Note: Descriptions are shown in the official language in which they were submitted.
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IN VITl~O FLUORESCENCE POLARIZATION ASSAY
Technical Field of the Invention
The invention relates to materials and methods for the identification of inhibitors
of protein:protein interactions, especially those involved in cellular signal transduction.
Back~round of the Invention
Cellular signal transduction, i.e., the series of events leading from extracellular
events to intracellular sequelae, is an aspect of cellular function in both normal and
disease states. Numerous proteins that function as signal transducing molecules have
been identified, including receptors, docking or recruiting proteins and enzymes such as
receptor and non-receptor tyrosine kinases, phosphatases and other molecules with
enzymatic or regulatory activities. These molecules generally demonstrate the capacity
to associate specifically with other proteins to form a si~n~ling complex that can alter
cell activity.
Signaling proteins often contain domain(s) of conserved sequence, which serve
as non-catalytic modules that direct protein-protein interactions during signal
transduction. Such domains include among others, SH2, phosphotyrosine interaction
("PI"), WW and SH3 domains. SH2 and PI domains recognize, i.e., bind to, proteins
containing characteristic peptide sequences which include one or more phosphorylated
tyrosine residues. WW and SH3 domains recognize proteins cont:lining characteristic
peptide sequences which need not contain phosphotyrosine residues. Significant
information related to such domains, proteins cont~ining them, the production ofproteins cont~ining such domains (including protein fragments and fusion proteins), the
characteristic peptide sequences which they recognize and the biological and/or clinical
role played by the interactions of such proteins has been described in the scientific
literature.
In cases in which the interaction of particular protein molecules is associated
with the cause or symptoms of a disease or pathological condition, compounds capable
of interfering with that protein:protein interaction may be useful in preventing or treating
the disease or condition in m~mm~ , including human patients.
Critical tools for the discovery of such inhibitors of protein:protein interactions
are binding assays. the well-known two-hybrid interaction/binding assay described by
Song and Fields, Nature, 340:245-247 (1989) has been used to study the interactions of
protein-protein interacting partners [See, Fields et al, US Patent No. 5,283,173 (I Feb
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1994)~. Such approaches have also been used to identify presumed SH2 dependent
interactions using yeast [Xing, Z. et al., Mol. Biol. Cell, 5:413-421 (1994); and Osborne,
M.A. et al., Biotechnol., 13: 1474 1478 (1995)] or to detect the inhibition of two-hybrid
formation in yeast [Chaudhuri, B. et al., FEBS Lett., 357: 221-226 (1995)]. See, also,
International patent application No. PCT/US95/03208, incorporated herein by reference
for background information on SH3 domains and their ligands including information on
the design and preparation of proteins containing various SH3 domains, preparation of
peptide ligands for an SH3 domain of interest, and biological/clinical roles of SH3
mediated interactions. See, PCT/US97/02635, incorporated herein by reference, for
information on receptor domains (e.g., SH2 and PI domains) for phosphotyrosine-
cont~ining lig~n(l~ including the design and ple~aldlion of proteins containing various
SH2 domains, plel)alalion of peptide ligands for an SH2 domain of interest, and
biological/clinical roles of SH2-mediated interactions.
Competitive binding assays have been described for detecting test substances
which interfere with the association of proteins cont~ining an SH2 domain with their
phosphotyrosine cont~inin~; ligands. See, e.g., Pawson, US Patent No. 5,352,660. More
recently reported binding assays have utili~ed surface plasmon resonance (Biacore) [see,
e.g., Panayotou et al, Mol. Cell. Biol., 13: 3567-3576 (1993)] or radioactive ligand based
assays. The former has a relatively low throughput, while the latter requires
cumbersome filtration manipulations and generates radioactive waste, an increasingly
difficult disposal issue.
The availability of materials and methods designed for the rapid and effective
identification of inhibitors of protein:protein interactions would be a boon for drug
discovery efforts aimed at a wide variety of target protein mediators. It would permit
higher-throughput and more efficient identification and development of new
ph~ eutical compositions cont~ining inhibitors of protein:protein interactions linked
to undesirable or pathological conditions.
Summary of the Invention
The present invention addresses this need by providing novel materials and
methods for in vilro competitive binding assays for identifying substances which inhibit
or interfere with the binding together of pairs of proteins capable of mutual association,
i.e., binding. to forrn binding complexes. Of particular interest are assays for identifying
compounds capable of inhibiting the binding of intracellular proteins or protein domains,
especially those involved in cellular signal transduction with their binding partners.
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Such proteins include, for instance, proteins which contain one or more SH2 domains, PI
domains, SH3 domains, or WW domains, each with its respective protein ligand.
In one aspect, the invention provides an in vitro assay method for identifying atest substance which inhibits the mutual association of a first protein to a second protein.
The method includes the steps of preparing a mixture containing the first protein, the
second protein bearing a covalently linked fluorophore, and at least one test substance.
The mixture is irradiated with polarized light of a suitable wavelength permitting
excitation of the fluorophore as indicated by emission of polarized light. The degree of
polarization of the emission is measured and the effect of the presence or concentration
of the test substance is determined. Inhibitory activity of the test substance is shown by
a decrease in the observed emission polarization values of the mixture of the first and
second proteins in the presence of the text substance as compared with the same protein
mixture in the absence of the test substance.
Inhibition of protein:protein association can result from binding a test substance
to the first protein or to the labeled ligand protein or peptide. Thus, the assay method
can be viewed as a method for identifying a test substance which competitively binds to
either member of the binding pair. As above, the degree of polarization of the emission
is measured, and the effect of the presence or concentration of the test substance in
decreasing the observed emission polarization is observed and compared with a mixture
in the absence of the test substance. Competitive binding of the test substance correlates
with decreased depolarization values.
In still another aspect, the invention provides an inhibitor of the association of a
first protein with a second protein, first identified by the methods above.
In yet another aspect, the invention provides components or reagents, e.g.,a
protein bearing a covalently linked fluorophore, useful in the methods of the invention.
The components or reagents can further be packaged in a kit with instructions for use in
the described methods.
Other aspects and advantages of the present invention are described further in the
following detailed description of the preferred embodiments thereof.
Brief Description of the Drawin~s
Fig. 1 depicts the structure of a fluorescent probe FMT 1, described in Example 2.
Fig. 2A is the saturation curve of FMTl binding to Src SH2, which plots bound
Src/total tracer vs. total Src concentration. Non-linear least squares fit for a saturation
experiment between probe FMTI and the Src-SH2 domain. Calculated Kd is 0.24 withan error of 0.008 ~M, (Chi)~ of 0.0027, R of 0.9987.
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Fig. 2B is the Scatchard analysis (transformation) of the data in Fig. 2A, plotting
Rb/Rf vs. Bound. Calculated Kd is 0.26 ~lM, with an R value of 0.992 for the linear fit.
Fig. 3 is an Src competition curve (2% DMSO) plotting % probe binding vs.
inhibitor concentration for the following tetrapeptides: Ac-pYEEI (open circle); Ac-
pYpYEEI (closed square); Ac-pYGGL (plus sign); Ac-pYEDL (open triangle); Ac-
DGVpYTGL (closed triangle).
Fig. 4A is a depiction of a 96 well plate of the experiment of Example 5, long
format, with an arrow illustrating the direction of dilution. Clear circles are sample
wells, gray circles are wells containing probe alone and dark circles are wells cont~ining
probe and protein
Fig. 4B is a depiction of a 96 well plate of the experiment of Example 5, short
format, with an arrow illustrating the direction of dilution per 4 rows of wells. Circles
are defined as in Fig. 4A.
Fig. 5 depicts the structure of an alternative fluorescent probe for use in the Src-
SH2 assay, described in Exarnple 2.
Fig. 6A is a depiction of performance of the FP assay with no inhibitor present.In this figure, the fluorescein-labeled second protein (or probe) binds IO its binding
partner (first protein having an SH2 domain). Light from the vertical polarized light
source remains polarized due to the slow rotation of the bound complex.
Fig. 6B is a depiction of performance of the FP assay with inhibitor present. Inthis figure, the inhibitor binds to the SH2 domain-containing first protein, thereby
preventing binding of the fluorescein-labeled second protein (or probe). The small
unbound probe rotates more quickly than does the complex of Fig. 6A. Light from the
vertical polarized light source becomes depolarized due to the quick rotation of the
unbound probe.
Detailed Description of the Invention
The present invention addresses the needs of the art by providing a fluorescencepolarization (FP)-based assay, for identifying and measuring the capacity of a test
substance to disrupt or inhibit the association between a pair of proteins. The novel
assay methods and materials disclosed herein have the advantages of being robust, non-
radioactive, and amenable to varying degrees of automation.
I. Components of the Assay
To facilitate underst~n(ling of this invention, the following descriptions of the
components of the assay are provided:
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~I The protein protein interaction
The assay of the present invention is designed to enable one to detect an inhibitor
of any protein:protein interaction. By the term "protein:protein interaction" or "mutual
association" of proteins is meant any complex or binding, covalent or non-covalent,
which naturally forms between two different proteins. One example of a protein:protein
association involves the complex formed between a receptor and its naturally-occurring
ligand. Interactions between fragments of proteins, i.e., peptides, with another protein or
peptide are also encompassed by the term protein:protein interaction. Examples of
protein:protein binding abound in the art, e.g., the binding between an antibody and a
protein antigen or epitope, the binding between a cell-surface receptor and its protein
ligand, the binding of various sign~lling proteins with their protein binding pairs, etc.
Specific examples of a protein:protein interaction, which are used herein to
demonstrate the method of this invention involve, as a first protein, a protein cont~ining
one or more SH2 domains, and/or SH3 domains (Syk, Zap, Src and Lck) and, as a
second protein, a ligand for that first protein. For additional background information on
Zap and Syk proteins and their SH2 domains, and peptide ligands, see, International
Patent Application Nos. PCT/US96/13918, incorporated herein by reference.
B The first protein~pep~ide molecule of the binding pair
For the purposes of the present invention, and for using currently conventional
detection e~uipment, it is preferred to use a first protein/peptide that has a significantly
greater molecular weight than the second protein or peptide, which naturally interacts
with it. For purposes of this invention, the larger protein which participates in the
protein:protein interaction is referred to as the first protein. It is the second protein or
peptide which is labelled with the fluorophore according to the assay method.
Generally, the first protein of the binding pair has a molecular weight of at least 2 to
about 100 times greater than the molecular weight of the labeled second protein/peptide
of the binding pair. Often, the first protein of the binding pair has a molecular weight of
at least 25 to about 50 times greater than the molecular weight of the labeled second
protein/peptide of the binding pair.
