6.2 1.0 15.4 3.8 4.1 2.0
15.6 6.0 2.6
[0193] In this experiment, the maximum fold difference in 460/530
emission ratios of non-phosphorylated substrate versus
phosphorylated substrate occurred at a chymotrypsin concentration
of about 0.3 .mu.M chymotrypsin. At this concentration the
difference in emission ratios of phosphorylated and
non-phosphorylated fluorescence samples was greater than 6 fold,
demonstrating that the present invention provides for highly
sensitive methods of measuring this class of serine/threonine
kinase activities in a screening format.
Measurement of ERK Kinase Activity
[0194] To demonstrate that the present invention could also be used
to detect the activity of a proline-directed serine/threonine
kinase, an optical probe (VAPFSPGGRAK, SEQ. ID. NO: 27) was
designed as a substrate for extracellular signal-regulated kinase
(ERK) containing the serine phosphorylated, (shown underlined in
SEQ. ID. NO: 27) in the P.sub.1' position relative to the
chymotrypsin cleavage site (F). This substrate (100 .mu.M) was
phosphorylated by incubation at 30.degree. C. for 3 hours in a 100
.mu.L reaction containing 500 ng ERK (Biomol) and 500 .mu.M ATP in
a buffer consisting of 50 mM Tris-Cl, pH 7.5, 10 mM MgCl.sub.2, 1
mM EGTA, 2 mM DTT, and 0.01% Brij-35. To test the proteolytic
sensitivity of phosphorylated and non-phosphorylated samples, 2
.mu.L of the mock (no kinase) or kinase reaction was diluted to 100
.mu.L in a buffer containing 50 mM HEPES, pH 7.5, 10 mM CaCl.sub.2,
and 0.01% Brij-35, and incubated with chymotrypsin at a
concentration of 4 nM. Cleavage reactions were monitored on the
Cytofluor for 1 hr. The results demonstrate that phosphorylated
optical probe was less sensitive to chymotrypsin than the
non-phosphorylated peptide (Table 16). The maximum fold difference
in 460/530 ratio was about 15.2 and occurred after 30 minutes of
cleavage. These data demonstrate that the present invention can be
used to monitor activity of a proline-directed kinase.
TABLE-US-00017 TABLE 16 Positive Control Time of Negative Control
(Active Kinase) Fold cleavage (No Active Kinase) 460/530 difference
in (min) 460/530 Emission Ratio Emission Ratio ratios 0 0.28 0.33
0.85 10 4.17 0.44 9.45 20 7.56 0.56 13.55 30 10.03 0.66 15.16 40
11.71 0.78 14.99 50 12.73 0.89 14.34 60 13.40 1.00 13.36
Measurement of Protein Kinase C Activity
[0195] To measure protein kinase C activity using the optical
probes of the present invention, a peptide (RRRKFSLRRKA, SEQ. ID.
NO: 12) was designed in which phosphorylation by protein kinase C
occurred at the P'.sub.1 position (underlined in SEQ. ID. NO: 12)
with respect to the scissile bond cleaved by chymotrypsin. This
enabled an optimal protein kinase C recognition motif to placed
within the optical probe sequence, and to create a site of
phosphorylation that modulated the proteolytic sensitivity of the
substrate towards chymotrypsin. Analysis of phosphorylated and
non-phosphorylated samples of the optical probe revealed that
phosphorylation by protein kinase C significantly modulated the
proteolytic susceptibility of the substrate. These results
demonstrate that the present invention can be used to develop
optical probes that can measure protein kinase C activity.