The first protein need not necessarily be rigorously purified in production, as
described below, but it is important to know the concentration of the first protein for
certain quantitative purposes, e.g., for the construction of a saturation curve or to
determine Kd values for the affinity of the two protein components.
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One specifically exemplified "first protein" of the examples contains an SH2
domain which serves as a receptor for a tyrosine phosphorylated peptide. Such a
receptor may be a protein, a fusion protein, polypeptide, peptide or fragment thereof
which contains a "phosphopeptide binding domain" (PBD). A PBD is a receptor domain
present, e.g., in certain sign~ling proteins, which is capable of binding to a
phosphorylated protein or phosphopeptide and thereby of directing protein-protein or
protein-peptide association. For example, an "SH2 domain" is one such receptor
domain. The receptor can be a polypeptide cont~ining one or more SH2, or other
phosphopeptide binding domains. The receptor may be located within a larger protein,
or may be a peptide fragment thereof. The receptor is preferably of human or other
anlmal orlgm.
Numerous proteins containing such receptors (e.g., SH2 domains, PI domains,
etc.) are known. See, e.g., US Patent No. 5,352,660.
Another specifically exemplified "first protein" contains an SH3 domain which
serves as a receptor for a corresponding peptide sequence or motif. Such a receptor may
be a protein, a fusion protein, polypeptide, peptide or fragment thereof which contains an
SH3 or SH3-like domain.
Another specifically exemplified "first protein" contains both an SH2 and an
SH3 domain.
C. The second protein/peptide of the binding pair
The second protein is a ligand (naturally-occurring or otherwise) of the first
protein or receptor. The second protein is generally the smaller of the two proteins in the
binding pair and is preferably the protein of the pair which is labeled with a fluorescent
moiety, thereby forming the "probe" component useful in the methods described herein.
In one example, the second protein or "ligand" is a protein, fusion protein,
peptide or fragment thereof which contains one or more tyrosine residues, which is
capable of binding selectively and with specificity to a phosphopeptide binding domain
when at least one of the peptide ligand's tyrosine residues is phosphorylated. An "SH2
ligand" is an example of such a ligand.
A considerable wealth of information on the sequence specificity of peptide
ligands for receptors, e.g., SH2, SH3, PI, and WW domains, etc. is also known. When it
is used as a probe in the assay of this invention, this ligand is labeled with a suitable
fluorophore as discussed in more detail below. Typically, the probe peptide or protein
binds to its (usually) larger binding partner with a Kd in the range of about 0.1 to about
1000 nM. More desirably, the two binding partners bind to each other with a Kd better
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than (i.e., numerically smaller than) about 300 nM, more preferably with a Kd in the
range of about 5 to about 50 nM.
D. Methods of producing the first and second proteins/peptides of the binding pair
DNA sequence information and expression technology is available which permits
recombinant production of any desired protein(s)/peptide(s) using a variety of expression
systems. To produce the proteins used in these assays, one may express DNAs encoding
the whole protein or a portion of the protein containing at least a domain of interest. The
protein or portion of the protein may be expressed as a fusion protein, also by
conventional techniques, especially in the case of the larger of the two binding proteins.
Any materials and methods conventional for producing a protein may be used
including both prokaryotic and eukaryotic systems. For example, such proteins/peptides
may be expressed by baculovirus, bacterial, yeast or m~mm~ n expression systems,whether as full-length proteins, fragments containing the receptor domain(s) or as fusion
proteins. Such expression systems are conventional in the art. See, for examples, the
descriptions in Sambrook et al, Molecular Cloning A Laboratory Manual., 2d edit.,
Cold Spring Harbor Laboratory, NY (1989).
The use of conventional protein/peptide expression technology permits the
production of any interacting protein pair of any size. The expression systems and the
conventional components thereof used to express the protein components of this
invention are well within the skill of the art and do not limit the scope of this invention.
By way of illustration, expression vectors for a protein or domain of interest can
be constructed by ligating into a conventional expression vector the DNA sequence
encoding the desired protein, protein domain or, if known, a consensus homology
domain for the domain of interest, alone or preferably with additional flanking sequence.
With routine experimentation, one can determine, if desired, whether such additional
fl~nking amino acids enhance stability, improve expression levels, improve its ability to
interact with ligands or other proteins or be necessary or desirable for linking to a fusion
protein for reasons discussed below. For example, for human Src the SH3 consensus
homology domain includes amino acids 91-140. We have prepared SH3 domain proteinfrom E. coli expression vectors using amino acids 84-145, which contain an additional 7
amino acids on the N-terminal side of the homology domain and S amino acids on the C-
terminal side.
The desired protein or protein domain may be expressed within all or part of itsnatural context, as an isolated domain, in a tandem array containing two or more of the
same or different domains, or as a fusion protein with other unrelated domains including
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but not limited to SH2-like domains, protein kinase domains, glutathinone S-transferase
(GST), epitope tags, kinase recognition sequences, maltose binding protein, signal
sequences, biotin-modification sequences, etc.
The proteins or protein domains may be modified to:
~ facilitate purification e.g. by expression as a fusion to glutathione-S-transferase,
maltose binding protein, metal-chelation sequences (poly-histidine), protein A or others;
~ facilitate identification or quantitation, e.g by covalent modification using biotin,
fluorophores, chromophores, scintillons, spin labels, radioactive or non-radioactive
isotope tags, magnetic particles, metal coloids, etc;
~ adhere to defined solid supports, e.g. by expression as a fusion to an epitope tag or
other antigenic domain; engineered to provide unique or uniquely accessible protein
features e.g. N-terminal serine, cysteine, Iysine or others, etc.
~ remove undesirable features that pose experimental complications, e.g by
mutation of cysteines that participate in unnatural domain dimerization
~ improve stability under conditions of binding assays (e.g. by altering the natural
coding sequence to encode cysteines that form stabilizing disulfides).
E. The "test substance"
A "test substance or inhibitor" is defined herein as a compound or composition
which binds selectively to either the first protein or the second protein which participate
in the protein:protein interaction. Alternatively, the test substance selectively blocks or
otherwise inhibits the interaction between these two proteins. For example, this inhibitor
can bind to the first protein with competitive avidity vis-a-vis its naturally occurring
binding protein, or it can bind the second protein. Where the two proteins are
exemplified as a tyrosine phosphorylated receptor and its naturally occurring ligand, the
inhibitor can bind to the receptor competitively with the ligand, or it can selectively
block or otherwise inhibit the interaction between the receptor and ligand normally
mediated by one or more tyrosine phosphorylated peptides or domains.
Test substances or compositions to be assessed for their ability to bind selectively
to the first or second protein of interest can be obtained from a variety of sources,
including, for exarnple, microbial broths, cellular extracts, conditioned media from cells,
synthetic compounds and combinatorial libraries. The assay method of this invention
may be used to screen natural product and test compound libraries or structurally-biased
diversity libraries to identify desired inhibitors. The test substance may be selected from
a mixture of one or more test peptides, wherein said mixture is provided in the form of a
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g
library of synthetic peptides or in the form of a phage library displaying the various
peptides.
F. ~1 "fluorescentmoiety"
A "fluorophore" or "fluorescent moiety" is a fluorescent molecule which, in
solution and upon excitation with polarized light, emits light back into a fixed plane (i.e.,
the light remains polarized). Numerous known fluorescent labeling moieties of a wide
variety of structures and characteristics are suitable for use in the practice of this
invention. Similarly, methods and materials are known for covalently linking them to
other molecules [see, e.~., Richard P. ~ugl~nd, Molecular Probes: Handbook of
Fluorescent Probes and Research Chemicals 1992-1994 (Sth edit, 1994, Molecular
Probes, Inc.)]. In choosing a fluorophore, it is preferred that the lifetime of the
fluorophore's exited state be long enough, relative to the rate of motion of the labeled
probe or peptide, to permit measurable loss of polarization following emission. Suitable
fluorophores include fluorescein, fluorescein isothiocyanate, rho~mine,
dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and umbelliferone.
It is typically preferred to use a fluorophore having an excitation wavelength and
emission wavelength in the visible rather than ultraviolet range of the spectrum to avoid
possible interference from test compound fluorescence. Preferably the fluorophore is
covalently linked to the smaller protein, i.e., the second protein, to be labeled, e.g., a
peptide ligand, using a sufficiently short linker to avoid introducing undue motion to the
fluorophore, i.e., motion not correlated to the motion of the labelled peptide.
More specifically, the examples below provide a description of the method used
by these inventors in which a fluorescent moiety is chemically attached by covalent
bonds onto a second protein molecule (a peptide ligand). In Example 2, the phospho-
Tyr cont~ining peptide is labelled with fluorescein in vitro. One of skill in the art will
understand that any method of production of such phosphopeptides is applicable.
G. "Fluorescence polarization"
FP, first described by Perrin, J. Phys. Rad., I :390-401 (1926), is based upon the
finding that the emission of light by a fluorophore can be depolarized by a number of
factors, the most predominant being rotational diffusion, or, in other words, the rate at
which a molecule tumbles in solution. "Polarization" is the measurement of the average
angular displacement of the fluorophore which occurs between the absorption and
subsequent emission of a photon. This angular displacement of the fluorophore is, in
turn, dependent upon the rate and extent of rotational diffusion during the lifetime of the
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excited state, which is influenced by the viscosity of the solution and the size and shape
of the diffusing fluorescent species. If viscosity and temperature are held constant, the
polarization is directly related to the molecular volume or size of the fluorophore. In
addition, the polarization value is a dimensionless number (being a ratio of vertical and
horizontal fluorescent intensities) and is not affected by the intensity of the fluorophore.
F. Additional Information Relating to Illustrative Signal Transducing Domains
of Particular Interest
(i) SH2 or SH2-like Domains
The term "SH2 domain" refers to a sequence which is substantially homologous
to a Src homology region 2 (SH2 region). The Src homology region 2 is a noncatalytic
domain of~l00 amino acids which was originally identified in the viral Fps and viral
Src cytoplasmic tyrosine kinases by virtue of its effects on both catalytic activity and
substrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I. Sadowski et al.,
Mol. Cell. Biol. 6, 4396 (1986)). SH2 domains have been found in a variety of
eukaryotic proteins, some of which function in intracellu}ar signal transduction. Many
are known in the art. Examples (including counterparts from various species) of SH2
domain-cont~ining proteins include (1) members of the src-family protein tyrosine
kinases (Src, Lyn, Fyn, Lck, Hck, Fgr, Yes), (2) Shc (3) Tsk, (4) Btk, (5) VAV, (6)
Grb2, (7) Crk, and (8) signal transducer and transcription (STAT) proteins. In addition,
a number of proteins, such as ZAP-70, p85 phosphatidylinositol 3' kinase (PI3K), Syk,
GTPase Activating Protein (GAP), and Phospholipase C g~mm~, have two SH2
domains. SH2 domain-contzlining proteins have been identified in human, rodent, sheep,
bovine, C. elegans, Drosophila, Xenopus, flatworm, freshwater sponge, and hydra.One way to identify new SH2 or SH2-like domains from unknown DNA, RNA
or protein sequence is by using one of many available computer alignment programs.
One example is pfscan, which can be run via the World Wide Web (WWW) site at
http://ulrec3.unil.ch/software/profilesc~n.html. To use the program, a protein sequence
is tested against a "profile" describing the SH2 domain motif. According to the program
inforrnation, the particular strength of profiles is that they can be used to describe very
divergent protein motifs. These profiles are normally derived from multiple alignments
of the initial sequence set. In addition to the sequences themselves, a profile identifies
which types of residues are allowed at what position within the domain, which amino
acids are conserved, which ones are not, which positions or regions can allow insertions,
and which regions may be dispensable. Additional information on Pfscan and PROSITE
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can be obtained at the web page http://ulrec3.unil.ch/index.html operated by theBioinformatics Group at the ISREC (Swiss Institute for Experimental Cancer Research).
As an example we analyzed the peptide sequence of human Src with the pfscan
program. The results are shown below. The program clearly identified the SH2 domain
of Src as encompassing the region from amino acids 150-247 of the Src peptide
sequence. In addition, the SH3 and kinase domains were identified by pfscan.
NScore raw from-to Profile ¦ Description
26.9695 1792 pos. 150-247 PS50001 ¦ SH2 Src homology 2 (SH2) domain
20.2947 1182 pos. 83-144 PS50002 ¦ SH3 Src homology 3 ~SH3) domain
43.4246 2912 pos. 269-522 PS50011 ¦ PROTEIN_KINASE_DOM Protein
kinase
The NScore of a match is the negative decadic logarithm of the expected number of
matches of the given quality (or better) in a random database of the given size. For
NScores ~1 this converges to the probability of finding the match in the database.
Since the number of expected matches depends on the size of the database, the decadic
logarithm of the ~l~t~b~ce size must be subtracted before the calculation:
-log(NExp) = NScore - log (DBsize)
where (NExp=Expected number of chance matches) and (DBsize=size of the database in
characters).
The following table gives somes examples on how to convert the NScores into
probabilities for the SwissProt database and the nonredundant (nr) protein database. The
calculation is based on a database size of
18,531,385 residues for SwissProt (log=7.27)
58,154,119 residues for the nr database (log=7.76)
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Expected chance matches in:
NScore SwissProt nonredundant
7.0 1.8 5.8
7.5 0.58 1.82
8.0 0.18 0.58
8.5 0.058 0.182
9.0 0.018 0.058
9.5 0.006 0.0182
10.0 0.0018 0.0058
10.5 0.0006 0.0018
...and so on...
The segment of a test sequence contains an SH2 domain with an SH2 profile NScorevalue > 7.5, preferably > 8, more preferably > 9, more preferably > 10.
As a second example, the N-terminal 160 amino acid sequence from human
ZAP-70 was applied to pfscan. The result indicated an SH2 domain bounded by amino
acids 10-102.
NScore raw from-to Profile ¦ Description
16.4402 1082 pos. 10-102 PS50001 ¦ SH2 Src homology 2 (SH2) domain
SH2 domains can be identified using other computer alignment programs, such
as MegAlign within the DNAstar computer package (Madison, WI). To do this, one or
more known SH2 domains and a test sequence are aligned by the clustal method. A
sequence having 3 25%, in some cases 30 - 50 %. in other cases > 50%, amino acids
identical to a known SH2 domain is identified as an SH2 homology domain. The
positions of identical amino acids between the test sequence and different known SH2
domains can vary, except for one position. All SH2 domains identified to date have a
conserved arginine residue approximately 25-40 residues from the start of the SH2
homology domain. In human src this arginine is found within the sequence FLVRES,where abbreviations for the amino acid residues are: F, Phe; L, Leu; V, Val; R, Arg; E,
Glu; S, Ser.
Another way to identify SH2 or SH2-like domains is by running a query in the
federated nucleotide or protein databases for the SH2 domain feature. In the SWISS-
PROT database, this is listed under the FT or "feature" heading. SWISS-PROT database
can be accessed over the WWW at EBI http://www.ebi.ac.uk. For example, in the file
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listed for human Src (P 1293 1 )? the region containing the SH2 domain is shown to be
1 50-247.
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SWISS-PROT: P12931
ID SRC_HUMANSTANDARD; PRT; 535 AA.
AC P12931;
DR MIM; 190090; -.
DR PROSITE; PS00107; PROTEIN_KINASE_ATP.
DR PROSITE; PS00109; PROTEIN_KINASE_TYR.
DR PROSITE; PS50001; SH2.
DR PROSITE; PS50002; SH3.
DR PROSITE; PS50011; PROTEIN_KINASE_DOM.
DR PRODOM [Domain structure / List of seq. sharing at least 1 domain]
DR SWISS-2DPAGE; GET REGION ON 2D PAGE.
KW TRANSFERASE; TYROSINE-PROTEIN KINASE; PROTO-ONCOGENE;
PHOSPHORYLATION;
KW ATP-BINDING; MYRISTYLATION; SH3 DOMAIN; SH2 DOMAIN.
FT INIT_MET O 0 BY SIMILARITY.
FT LIPID 1 1 MYRISTATE (BY SIMILARITY).
FT DOMAIN83 144 SH3.
FT DOMAIN150 247 SH2.
FT DOMAIN269 522 PROTEIN KINASE.
FT NP_BIND275283 ATP (BY SIMILARITY).
FT BINDING297297 ATP (BY SIMILARITY).
FT ACT_SITE 388 388 BY SIMILARITY.
FT MOD_RES419419 PHOSPHORYLATION (AUTO-) (BY
SIMILARITY).
FT MOD_RES529529 PHOSPHORYLATION (BY SIMILARITY).
Yet another way to identify SH2 or SH2-like domains may be accomplished by
screening a cDNA expression library with a phosphorylated peptide ligand for a known
SH2 domain to isolate cDNAs for SH2 proteins. One could use PCR or low stringency
screening with an SH2-specific probe. The SH2 domain or protein cont~ining the SH2
domain may be isolated from naturally occuring sources (e.g. cells, tissues, organs, etc);
produced recombinantly in bacteria, yeast or eukaryotic cells; produced in vitro using
cell free translation systems; or produced synthetically (e.g. peptide synthesis).
Certain SH2 or SH2-like domains may not be identified via the pfscan program
nor exhibit significant homology with known SH2 domain sequences to be detected by
computer alignment programs. These sequences may, nevertheless, exhibit the same or
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similar three-dimensional structure as known SH2 domains and function as an SH2-like
domain and function to bind phosphotyrosine-cont~inin~ peptides or proteins. The three-
dimensional structure of several known SH2 domains have been determined. S~I2
domains are characterized as two anti-parallel beta sheets composed of 5 or 6 beta
strands. Regions forming an alpha helix may or may not be present within the domain.
SH2 or SH2-like domains may be recognized as having an SH2-like domain structurewhen solved by x-ray crystallography or NMR spectroscopy. Alternatively, a predicted
structure by homology modeling may be used to identify a particular protein sequence as
an SH2-like domain.
The alignment of SH2 domains used to generate the SH2 profile for pfscan, as
taken from http://ulrec3.unil.ch/prf_details/alignments/SH2.msf (profile matrix can be
obtained from http://ulrec3.unil.ch/cgi-bin/get_pstpr~?SH2) is based on alignment o3~
approximately 390 SH2 domains from proteins of various species. The list of proteins
cont~inin~; SH2 domains used in the alignments in the Swiss-Prot Database includes the
following (P# is the Swiss-Prot Database Accession number):
P00519, A}3Ll_HUMAN P00520, ABL_MOUSE P00521, F~3L_MLVA}3
P00522, A~3L_DROME P00523, SRC_CHICK P00524, SRC_RSVSR
P00525, SRC_AVISR P00526, SRC_RSVP P00527, YES AVISY
P00528, SRCl_DROME P00530, FPS_FU~SV P00541, FPS_AVISP
P00542, FES_FSVGA P00543, FES_FSVST P00544, FGR_FSVGR
P03949, ABLl_CAEEL P05433, GAGC_AVISC P05480, SRCN_MOUSE
P06239, LCK_HUMAN P06240, LCK_MOUSE P06241, FYN_HUMAN
P07332, FES_HUMAN P07947, YES_HUMAN P07948, LYN_HUMAN
P08103, HCK_MOUSE P08487, PIP4_BOVIN P08630, SRC2_DROME
P08631, HCK_HUMAN P09324, YES_CHICK P09769, FGR_HUMAN
P09851, GTPA_BOVIN P10447, A}3L_FSVHY P10686, PIP4_RAT
P10936, YES_XENLA P12931, SRC_HUMAN P13115, SRCl_XENLA
P13116, SRC2_XENLA P13406, FYN_XENLA P14084, SRC_AVISS
P14085, SRC_AVIST P14234, FGR_MOUSE P14238, FES_FELCA
P15054, SRC_AVIS2 P15498, VAV_HUMAN P16277, BLK_MOUSE
P16333, NCK_HUMAN P16591, FER_HUMAN P16879, FES_MOUSE
P16885, PIP5_HUMAN P17713, STK_HYDAT P18106, FPS_DROME
P19174, PIP4_HUMAN P20936, GTPA_HUMAN P23615, SPT6_YEAST
P23726, P85B_BOVIN P23727, P85A_BOVIN P24135, PIP5_RAT
P24604, TEC_MOUSE P25020, SRC_RSVHl P25911, LYN MOUSE
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P26450, P85A_MOUSE P27446, FYN_XIPHE P27447, YES_XIPHE
P27870, VAV_MOUSE P27986, P85A_HUMAN P29349, CSW_DROME
P29350, PTN6_HUMAN P29351, PTN6_MOUSE P29353, SHC_HUMAN
P29354, GRB2_HUMAN P29355, SEM5_CAEEL P31693, SRC_RSVPA
P32577, CSK_RAT P34265, YKFl_CAEEL P35235, PTNB_MOUSE
P35991, BTK_MOUSE P39688, FYN_MOUSE P40763, STA3_HUMAN
P41239, CSK_CHICK P41240, CSK_HUMAN P41241, CSK_MOUSE
P41242, CTK_MOUSE P41243, CTK_RAT P41499, PTNB_RAT
P42224, STAl_HUMAN P42225, STAl_MOUSE P42226, STA2_HUMAN
P42227, STA3_MOUSE P42228, STA4_MOUSE P42229, STA5_HUMAN
P42230, STA5_MOUSE P42231, STA5_SHEEP P42232, STAB_MOUSE
P42679, CTK_HUMAN P42680, TEC_HUMAN P42681, TXK_HUMAN
P42682, TXK_MOUSE P42683, LCK CHICK P42684, ABL2_HUMAN
P42685, FRK_HUMAN P42686, SRKl_SPOLA P42687, SPKl_DUGTI
P42688, SRK2_SPOLA P42689, SRK3_SPOLA P42690, SRK4_SPOLA
P43403, ZA70_HUMAN P43404, ZA70_MOUSE P43405, SYK_HUMAN
P46108, CRK_HUMAN P46109, CRKL_HUMAN Q00655, SYK_PIG
Q02977, YRK_CHICK Q03526, ITK_MOUSE Q04205, TENS_CHICK
Q04736, YES_MOUSE Q04929, CRK CHICK Q05876, FYN_CHICK
Q06124, PTNB_HUMAN Q06187, BTK_HUMAN Q07014, LYN_RAT
Q07883, GRB2_CHICK Q08012, DRK_DROME Q08881, ITK_HUMAN
A general method to identify an SH2 domain within a test peptide or nucleotide
sequence follows:
I . Translate the cDNA or RNA into single letter code protein sequence. This could be
accomplished using a computer program such as DNA strider or EditSeq in the DNAstar
package.