Example 9
Measurement of Serine/Threonine Phosphatase Activities
Protein Phosphatase I Activity
[0196] To determine if the optical probes could be used to detect
serine/threonine phosphatase activities, samples of the casein
kinase 1 specific phosphorylated optical probes were prepared and
treated with various serine/threonine protein phosphatases. To do
this, the casein kinase 1 optical probe (GDQDYLSLDK, SEQ. ID. NO:
17) (100 .mu.M) was phosphorylated to completion with CKI (2000
units) in a 100 .mu.L reaction containing 200 .mu.M ATP for 5 hours
at 30.degree. C. A mock kinase reaction was performed which
contained no ATP for preparation of control, non-phosphorylated
optical probes. The resistance of the phosphorylated optical probe
to chymotrypsin cleavage (data not shown) confirmed complete
phosphorylation of the CKI-treated optical probe. Phosphorylated
and non-phosphorylated samples of the optical probe (2 .mu.M) were
incubated in a 50 .mu.L volume with or without 1 unit of the
serine/threonine phosphatase protein phosphatase I (PPI) for 2
hours at 30.degree. C. in a buffer consisting of 50 mM Tris-Cl, pH
7.0, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij-35, and 1 mM MnCl.sub.2. The
reactions were diluted to 100 .mu.L with 50 mM HEPES, pH 7.5,
incubated with chymotrypsin (0.2 .mu.M) for 1 hour at room
temperature, and fluorescence values were measured on the Cytofluor
as described above. Before chymotrypsin addition all reactions had
similar 460/530 ratios of about 1.2.
TABLE-US-00018 TABLE 17 Non- Phosphorylated phosphorylated
Phosphorylated Control + Control Control Phosphatase (PP1) 460/530
Emission 9.8 2.0 10.0 Ratio After Chymotrypsin
After chymotrypsin addition (Table 17) the 460/530 ratio of the
non-phosphorylated optical probe was 9.8, whereas the 460/530 ratio
of the phosphorylated optical probe was 2.0. However, when the
phosphorylated optical probe was first treated with PP1 and then
with chymotrypsin, the 460/530 ratio was 10.0, indicating that PP1
dephosphorylated nearly all of the optical probe.
Protein Phosphatase 2A Activity
[0197] The optical probes were also evaluated to determine if they
could be used to measure protein phosphatase 2A (PP2A) activity.
Phosphorylated and non-phosphorylated samples of the CKI optical
probes (SEQ. ID. NO: 23) were prepared and incubated with 0.03
units of PP2A as described above, except MnCl.sub.2 was not
included. After dilution and addition of chymotrypsin as described
above, fluorescence values were measured using a 96 well plate
reader (Cytofluor). After a 1.5 hour chymotrypsin incubation, the
460/530 emission ratio of the phosphorylated optical probe was
1.8.+-.0.2 (Table 18).
TABLE-US-00019 TABLE 18 Non- Phosphorylated phosphorylated
Phosphorylated Control + Control Control Phosphatase (PP2A) 460/530
Emission 11.0 .+-. 0.1 1.8 .+-. 0.2 8.8 .+-. 0.1 ratio After
Chymotrypsin
[0198] However, when the phosphorylated optical probe was first
incubated with PP2A followed by chymotrypsin, the 460/530 ratio was
8.8.+-.0.1. This value is approximately 80% of that obtained when
the non-phosphorylated optical probe was treated with chymotrypsin
(11.0.+-.0.1). Thus, a majority of the phosphorylated optical probe
was dephosphorylated under the conditions used. Taken together,
these data indicate that the present invention can also be used as
an assay for the activity of the serine/threonine phosphatase
PP2A.
Example 10
Validation of Optical Probes for Screening for Serine or Threonine
Protein Phosphatase Inhibitors
Identification of Protein Phosphatase 1 Inhibitors
[0199] To determine if the optical probe protein phosphatase assay
could detect inhibitors of PP1, the phosphatase assay was performed
in the presence or absence of 1 .mu.M microcystin-LR, a potent
inhibitor of PP1. Phosphatase assays were set up as described above
except the phosphatase was allowed to pre-incubate with
microcystin-LR for 10 minutes before the addition of phosphorylated
or non-phosphorylated optical probes (SEQ. ID. NO: 23). PP1
reactions were incubated at 30.degree. C. for 1 hour and then
diluted to 100 .mu.L in 50 mM HEPES, pH 7.5 followed by the
addition of chymotrypsin to 0.2 .mu.M. Fluorescence values were
measured on the Cytofluor after a 2 hour incubation at room
temperature as described above. After treatment of the
phosphorylated optical probe with PP1 followed by chymotrypsin, the
460/530 ratio was 13.2.+-.0.2. This value was identical to that of
the non-phosphorylated optical probe (13.2.+-.0.1) indicating that
PP1 completely dephosphorylated the CKI-treated optical probe in
this experiment (Table 19). However, in the presence of 1 .mu.M
microcystin-LR, PP1 activity was almost completely inhibited as
demonstrated by the 460/530 ratio, which was 2.8.+-.0.1. Control
samples, in which non-phosphorylated optical probe was treated with
microcystin-LR gave a final 460/530 ratio of 13.3.+-.0.2,
demonstrating that microcystin-LR did not inhibit chymotrypsin
cleavage. Thus, the present invention could be used to detect
inhibitors of PP1 activity.
TABLE-US-00020 TABLE 19 Non- phosphorylated Phosphorylated
Non-phosphorylated Phosphorylated Optical Optical Optical probe +
PP1 + Optical probe + PP1 + probe + PP1 probe + PP1 microcystin-LR
microcystin-LR After 13.2 .+-. 0.1 13.2 .+-. 0.2 13.3 .+-. 0.2 2.8
.+-. 0.1 Chymotrypsin
Identification of Protein Phosphatase 2A Inhibitors
[0200] To determine if the optical probe phosphatase assay could
detect inhibitors of PP2A, the phosphatase assay was performed in
the presence or absence of 100 nM microcystin-LR. PP2A assays were
set up as described above, and were incubated at 30.degree. C. for
2 hours. Reactions were diluted to 100 .mu.L with 50 mM HEPES, pH
7.5 followed by the addition of chymotrypsin to 0.2 .mu.M.
Fluorescence values were measured on the Cytofluor after a 1.5 hour
incubation at room temperature. As described above, treatment of
the phosphorylated optical probe (SEQ. ID. NO: 23) with PP2A
followed by chymotrypsin gave a final 460/530 ratio of 8.8.+-.0.1.
However, in the presence of 100 nM microcystin-LR, PP2A activity
was completely inhibited as demonstrated by the 460/530 ratio of
1.9.+-.0.1 (Table 20). Control samples in which non-phosphorylated
optical probe was treated with microcystin-LR and chymotrypsin gave
a final 460/530 ratio of 11.1.+-.0.2, demonstrating that
microcystin-LR did not inhibit chymotrypsin cleavage. Thus, the
optical probe based phosphatase assay can detect inhibitors of PP2A
activity.
TABLE-US-00021 TABLE 20 Non-phosphorylated Phosphorylated
Non-phosphorylated Phosphorylated optical probe + PP2A + optical
probe + PP2A + optical probe + PP2A optical probe + PP2A
microcystin-LR microcystin-LR Before 1.4 .+-. 0.0 1.3 .+-. 0.0 1.4
.+-. 0.1 1.4 .+-. 0.1 Chymotrypsin After 11.1 .+-. 0.1 8.8 .+-. 0.1
11.1 .+-. 0.2 1.9 .+-. 0.1 Chymotrypsin
Example 11
Use of Other Proteases to Measure Kinase Activity
Measurement of ERK Kinase Activity
[0201] To determine how phosphorylation on serine in the optical
probe (SEQ. ID. NO: 24) effected the rate of caspase-3 cleavage,
samples of phosphorylated and control (non-phosphorylated) optical
probes were treated with the protease caspase-3. Phosphorylated
samples of the optical probe (70 pmol) were prepared by 8 hour to
overnight incubation with ERK2 kinase (Biomol). Reactions were
typically performed in 10 .mu.l using 200-500 .mu.M ATP and 50-200
ng ERK2 in a buffer consisting of 50 mM HEPES, pH 7.5, 10 nM
MgCl.sub.2, 1 mM EGTA, and 1 mM DTT at 30.degree. C. Mock kinase
reactions were performed for preparation of control
(non-phosphorylated) optical probe as above, except ATP was
omitted. To monitor cleavage of the optical probes by caspase-3, 10
.mu.L volumes of phosphorylated and control (non-phosphorylated)
samples of the optical probe were placed in individual wells of a
96-well multiwell plate. Caspase-3 cleavage reactions were carried
out in these samples after dilution to 100 .mu.l in a buffer
consisting of 100 mM HEPES, pH 7.5, 5 mM DTT, 0.5 mM EDTA, 20%
glycerol, 0.01% Brij-35, and 50-100 ng caspase-3 (Upstate
Biotechnology), and incubated at room temperature. Emission
readings were taken at 5 minute intervals during the course of the
caspase-3 incubation using a Cytofluor plate reader as described in
Example 1.