2. Go to the WWW site at http://ulrec3.unil.ch/software/profilescan.html
3. Copy the test sequence into the appropriate box in the pfscan forrn
4. Submit the form to the pfscan server
5. The results are sent back through the web browser or via e-mail.
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SH2 and SH2-like domains as described in the foregoing paragraphs may be used
in the practice of this invention. Using information provided herein, and by analogy to
the examples provided below, one may carry out this invention with any SH2 domain,
SH2-like domain, PID or PID-like domain and a peptide ligand therefor, e.g. in place of
ZAP, Syk, Src or Fyn SH2 domains.
(ii) PID or PID-like Domains
An alternative phosphotyrosine binding domain to SH2 domains is the so-called
phosphotyrosine interaction domain (PID). This domain, con~ining on average about
160 amino acid residues, was originally identified in the Shc protein. In contrast to SH2
domains, which recognize sequences having a consensus pTyr-Xaa-Xaa-Xaa-Xaa (a
phosphotyrosine followed by three or more amino acids), PID domains recognize
se~uences with the consensus Asn-Xaa-Pro-pTyr (also called NPXY in single lettercode). The invention described in this application is also relevant to PID and PID-like
domains. In this case, the coding sequence for a PID domain is substituted in the
appropriate vector for the SH2 domain coding sequence and a ligand that recognizes the
PID domain replaces the SH2 domain ligand. Phosphorylation of the PID ligand could
be accomplished using v-Src, as described herein. Alternative protein kinases could be
used to phosphorylate the PID ligand. In addition, a protein kinase endogenous within
the cell could catalyze phosphorylation of the PID ligand.
Significant information concerning these domains is known in the art. A detaileddescription of the PID domains can also be found on the WWW at the site
http://w~,vw.bork.embl-heidelberg.de/Modules/pid-gif.html. The following information is
taken from that site:
Documentation - PROSITE description
Beside SH2,the phosphotyrosine interaction domain (PI domain or PID)[3] is the second
phosphotyrosine-binding domain found inthetransformingproteinShc[1,2]. Shc
couples activated growth factor receptors to a sign~lling pathway that regulates the
proliferation of m~mm~ n cells and it might participate in the transforming activity of
oncogenic tyrosine kinases. The PI domain specifically binds to the Asn-Pro-Xaa-Tyr(p) motif found in many tyrosine-phosphorylated proteins including growth factor
receptors. PID has also been found in the Shc related protein Sck [I] and several
otherwise unrelated regulatory proteins [3] which are listed below.
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~ M~mm~ n Shc (46 kD and 52 kD isoforms) contains one N-terminal PID, a
collagen-like domain and a C-terminal SH2 domain.
~ Human Shc related protein Sck contains one PI domain and a SH2 domain.
~mm~ n X11 is expressed prominently in the nervous system. It contains 2
disc homologous regions (DHR) of about 100 AA downstream of the PID.
~ Drosophila nuclear Numb protein is required in deterrnination of cell fate during
sensory organ formation in drosophila embryos. It has one PID.
~ Caenorhabditis hypothetical protein F56D2.1 contains an N-terminal
metalloproteinase domain followed by one PID.
~ Rat FE65. The WW domain as well as the 2 PIDs found in the sequence of FE65
indicate that this protein is probably involved in signal transduction.
~ Drosophila protein disabled is a cytoplasmic, tyrosine phosphorylated protein
found in CNS axons and body wall muscles. Itis involvedinembryonic
neural development. It contains one N-terminal PI domain.
~ Mouse mitogen responsive phosphoprotein isoforms P96, P93 and P67 which are
produced by alternative splicing, contain one N-terminal PID. This is also true
for the differentially expressed human ortholog Doc-2.
Human EST05045 protein fragment has one PID.
References:
[ 1] Kavanaugh W.M., Williams L.T. Science 266:1862-1865(1994)
[ 2] Blaiki, P. et al., J.Biol.Chem. 269, 32031-32034 (1994)
[ 3] Bork P., Margolis B. Cell 80, 693 (1995)
A PI domain alignment based on a number of PI domains from various species is
illustrated in the WWW site at http://ulrec3.unil.ch/prf_details/alignments/PID.msf.
Another such alignment is shown at the web site at http://www.bork.embl-
heidelberg.de/Modules/pi-~li html
(iii) SH3 and SH3-like domains
The term "SH3-like domain" refers to a sequence which is substantially
homologous to a Src homology region 3 (SH3 region). The Src homology 3 region is a
noncatalytic domain of ~60 amino acids which was originally identified in the viral Fps
and viral Src cytoplasmic tyrosine kinases by virtue of its effects on both catalytic
activity and substrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I.
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Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986)). SH3 domains have been found in a
variety of eukaryotic proteins, some of which function in intracellular signal
transduction. Examples (including counterparts from various species) of SH3 domain-
containing proteins include ( 1 ) members of the src-family protein tyrosine kinases (Src,
Lyn, Fyn, Lck, Hck, Fgr, Yes), (2) Grb-2, which has two SH3 domains, (3) Sprk, a-threonine/serine protein kinase, (4) Tsk, (5) Btk, (6) Vav, (7) GTPase Activating Protein
(GAP), (8) p40, p47, and p67 proteins of the neutrophil oxidase complex, and (9)phosphatidylinositol 3' kinase, (10) Crk, (11) phospholipase C g~mrn~, (12) Abl. SH3
domain-cont~ining proteins have been identified in human, rodent, bovine, C. elegans,
and yeast. Other SH3 domains may be selected from the scientific literature or identified
by sequence analysis or cloning by the methods described above. See e.g.
PCT/US95/03208 for a wealth of background information relating to SH3 domains and
their ligands.
Certain SH3 or SH3-like domains may not match any of the 18 conserved amino
acids nor exhibit significant homology with known SH3 domain sequences to be
detected by computer alignment programs. These sequences may, nevertheless, exhibit
the same or similar three-dimensional structure as known SH3 domains and function as
an SH3-like domain. The three-dimensional structure of several known SH3 domainshave been determined. SH3 domains are characterized as two anti-parallel beta sheets
composed of 5 or 6 beta strands. Regions forming an alpha helix may or may not be
present within the domain. SH3 or SH3-like domains may be recognized as having an
SH3-like domain structure when solved by x-ray crystallography or NMR spectroscopy.
Alternatively, a predicted structure by homology modeling may be used to identify a
particular protein sequence as an SH3-like domain.
Il. The Assay Protocol
The in vitro assay method of this invention utilizes FP for identifying a test
substance which competitively binds to, or inhibits the mutual association of, a first
protein molecule to a second protein molecule. Fluorescence polarization is an
extremely useful method for studying ligand-protein and protein-protein interaction.
The present invention is based upon the observation that changes in polarization will
occur if a fluorescent molecule undergoes a molecular weight change due to cleavage or
binding to another molecule. Fluorophores that are of a low-molecular weight, and/or
are very flexible, have low polarization values, while those that have a high molecular
weight, and/or are rigid, have higher polarization values.
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This intrinsic property of the fluorescent moiety is utilized in the assay of this
invention. According to this method, a mixture is made which contains the following
components:
(a) a selected amount of a first protein molecule, e.g., a tyrosine phosphorylated
receptor, which is capable of binding or otherwise mutually associating with a smaller
second protein molecule;
(b) a selected amount of a smaller, second protein molecule, which is
covalently linked to, or labeled with, a fluorophore. In the examples below, it is the
peptide ligand that is labelled with a fluorophore by covalent linkage, thereby forming
fluorophore-labeled probes of low molecular weight; and
(c) a selected, potentially competitively-inhibiting test substance.
This mixture is accomplished under conditions suitable to permit complex
formation between the first and second proteins, if they were admixed in the absence of
test substance. Thus, according to this method, in the event that the test substance is, in
fact, an inhibitor of the protein:protein complex formation, the conditions are also
suitable to permit its competitive binding to the first or second proteins.
This mixture of (a), (b) and (c) is irradiated with plane polarized light of a
wavelength which is sufficient to excite the fluorophore. The light subsequently emitted
by the fluorescent second protein is polarized to varying degrees depending on the
molecular volume of the fluorescent second protein. In the unbound state in solution,
low molecular weight peptides rotate rapidly, and give low polarization readings.
When in the presence of its binding/interacting first protein partner, e.g., a
receptor, the lower-molecular weight fluorescent second protein binds to the higher
molecular weight first protein, e.g., a tyrosine phosphorylated protein or peptide
receptor. When the labeled second protein binds to its target first protein and is
min~ted by plane polarized light, the large first protein:second protein complextumbles more slowly, and the polarization readings increase. The method of this
invention thus follows changes in the ratio of polarization in the horizontal and vertical
planes of the emission wavelength range. This is in distinct contrast to following
changes in the intensity of absorbance within a particular wavelength range, which is the
way conventional fluorescent labels are used. The change measured by the presentinvention is a direct measure of the binding of the labeled second protein to the first
protein.
This difference in polarization values of free labeled second protein vs. bound
second protein:first protein complex is used to measure the bound and free ratios of the
second protein and analyze its binding to the first protein when in the presence of a test
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substance. Such measurement may occur in either saturation or competition
experiments. The FP assay of this invention can thus be used in many solutions,
including in the cytoplasm of the cell.
The degree of polarization of the emission is measured without the necessity to
separate the components in the mixture. Finally, the effect of the presence or
concentration of the test substance is determined by comparing the ratio of the
polarization levels of the mixture with the polarization levels of the same amounts of the
first and second proteins/peptides in the absence of test compound.
If competitive binding occurs between the first or second protein and the test
substance instead of between the two proteins, so that the protein:protein complex is not
formed, the second protein will remain free in solution and low polarization will be
measured. If the test substance is not an inhibitor or a good inhibitor, the complex will
be formed and the polarization of the mixture will increase. Thus a decrease in the
observed emission polarization depolarization values from known polarization levels of
the first protein:second protein complex in the absence of test compound is noted in the
presence of an inhibitor test substance.
Since the method of this invention follows changes in the ratio of polarization in
the horizontal and vertical planes of the emission wavelength range, rather than changes
in the intensity of absorbance within a particular wavelength range, the method is less
vulnerable to interference from high absorbance of test compounds in solution.
The methods of this invention are susceptible to automation. For example, all orseveral of the steps outlined above may be performed by an apparatus programmed to
conduct automatically two or more steps for a given test substance or one or more steps
for a plurality of test substances or test substance concentrations. As one example, any
standard fluorometer equipped for polarization experiments or measurements may be
used in practicing this invention to both irradiate the mixture and measure the
polarization. Wavelengths suitable to excite the fluorophore depend on the nature of the
fluorophore, as described above. Typically, one uses cut off filters to define awavelength range which is determined by the excitation and emission wavelengths of the
fluorophore. For fluorescein carboxyamide peptides, one would typically use an
excitation cutoff filter of 485nM. Also, non-polarizing material should be used for any
component of the ap~aldLus, including the test chambers in which samples are evaluated,
which will be in the light path. Plastics and fiber optics are generally avoided in such
uses in favor of optical glasses, quartz, etc.
In addition to using standard fluorometers, one can also use specialty
fluorometers such as the Jolley FPM1 (for individual samples) or the Jolley FPM2 (for
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high-throughput assays in 96-well format). Such fluorometers have been optimized for
polarization measurements and have much higher sensitivity than standard fluorometers.
Other automated equipment may provide both the admixing step combined with
the other steps, and/or the comparison of the polarization of the control mixture without
the test substance and the test mixture with the test substance. One of skill in the area of
automation may use various apparatus to substantially automate the assays of this
invention.
In yet another aspect, the invention provides components or reagents, e.g.,a
protein bearing a covalently linked fluorophore, useful in the methods of the invention.
The components or reagents can further be packaged in a kit with instructions for use in
the described methods.
III. The Inhibitors
Once a compound has been identified as an inhibitor, it can be produced using
known methods, such as by recombinant methods of protein production or chemical
synthesis. It can also be obtained from the source in which it was initially identified.
A. Counterscreens
Having identified an inhibitor of a protein:ligand association by means of the
assay of this invention, one may use counterscreens against one or more other protein:
ligand pairs to identify nonspecific inhibitors, or confirm inhibitor specificity. Test
compounds identified as inhibitors by the method of this invention may be further
evaluated for binding activity with respect to one or more additional proteins of interest,
or with respect to additional proteins containing the domain(s), using various
approaches, a number of which are well known in the art. The counterscreen may be
carried out using the methods and materials of the subject invention, or may be
conducted using alternative approaches for the detection of direct or competitive
binding, including, e.g., cell-based assays or surface plasmon resonance (BIAcore~)
technology [see, e.g., Panayotou et al, Mol. Cell. Biol.. I3: 3567-3576 (1993)].The inhibitors identified in the assay system of this invention can be further
evaluated by conventional methods for assessing toxicological and pharmacological
activity. For example, test compounds identified as inhibitors may further be evaluated
for activity in inhibiting cellular or other biological events mediated by a pathway
involving the protein:ligand interaction of interest using a suitable cell-based assay or an
animal model. Cell-based assays and animal models suitable for evaluating inhibitory
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activity of a test compound with respect to a wide variety of cellular and other biological
events are known in the art. New assays and models are regularly developed and
reported in the scientific literature.
By way of nonlimiting example, compounds which bind to an SH2 domain
involved in the transduction of a signal leading to asthma or allergic episodes may be
evaluated in a mast cell or basophil degranulation assay. The inhibitory activity of a test
compound identified as an SH2 inhibitor by the method of this invention with respect to
cellular release of specific mediators such as hi~t~mine, leukotrienes, hormonalmediators and/or cytokines, as well as its biological activity with respect to the levels of
phosphatidylinositol hydrolysis or tyrosine phosphorylation can be characterized with
conventional in vitro assays as an indication of biological activity. [See, e.g., Edward L.
Barsumian et al, Eur. J. Immunol.. 11:317 323 (1981); M. J. Forrest, Biochem.
Pharmacol., 42:1221-1228 (1991) (measuring N-acetyl-betagluco.s~min~ e from
activated neutrophils); and V. M. Stephan et al., J. Biol. Chem., 267:5434-5441 (1992)].
For example, histamine release can be measured by a radioimmllnf)assay using a
kit available from AMAC Inc. (Westbrook, ME). One can thus evaluate the biological
activity of inhibitors identified by the method of this invention and compare them to one
another and to known active compounds or clinically relevant compounds which can be
used as positive controls.
Generally speaking, in such assays IC50 scores of 150-300 uM are considered of
interest, scores of 50-150 uM are considered good, and scores below about 50 uM are of
high interest. Prior to or in addition to in vil~o models, inhibitors identified by this
invention may also be tested in an ex vivo assay for their ability to block
antigen-stimulated contraction of sensitized guinea pig tracheal strip tissue. Activity in
this assay has been shown to be useful in predicting the efficacy of potential anti-asthrna
drugs.
Numerous animal models of asthma have been developed and can be used [for
reviews, see Larson, "Experimental Models of Reversible Airway Obstruction", in THE
LUNG, Scientific Foundations, Crystal, West et al. (eds.), Raven Press, New York, pp.
953-965 (1991); Warner et al., Am. Rev. Respir. Dis., 141:253 257 (1990)]. Species
used in animal models of asthma include mice, rats, guinea pigs, rabbits, dogs, sheep and
- primates. Other in vivo models available are described in Cross et al., Lab Invest.,
63:162-170 (1990); and Koh, et al., Science, 256:1210-1213 (1992).
By way of further example, inhibitors identified by the method of this inventionwhich bind to a protein involved in the transduction of a signal involved in the initiation,
maintenance or spread of cancerous growth may be evaluated in relevant conventional in
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vitro and in vivo assays. See e.g., Ishii et al., J. Antibiot., XLII:1877-1878 (1989); and
US Patent 5,206,249 (issued 27 April 1993).
B. Uses of Inhibitors Identified by This Invention
Inhibitors identified by this invention may be used as biological reagents in
assays as described herein for functional classification of a particular protein,
particularly a newly discovered protein. Families or classes of proteins may thus be
defined functionally, with respect to ligand specificity. Moreover, inhibitors identified
by this invention can be used to inhibit the occurrence of biological events resulting
from molecular interactions mediated by a the protein or protein:ligand pair of interest.
Inhibiting such interactions can be useful in research aimed at better understanding the
regulation and biological significance of such events.
Such inhibitory agents would be useful, for example, in the diagnosis, prevention
or treatment of conditions or diseases resulting from a cellular process(es) mediated by a
targeted interaction. For example, a patient can be treated to prevent the occurrence or
progression of osteoporosis or to reverse its course by ~(1mini~tering to the patient in
need thereof an SH2 binding or blocking agent which selectively binds Src SH2.
There are many other conditions for which phosphopeptide binding or blocking
agents may be useful therapeutically, including, e.g., breast cancer where the SH2
domain-cont~inin~ proteins Src, PLCgamma and Grb7 have been implicated. Other
relevant conditions include prostate cancer, in which case targeting Grb2, PLCg, and
PI3K, all of which contain SH2 domains, may be useful in treatment or prevention of the
disease. Inhibition of the interaction of Grb2 or Abl SH2 domains with Bcr-abl may be
useful to treat chronic myelogenous leukemia (CML) or acute myelogenous leukemia(AML).
Still other relevant applications of an PBP inhibitor would be to prevent
interferon-, growth factor-, or cytokine-mediated diseases (e.g. infl~mm~tory diseases)
by targeting the PBDs of STAT proteins. Agents that block the SH2 domains of
ZAP-70, which is involved in activation of T-cells, would be useful in the treatment of
autoimmune diseases. An inhibitor that blocks one or both SH2 domains of ZAP-70
would also be useful as an immunosuppressant to prevent rejection of skin and organ
transplants.
Likewise, by further way of example, SH3 inhibtors would be useful in the
diagnosis, prevention or treatment of conditions or diseases resulting from a cellular
processes mediated by an SH3-based interaction. For example, a patient can be treated to
prevent the occurence or progression of osteoporosis or to reverse its course by
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~rlmini~tering to the patient in need thereof an SH3 inhibitor which selectively binds to
or inhibits interactions with src SH3. There are many other conditions for which SH3
inhibitors can be used therapeutically, including restenosis, rheumatoid arthritis, gout,
asthma, emphysema, immune vasculitis, ulcerative colitis, psoriasis and acute
respiratory distress syndrome, in which an SH3 of neutrophil oxidase p47 and p67complex has been implicated. Other relevant conditions include chronic myelogenous
leukemia, in which case SH3 domains of Grb-2 are targeted. It has recently been shown
that the BCR-abl oncogene in CML participates in the ras pathway for growth
stimulation through its interaction with Grb-2. In these cells, inhibition of the interaction
of Grb-2 SH3 domains with the SOS oncogene will block its ability to stimulate cell
proliferation. Still other relevant conditions include cancers such as breast cancer,
glioblastomas, head and neck tumors and ovarian tumors, for which the SH3 domain of
Grb-2 would be targeted. For example, tumors with associated amplification of receptors
for EG~ and PDGF could be inhibited by blocking activation of the Ras pathway
through inhibition of the interaction between Grb-2 (SH3)and Ras. Furthermore, since
the SH3 domain of Src family kinases are believed to be involved in activation of T-
cells, B-cells, mast cells, and NK cells and since the SH3 domains of the tyrosine
kinases Tsk and Btk are believed to be involved in T-cell (Tsk SH3) and B-cell (Btk
SH3) function an SH3 inhibitor identified by the subject invention could be ~(lmini~tered
to a patient in need thereof to suppress immune function.