[0202] As shown in FIG. 7, the 460/530 emission ratio, which as
described above, (Example 1) indicates increased cleavage of the
optical probe, changes more rapidly for the control
(non-phosphorylated) optical probe than it does for the
phosphorylated substrate. These results demonstrate that
phosphorylation of the optical probe by a serine/threonine directed
protein kinase results in a modulation of the rate of cleavage of
that substrate by, caspase-3. The maximal differences in
fluorescence emission ratio occurred in this case after 30 minutes
exposure to caspase-3, and resulted in over a three fold difference
in emission ratio of phosphorylated and non-phosphorylated optical
probes.
Measurement of Serine or Threonine Kinase Inhibitors
[0203] To confirm that the assay method could be used to detect
inhibitors of ERK kinase activity, the effect of roscovitine (a
known ERK kinase inhibitor) were examined using the present
invention. To do this, ERK kinase (50 ng) was pre-incubated with
the indicated amounts of roscovitine (Calbiochem), in the presence
of 100 .mu.M ATP. After 10 minutes optical probes (to a final
concentration of 0.7 .mu.M) were added and the incubations
continued for an additional 2 h at 30.degree. C. After incubation,
reactions were diluted to 100 .mu.l and fluorescence measurements
made as described above in Example 6.
[0204] The results show FIG. 8, that the assay was able to detect
the presence of the kinase inhibitor. The calculated IC.sub.50 for
roscovitine using the optical probe based assay was 45 .mu.M. These
experiments therefore demonstrate that the present invention
provides for a sensitive and convenient system of measuring Erk
serine/threonine kinase inhibitor activity.
[0205] The present invention provides novel optical probes and
methods for their use. While specific examples have been provided,
the above description is illustrative and not restrictive. Many
variations of the invention will become apparent to those skilled
in the art upon review of this specification. The scope of the
invention should, therefore, be determined not with reference to
the above description, but instead should be determined with
reference to the appended claims along with their full scope of
equivalents.