An inhibitor of a protein:ligand interaction identified by the method of this
invention can be formulated into a ph~rm~reutical composition containing a
pharmaceutically acceptable carrier and/or other excipient(s) using conventionalmaterials and means. Such a composition can be a~mini~tered to an animal, eitherhuman or non-human, for therapy of a disease or condition resulting from cellular events
involving the targeted protein-ligand interaction. Administration of such composition
may be by any conventional route (parenteral, oral, inhalation, and the like) using
apl)lo~,iate formulations as are well known in this art. The inhibitor can be employed in
admixture with conventional excipients, ie, pharmaceutically acceptable organic or
inorganic carrier substances suitable for parenteral ~-lmini~tration.
C. Pharmaceutical Compositions and Methods
i. Compositions
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Inhibitors identified by this invention can be formulated into pharmaceutical
compositions cont~ining a therapeutically (or prophylactically) effective amount of the
inhibitor in admixture with a pharmaceutically acceptable carrier and/or other excipients
(i.e., pharrnaceutically acceptable organic or inorganic carrier substances suitable for
parenteral ~Aminictration) using conventional materials and means. Such a carrier
includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol,
and combinations thereof. The carrier and composition can be sterile. The formulation
should suit the mode of a-lmini.~tration.
The composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. The composition can be a liquid solution,
suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, e~c.
In a specific embodiment, the composition is formulated in accordance with routine
procedures as a pharmaceutical composition adapted for intravenous a-lmini~tration to
human beings. Typically, compositions for intravenous ~llmini.~tration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ease pain at the side of the injection.
Generally, the ingredients are supplied either separately or mixed together in unit dosage
form, for example, as a Iyophilized powder or water free concentrate in a herrnetically
sealed container such as an ampoule or sachette indicating the quantity of active agent.
Where the composition is to be a~1ministered by infusion, it can be dispensed with an
infusion bottle cont~ining sterile pharmaceutical grade water or saline. Where the
composition is ;l(lmini~tered by injection, an ampoule of sterile water for injection or
saline can be provided so that the ingredients may be mixed prior to ~-imini~tratjon.
Topical compositions include a ph~rmaçologically acceptable topical carrier~ such
as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such
carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty
acid esters, or mineral oils. Other topical carriers include liquid petroleum, isopropyl
palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in
water, or sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants,
humectants, viscosity stabilizers, and similar agents may be added as necessary.Materials and methods for producing the various formulations are well known in
the art [see e.g US Patent Nos. 5,182,293 and 4,837,311 1.
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ii. Methods
The invention provides methods of treating, preventing and/or alleviating the
symptoms and/or severity of a disease or disorder referred to above by a(lmini.~tration to
a subject of the inhibitor in an amount effective therefor. The subject will be an animal,
including but not limited to anim~l~ such as cows, pigs, chickens, etc., and is preferably
a m~mm~l, and most preferably human. By "m~mm~l~" is meant rodents such as mice,rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, nonhuman primates and
humans. Such effective amounts can be readily determined by evaluating the inhibitors
identified by this invention in conventional assays well-known in the art, including
assays described herein.
Administration of such composition may be by any conventional route using
appl()p"ate formulations as are well known in this art. Various delivery systems are
known and can be used to ~1mini.~ter the inhibitor, e.g., encapsulation in liposomes,
microparticles, microcapsules. One mode of delivery of interest is via pulmonarya-lmini.stration. Other methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, nasal and
oral routes. The inhibitor may be ~lmini~tered by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g, oral mucosa, rectal and
intestinal mucosa, etc.) and may be ~flmini.~tered together with other biologically active
agents.
Administration can be systemic or local. For treatment or prophylaxis of nasal,
bronchial or pulmonary conditions, preferred routes of atlmini~tration are oral, nasal or
via a bronchial aerosol or nebulizer. In specific embodiments, it may thus be desirable to
aflminicter the inhibitor locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during surgery, topical
application, by injection, by means of a catheter, by means of a suppository, or by means
of a skin patch or implant, said implant being of a porous, nonporous, or gelatinous
material, including membranes, such as sialastic membranes, or fibers.
Administration to an individual of an effective amount of the inhibitor can also be
accomplished topically by a(lmini~tering the compound(s) directly to the affected area of
the skin of the individual. In certain instances, it is expected that the inhibitor may be
disposed within devices placed upon, in, or under the skin. Such devices include patches,
implants, and injections which release the compound into the skin, by either passive or
active release mech~ni~m~
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The amount of the inhibitor which will be effective in the treatment or prevention
of a particular disorder or condition will depend on the nature of the disorder or
condition, and can be deterrnined by standard clinical techni~ues. In addition, in vitro or
in vivo assays may optionally be employed to help identify optimal dosage ranges.
Effective doses may be extrapolated from dose-response curves derived from in vitro or
animal model test systems. For example, a typical effective dose of the inhibitor is in the
range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of
m~mm~ n body weight, a(lministered in single or multiple doses. Generally, the
inhibitor may be a~mini.stered to patients in need of such treatment in a daily dose range
of about 1 to about 2000 mg per patient.
The precise dosage level of the inhibitor, as the active component(s), should bedetermined by the attending physician or other health care provider and will depend
upon well known factors, including the phosphopeptide binding interaction under
consideration, the route of atlmini.stration, and the age, body weight, sex and general
health of the individual; the nature, severity and clinical stage of the disease, and the use
(or not) of concomitant therapies.
C. Kits
The invention also provides a pharmaceutical pack or kit comprising one or more
containers filled with one or more of the ingredients of the pharmaceutical compositions
of the invention. Optionally associated with such container(s) can be a notice in the form
prescribed by a governrnental agency regulating the manufacture, use or sale of
pharmaceutical or biological products, which notice reflects approval by the agency of
manufacture, use or sale for human ~mini.stration.
Other components such as physiologically acceptable surfactants (e.g., glycerides),
excipients (e.g., lactose), carriers, and diluents may also be included.
The following examples illustrate various aspects of this invention. These
examples do not limit the scope of this invention which is defined by the appended
claims. The contents of all references, pending patent applications, published patent
applications, issued patents and information contained in web sites, cited throughout this
application (including the "Back~round" Section) are hereby expressly incorporated by
reference.
IV. Examples
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The following examples illustrate various aspects of this invention. These
examples do not limit the scope of this invention which is defined by the appended
claims.
The introduction of a 96-well plate reader (FPM2, Jolley Instruments) with a
high sensitivity towards fluorescein and fluorescein conjugates (in the low nanomolar
probe concentration range) has allowed the development of 96-well based FP assays.
These examples describe an FP assay and the necessary components for measuring the
binding of compounds to the Src-SH2 domain.
ExamPle 1 - PEPTIDE SYNTHESIS
Peptide synthesis was performed manually using Fmoc-Rink amide resin [4-
(2',4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy resin; (Advanced Chemtech)] with
substitution levels of 0.3-0.6 mmole/g. Standard FMOC synthesis methods were used.
The wash and deprotection solvent used was dimethyl acetamide (DMA); the coupling
solvent used was N-methylpyrrolidone (NMP). For amino acid couplings, four
equivalents of amino acid, four equivalents of coupling reagent, 2-(1 H-benzotriazole- 1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and eight equivalents of N-
methylmorpholine (NMM) were used per equivalent of amine on the resin. Amino acids
used were Fmoc-Gly, Fmoc-Glu(Tbu), Fmoc-lle, Fmoc-Thr(Tbu), and Fmoc-Tyr(Tbu).
Fmoc deprotection was done using 20% piperidine in DMA.
Peptides were phosphorylated on the solid-phase using standard methodology
[see, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. Resins and l-l,H-
tetrazole were dried under vacuum over P2O5 overnight. To a portion of resin (0.1-0.5 g)
mixed with 50 equivalents of 1-1 ,H-tetrazole was added I ml/0. I g of dry DMA. The
resin was stirred and swelled for 20 minutes. 10 equivalents of dibenzyl N,N-
diethylphosphoramidite (Toronto Research Chemicals, Inc.) was then added, the resin
mixture was stirred for 15 minutes, sonicated for 30 minutes, and stirred for 15 minutes.
Resin was washed 5x with DMA. Oxidization was performed by adding 3 equivalents
of chlorobenzylperoxide in DMA, with stirring for 30 minutes, and sonication for 30
minutes. Finally, peptides were washed 5X DMA, 3X CH2CI2, and 3X MeOH, followed
by vacuum drying overnight.
Final cleavage from the resin and side chain deprotection was done using
90:10:10:5 ratios oftrifluoroacetic acid (TFA):H2O:ethane dithiol (EDT):tri-isopropyl
silane (TIPS). Scavengers were added to resin, followed by addition of TFA with
stirring for 2.5 hours. Resin was filtered off. TFA was removed by blowing under N2
for several hours. Crude peptide slurry was resuspended in water, and extracted three
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times with an equal portion of ice-cold diethyl ether. Excess ether was removed by
blowing under N2. The crude peptide mix was Iyophilized overnight.
All peptides were purified in the following manner. Crude Iyophilized peptides
were dissolved in DMSO at a concentration of 100-300 mgs per ml. Peptides were
purified on a Semi-Preparative reverse phase HPLC column (Vydac). A series of 15-30
ul injections of crude peptides in 100% DMSO were used. Purity was checked using an
analytical reverse phase HPLC column, with a diode-array spectrophotometer. One pass
was adequate to give greater than 90% purity.
Example 2 - PROBE SYNTHESIS
An exemplary fluorescent probe (Fig. 1 or Fig. 5) was designed to consist of thefluorescent moiety, 5-carboxyfluorescein, coupled to a pentapeptide ligand based on the
known Src-SH2 high affinity tetrapeptide sequence derived from the core middle-Tantigen. The peptide ligand sequence is GpYEEI, cont~ining the core middle-T antigen
high-affinity Src-SH2 sequence (pYEEI), with an N-terminal glycine for ease of
coupling.
5-Carboxyfluorescein was chosen for several reasons. It is one of the few
defined isomer fluoresceins available. It yields a conjugate with less flexibility than
many available fluoresceins, which is important for minimi7.ing the "propeller effect"
that can interfere with FP based measurements and an activated version is commercially
available (Molecular Probes, Inc.).
The probe sequence was prepared as follows: The peptide GpYEEI was coupled
to the fluorescein moiety directly on the resin. The peptide sequence was assembled on
Rink amide resin~ and phosphorylated as described above. The resultant sequence was
Fmoc-GpYEEI-RlNK. The peptide/resin was deprotected with 20% piperidine in DMA,
removing the FMOC protecting group, and leaving the free-amino terrninus available for
coupling. After thorough washing with DMA, 1.1 equivalent of 5-carboxyfluorescein
succinimidyl ester (Molecular Probes) was added with 6 equivalents of
diisopropylethylamine. Coupling was carried out for 1.5 hours, followed by I NMP and
3 DMA washes, and by a repeat coupling as above. The completed probe was cleavedand worked up as described above, yielding a probe termed FMTI.
Two exemplary labeled probes for Src SH2 domain, prepared as described above
are:
fluorescein-G-pYEEI-NH2 and
fluorescein-pYpYpYIE-NH2
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Example 3 - PROTEIN PRODUCTION - Src
Human Src encoding residues 145-251 [Tanaka, A. and Fujita, D.J., Mol. Cell.
Bioh 6:3900-3909 (1986)] was cloned into the pT7 expression vector and transformed
into ~ coli BL21 (DE3). Protein was produced ~rom the growth and induction of 27liters of culture in minim~l medium. In a typical preparation, the culture was grown at
37~C to an optical density (OD) of 1.0 at 595 nm. The culture was induced with 1 mM
isopropyl-~-D-thiogalactopyranoside (IPTG) and the temperature was dropped to 25~C.
The culture was harvested 21 hours later.
The cells were Iysed in 50 mM potassium phosphate, 250 mM NaCI, 5 mM DTT,
2 mM E;DTA, 1 mm PMSF, pH 7.0 using a French pressure cell at 16,000 psi. The
protein was purified over carboxy-sulfon (J. T. Baker), a weak-strong cation exchanger.
The column was equilibrated with 50 mM potassium phosphate, 5 mM DTT, 0.02%
NaN3, pH 7.0 and loaded with filtered bacterial lysate at 2 ml/minute. The Src protein
was eluted with a 1 M NaCl gradient. The eluate was concentrated using a Centriprep
10 concentrator (Amicon, 10,000 MW cutoff) and centrifuged at 3000 x g. The protein
was then purified by gel filtration on a Sephacryl S-100 (Pharmacia) column equilibrated
with 20 mM potassium phosphate,50 mM NaCl,5 mM DTT, 1 mM EDTA, 0.02%
NaN3, pH 7.4. Purity, as measured by SDS gel electrophoresis and RP-HPLC, is >95%.
The purified protein is stored frozen in 50 mM potassium phosphate,500 mM NaCI,
10% glycerol, 5 mM DTT, 5 mM EDTA, 0.02% NaN3, pH 7.4.
Example 4 - A FLUORESCENCE-POLARIZATION BASED SRC-SH2 BINDING
ASSAY
A. FP - General
All polarization methods were performed on an FPM2 96-well plate reader
(Jolley), with standard cutoff filters (excitation = 485 nm; emission = 530 nm).Saturation experiments were used to explore various conditions for the assay, with the
aim of maximi7in~ the protein and assay stability, and of determining Kds of theprotein/probe interaction.
B. Saturation Experiments
For saturation experiments, fixed concentrations of probe were used, and
increasing concentrations of Src-SH2 were added. This is the reverse of the way such
assays are commonly done with radioactive ligands.
A saturation experiment was conducted in which the Src-SH2 domain is varied in
concentration from a high of 40 uM to a low of 39 nM. The fluorescent probe FMT1(Fig. I ) was kept at a fixed concentration of 20 nM. Amino acid analysis was used to
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quantitate both the protein and probe concentrations. The results are illustrated in the
saturation curve and Scatchard analysis of Figs. 2A and 2B, respectively from this
experiment.
The data shown in Figs. 2A and 2B show that the affinity of the probe for the
receptor domain is applo~l,ate for conducting competitive binding assays and that
saturable binding to a single site is observed, consistent with the assay of this invention
and with competitive, reversible binding to a single site.
C. Competition Experiments
For competition experiments, fixed concentrations of probe and protein were
used, and increasing concentrations of peptides were added.
The designed probe FMT- 1 has a Kd of ~0.3 uM towards Src-SH2 in the
standard buffer conditions. Some variation in observed Kd values will occur withchanges in buffer conditions.
As illustrated in Fig. 3, a Src competition assay (2% DMSO buffer) was
conducted for the following tetrapeptides: Ac-pYEEI (open circle); Ac-pYpYEEI
(closed square); Ac-pYGGL (plus sign); Ac-pYEDL (open triangle); Ac-DGVpYTGL
(closed triangle). Fig. 3 shows the competition curve plotting % probe binding vs.
inhibitor concentration obtained in one set of experiments.
Table I
Representative Peptide competitive IC50s
Sequences Number IC50
Ac-pYEEI I 8
Ac-pYpYEEI 2 1.5
Ac-pYpYpYIE 3 0.5
Ac-pYTGL 4 100
Ac-pYGGL 5 700
Data of this sort demonstrates that the materials and methods of this invention
can be used to conduct a competitive binding assay with inhibitory substances having a
range of IC50 values.
Illustrative protocols according to this invention are both manual and automatedand are performed as follows:
D. Manual Assays
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Two different plates can be run for each experiment - a 2% DMSO and 20%
DMSO plate.
Buffers
All buffer components were low-fluorescence grade (Panvera Corporation).
Standard (STD) buffer contains 20 mM phosphate (pH 7.4), 100 mM NaCl, 2 mM DTT,
1 mM EDTA, and 100 ug/ml BGG. Standard buffer is prepared by ple~ g NaCI,
EDTA and phosphate stocks in Millipore water or the cleanest available water supply.
The buffer is brought to volume in the same clean water.
The five buffers needed for these experiments are standard (STD) buffer, STD
Buffer + 4% DMSO, 100% DMSO, STD buffer with labelled probe alone and STD
buffer with Src protein and labelled probe.
One liter is made up of 100 ml of I M NaCI,
20 ml of I M phosphate (pH 7.4), 2 ml of 500 mM EDTA, 20 ml of 5 mg/ml BGG, and
858 ml of Clean Water.
The standard buffer is made up and stored at 4~C. Before each use, the DTT is
added at 2 mM. This standard buffer is employed to make up the Standard Buffer + 4%
DMSO stock also. This can be stored at 4~C as well and DTT can be added before use.
Protein and Control Peptide
The SRC protein is used at a concentration of 0.75 uM final. An example of
SRC stock solution is 416.6 uM in STD buffer. The peptide Ac-pYEEI is used at 100
uM final. It is desirable to make a 2X stock of this.
Probe
Fluor-GpYEEI is used as the probe at a concentration of 20 nM final. The probe
stock sent is 10 uM in STD buffer.
As one exarnple, to prepare 25 mls of protein and only 2 mls of probe alone, thefollowing steps are followed:
(a) 27 mls Standard buffer (add 2 mM DTT)
(b) Add Probe-1515 at 2X or 40 nM = 108uL
(c) Remove 2 mls for Probe Alone
(d) To the rem~inin~ 25 mls add SRC at 2X or 1.50 uM = 90 uL
This is now the Protein and Probe solution.
Secondary Stocks
Each compound/peptide and the control peptide is in separate tubes. They are at
2X stocks.
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It is desirable to make primary stock at 50 mM in 100% DMSO, then make the
secondary stocks in STD Buffer + 4% DMSO at the appropriate concentration for the
desired experiment.
One example of a secondary stock is made by combining 2 mM stock in STD
Buffer + 4% DMSO for one embodiment of an assay (Long Format, 2% DMSO).
Another is 5 mM stock in 100% DMSO for Lon~ Format assay with 20% DMSO.
Another secondary stock solution is 400 uM stock in STD buffer + 4% DMSO for Short
Format assay #1 and #2 -2% DMSO. 1 mM stock in 100% DMSO for Short Format #1
and #2 -20% DMSO
Lon~ Format Assay
A 1:2 dilution is used with first well at 1 mM final. For Plate 1 - 2% DMSO, theassay protocol is as follows:
(a) 50 ul Standard Buffer + 4% DMSO in column 2-12.
(b) 100 ul of 8 different compounds (2 mM stock/SB+4% DMSO) in each
well in column 1 row A-H.
(c) Serially dilute 50 ul (1:2) horizontally down plate to column 10.
(d) Add 50 ul probe alone to column 12.
(e) Add 50 ul protein and probe to column 1-11.
(fl 1 oo ul final volume per well.
(g) Read plate on FPM2.
For Plate 1 - 20% DMSO, the assay protocol is as follows:
(a) 20 ul 100% DMSO in columns 2-12.
(b) 40 ul of 8 different compounds (5 mM stock/100% DMSO) in each well
in column 1 row A-H.
(c) Serially dilute 20 ul (1:2) horizontally down plate to column 10.
(d) Add 50 ul probe alone to column 12.
(e) Add 50 ul protein and probe to column 1-11.
(fl Add 50 ul STD buffer to entire plate.
(g) 100 ul final volume per well.
(h) Read plate on FPM2.
Short Format #l Assay
A 1:3 dilution with first well at 200 uM final. Compounds/peptides are used in
duplicate. These examples are for a 1:3 dilution but volumes may be changed to
accommodate any desired dilution.
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For Plate I - 2% DMSO, the assay protocol is as follows:
(a) 50 ul Standard Buffèr + 4% DMSO in row B-D and F-G.
(b) 75 ul of 11 different compounds (400 uM/SB+4% DMSO) in duplicate
each well in column 1-10, row A and column 1-12, row E.
(c) Serially dilute 25 ul (1:3) vertically down plate from A-D then from E-H.
(d) Add 50 ul probe alone to column 12.
(e) Add 50 ul protein and probe to column 1-11 A-D and 1-12 E-H.
(f) 100 ul final volume per well.
(g) Read plate on FPM2.
For Plate 2 - 20% DMSO, the assay protocol is as follows:
(a) 20 ul 100% DMSO in row B-D and F-G.
(b) 30 ul of 11 different compounds (1 mM/100% DMSO) in duplicate each
well in column 1-10, row A and column 1-12, row E.
(c) Serially dilute 10 ul (1 :3) vertically down plate from A-D then from E-H.
(d) Add 50 ul probe alone to column 12.