[0206] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
Sequence CWU 1
1
6019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Met Glu Glu Ile Tyr Gly Ile Leu Ser1
529PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Asp Glu Glu Ile Tyr Glu Ser Leu Glu1
5311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Gly Glu Glu Glu Ile Tyr Gly Glu Ile Glu Lys1 5
1049PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Ala Glu Ala Ile Tyr Ala Ala Pro Leu1
558PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Glu Pro Ile Tyr Met Leu Ser Leu1
568PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Glu Glu Glu Tyr Met Met Met Met1
579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Glu Glu Glu Glu Tyr Val Val Ile Xaa1
589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Glu Glu Glu Glu Tyr Val Leu Leu Val1
599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Ala Glu Glu Glu Tyr Phe Val Leu Met1
5109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Arg Arg Arg Phe Ser Ile Ile Ile Ile1
5119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Arg Arg Phe Arg Ser Ile Ile Ile Ile1
51211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Arg Arg Arg Lys Phe Ser Leu Arg Arg Lys Ala1 5
101310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Leu Arg Arg Arg Phe Ser Ala Ser Asn Leu1 5
10149PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Lys Arg Gln Phe Ser Ile Asp Leu Lys1
5159PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Lys Arg Phe Gln Ser Ile Asp Leu Lys1
51611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gly Asp Gln Asp Thr Tyr Ser Leu Leu Asp Lys1 5
101710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Gly Asp Gln Asp Tyr Leu Ser Leu Asp Lys1 5
10189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Glu Asp Glu Phe Ser Glu Asp Glu Glu1
5199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Glu Asp Phe Glu Ser Glu Asp Glu Glu1
5209PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20His His His Phe Ser Pro Arg Lys Arg1
5219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21His His Phe Arg Ser Pro Arg Lys Arg1
5229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22His His His Phe Ser Pro Arg Arg Arg1
5239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23His His Phe Lys Ser Pro Arg Arg Arg1
52411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Arg Val Asp Glu Pro Phe Ser Pro Gly Glu Lys1 5
10258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Pro Arg Pro Phe Ser Val Pro Pro1
5269PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Arg Arg Arg Phe Ser Leu Arg Arg Ile1
5279PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Arg Arg Phe Gly Ser Leu Arg Arg Ile1
5289PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Arg Arg Arg Phe Ser Arg Arg Arg Arg1
5299PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Arg Arg Phe His Ser Arg Arg Arg Arg1
5309PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Met Glu Glu Ile Tyr Gly Ile Phe Phe1
5319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Asp Glu Glu Ile Tyr Glu Glu Leu Glu1
53211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Gly Glu Glu Glu Ile Tyr Gly Glu Phe Glu Lys1 5
10 339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Ala Xaa Val Ile Tyr Ala Ala Pro Phe1
5349PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Xaa Glu Pro Ile Tyr Met Phe Phe Phe1
5359PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Xaa Glu Glu Glu Tyr Met Met Met Phe1
5369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Glu Glu Glu Glu Tyr Val Phe Ile Xaa1
5379PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 37Glu Glu Glu Glu Tyr Phe Glu Leu Val1
5389PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Ala Glu Glu Glu Tyr Phe Phe Leu Phe1
5399PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Arg Arg Arg Arg Ser Ile Ile Phe Ile1
54010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Arg Arg Arg Lys Phe Ser Phe Arg Arg Lys1 5
104110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Leu Arg Arg Arg Leu Ser Asp Ser Asn Leu1 5
10429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Lys Arg Gln Gln Ser Phe Asp Leu Phe1
54310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Phe Asp Thr Gly Ser Ile Ile Ile Phe Phe1 5
10449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Glu Asp Glu Glu Ser Glu Asp Glu Glu1
5459PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45His His His Arg Ser Pro Arg Lys Arg1
5469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46His His His Lys Ser Pro Arg Arg Arg1
54711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Arg Val Asp Glu Pro Asp Ser Pro Gly Glu Lys1 5
10488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Pro Arg Pro Ala Ser Val Pro Pro1
5499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Arg Arg Phe Gly Ser Leu Arg Arg Phe1
5509PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Arg Arg Arg His Ser Arg Arg Arg Arg1
5514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Cys Ala Ala Xaa1524PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Cys
Ala Ala Leu1535PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Xaa Thr Pro Xaa Pro1 5545PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Glu
Glu Glu Tyr Phe1 5555PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 55Glu Glu Glu Tyr Val1
5565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Glu Xaa Tyr Xaa Phe1 5574PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Tyr
Met Met Met1585PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 58Asp Xaa Xaa Asp Xaa1
55911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Gly Glu Glu Glu Ile Tyr Gly Val Ile Glu Lys1 5
106011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Val Ala Pro Phe Ser Pro Gly Gly Arg Ala Lys1 5
10
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References