(e) Add 50 ul protein and probe to column 1-11 A-D and 1-12 E-H.
(f) 100 ul final volume per well.
(g) Add 30 ul STD buffer to entire plate.
(h) Read plate on FPM2.
Short Format #2 AssaY
A 1 :3 dilution with first well at 200 uM final. Compounds/peptides are used in
singly. These examples are for a 1 :3 dilution but volumes may be changed to
accommodate whatever dilution you wish.
For Plate I - 2% DMSO, the assay protocol is as follows:
(a) 50 ul Standard Buffer + 4% DMSO in column 2-4 and 6-8 in 10-12.
(b) 75 ul of 16 different compounds in (400 uM/SB+4% DMSO) each well in
column 1 and 5.
(c) 75 ul of 6 different (400 uM/SB+4% DMSO) compounds to column 9, A-
F only. Total of 22 compounds.
(d) Serially dilute 25 ul (1 :3) horizontally down plate from 1 -4 then from 5-8,
then from 9-12.
(e) Add 50 ul probe alone to row H, columns 9-12.
(f) Add 50 ul protein and probe row A-H, columns 1-8 and A-G, columns 9-
12.
(g) There should be 100 ul final volume per well.
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(h) Read plate on FPM2.
For Plate 2 - 20% DMSO assay, the following protocol is followed:
(a) 20 ul 100% DMSO in column 2-4 and 6-8 and 10-12.
(b) 30 ul of 16 different compounds (1 mM/100% DMSO) in each well in
column 1 and 5.
(c) 30 ul of 6 different compounds (1 mM/100% DMSO) to column 9, A-F
only. Total of 22 compounds.
(d) Serially dilute 10 ul (1:3) horizontally down plate from 1-4 then from 5-8,
then from 9-12.
(e) Add 50 ul probe alone to row H, column 9-12.
(f) Add 50 ul protein and probe row A-H, columns 1-8 and A-G, columns 9-
12.
(h) Add 50 ul STD buffer to entire plate.
(i) There should be 100 ul final volume per well.
(j) Read plate on FPM2.
E. Automated Assays
( 1 ) Short dilution
This assay is run with l/3 dilution steps with first well at 200 uM, 4 wells
down, 22 cpds/plate, "landscape" orientation of plate with respect to Genesis deck.
(i) 20% DMSO
(ii) 2% DMSO
(2) Lon~ dilution
This assay is run with 1/2 dilution steps with first well at 1 mM, 10 wells
down, 8 cpds/plate, "landscape" orientation of plate with respect to Genesis deck.
(i) 20% DMSO
(ii) 2% DMSO
Dilution
5 Buffers:
- Buffer + 4% DMSO
- 100% DMSO
- Buffer with probe alone
- Buffer with protein and probe
- Buffer
(3) Short Format
Each compound is in a separate well of a 96 well plate.
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Plate 1 - 2% DMSO
- 50 ul 4~/0 DMSO in columns 2? 3. 4, 6, 7, 8, 10, 11 and 12.
- 75 ul of 22 different compounds (400 uM stock, 4% DMSO) each well
in all rows of columns I and 5 and rows A through F of column 9.
- 75 ul of Buffer ~ 4% DMSO to wells G9 and H9.
- Serially dilute 25 ul (1 :3) across plate from I to 4, throwing away last
25 ul, then from 5 to 8, and 9 to 12. This leaves 50 ul of liquid/well.
- Add 50 ul protein and probe to the entire plate except wells H9, 10, 11
and 12.
- Add 50 ul probe alone to wells H9, 10, 11 and 12.
- 100 ul final volume per well.
- Read plate.
Plate 2 - 20-% DMSO
- 20 ul 100% DMSO in columns 2, 3, 4, 6, 7, 8, 10, 11 and 12.
- 30 ul of 22 different compounds (1 mM stock, 100% DMSO) each well
in all rows of columns I and 5 and rows A through F of column 9.
-30uloflO0%DMSOtowellsG9andH9.
- Serially dilute 10 ul (1 :3) across plate from 1 to 4, throwing away last
25 ul, then from 5 to 8, and 9 to 12. This leaves 50 ul of liquid/well.
- Add 50 ul protein and probe to the entire plate except wells H9, 10, 11
and 12.
- Add 50 ul probe alone to wells H9, 10, 11 and 12.
- Add 30 ul STD buffer to entire plate.
- 100 ul final volume per well.
- Read plate.
(4) Lon~ Format
Each compound is located in a separate well of a 96 well plate.
Plate I - 2% DMSO
- 50 ul 4% DMSO in columns 2-12.
- 100 ul of 8 different compounds in each well in column I, row A-H.
- Add 50 ul of std. 3 to well A11.
- Serially dilute 50 ul (1 :2) across plate to column 10, throwing away last
50 ul (all rows).
- Serially dilute 50 ul from well Al l down to Hl l, throwing away last 50
ul.
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- Add 50 ul protein and probe to the entire plate except A12, B12, C12
and D12.
- Add 50 ul probe alone to A12, B12, C12 and D12.
- 100 ul final volume per well.
- Read plate.
Plate 2 - 20-% DMSO
- 20 ul 100% DMSO in rows 2-12.
- 40 ul of 8 different compounds in each well in column 1, row A-H.
- Add 20 ul of std. 4 to well Al l .
- Serially dilute 20 ul (1 :2) across plate to column 10 throwing away last
20 ul (all rows).
- Serially dilute 20 ul from well A11 down to H11, throwing away last 20
ul.
- Add 50 ul protein and probe to the entire plate except A 12, B 12, C 12
and D12.
- Add 50 ul probe alone to A12, B12, C12 and D12.
- Add 30 ul STD buffer to entire plate.
- 100 ul final volume per well.
- Read plate.
* Once diluted, the plate can be read between S and 30 minutes.
* Assay plate is transferred to FPM2 machine for 3 minute read.
Example 5 - FP-BASED ZAP, SYK, and LCK ASSAYS
In addition to the example of the Src SH2 domain and its phosphoTyr-containing
peptide ligand which is linked to the fluorophore which are exemplified in Examples 1-4
above, the assay has also been used for the proteins Zap, Syk and Lck. Those three
proteins are produced analogously to the production of Src in E. coli, as described above
with slight variations in production parameters such as salt concentration, DTT
concentration, protein concentration, temperature and the like.
While Src has a single SH2 domain, ZAP and Syk comprise two SH2 domains and the
Lck protein comprises one SH2 domain and one SH3 domain.
The "first proteins" produced for these assays are produced generally as
described for Src aal45-251 in Example 3. The proteins are represented in the table
below. The term "NC" means that the first protein contains both the N terminal and C
terminal SH2 domains. The sequences of Zap, Syk and Lck are known in the art. See
also PCT/US96/13918
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Table I
First Amino Acid Residues of the
Protein Naturally Occurrin~ Protein
- NC-ZAP aal-259
NC-Syk aal-260
Lck aa 109-266
The probes for each assay are designed and used as described above. For
example, an exemplary probe for N,C-ZAP proteins7 i.e., proteins cont~inin~ the two
SH2 domains of human ZAP in series, is fluorescein-GpYNELNLGRRGEEpYEVL-
NH,. As another example, an exemplary probe for N,C-Syk proteins, i.e., proteinscont~ining the two SH2 domains of human Syk in series, is fluorescein-
ApYTGLSTRNQETpYETL-NH2. The SRC probe was also used with Lck.
The Lck protein has been assayed against fluorophore-labeled phosphopeptide
ligand for the SH2 domain, fluorophore-labeled peptide ligand for the SH3 domain and
fluorophore-labeled phosphopeptide double ligand for the SH2 and SH3 domains.
The assay format described in Example 4 is reproduced for these binding pairs
and results obtained.
The data shown in Figs. 2A and 2B show that the affinity of the probe for the
receptor domain is appropriate for conducting competitive binding assays and that
saturable binding to a single site is observed, consistent with the assay of this invention
and with competitive, reversible binding to a single site. The data obtained from the
saturation assay can, within the methods of the present invention, be used to assess
whether a particular probe would be useful. For example, if a probe performs
at a particular level in a saturation assay (e.g." Kd <I 0uM and mP difference values >50
(difference in observed polarization in the presence and absence of protein)), it is
indicative of its suitability for use in a competition assay of this invention.
Example 6 - A FLUORESCENCE-POLARIZATION BASED HUMAN
GRB2-SH2 BINDING ASSAY
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A. Assay- General
All assay methods, including buffers and instrumental usage, were perforrned
generally as in the case of Src SH2 assay described in the previous examples, but
substituting a human GRB2 protein (for Src SH2) and a probe specific for the GRB2
Sl I2 domain (in place of the Src-specific probe). The human GRB2 protein tested in the
assay comprised amino acid residues 55 - 152 of huGRB2 [see Cell 70: 431-442 (1992)],
although longer sequences encompassing the SH2 domain may also be used. Production
and purification of the protein domain was similar to methods described above,
especially the use of a phosphotyrosine column to purify the GRE~2 SH2domain.
B. Saturation Experiments
As in previous examples, saturation experiments were perforrned with fixed
concentrations of probe and increasing concentrations of Grb-2-SH2.
The following Grb-2-specific SH2 probe (" FPgb2") was synthesized using the
techniques described in Example 1
(~H O
J~ I OH ~p-P-OH
f ~ b~NJ~N~ NJ~NHz
FPgb2, Fluor-GpYVNV-NH2
in which "Fluor" represents 5-carboxyfluorescein conjugated through an amide bond to
the peptide containing the sequence GpYVNV. That tetrapeptide sequence is specific for
Grb-2 SH2 domain binding. This probe had applop-iate characteristics and an
appropriate saturation curve with the GRB2 SH2 protein to (e.g., appropriate Kd and
total mP difference on protein binding) to serve as a probe for a competition assay.
Example 7- A FLUORESCENCE-POLARIZATION BASED Src SH3 BINDING
ASSAY
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A. Assay- General
All assay methods, including buffers and instrumental usage, were performed
generally as in the case of Src SH2 assay described in a previous example(s), but
substituting a protein cont~ining peptide sequence spanning the Src SH2 and SH3
domains (amino acid residues 84-251) [see Mol. Cell. Biol. 7(5): 1978-1983 (1987)], and
a probe specific for the Src SH3 domain (in place of the Src-specific SH2 probe). Note
that larger or smaller Src proteins may be used, so long as they include the Src SH3
domain (with or without the Src SH2 domain). Production and purification of the
protein were essentially as described above, e.g., see Example 3. The following details
are provided:
Src-SH2-SH3 Domain Purification:
I) French press Iysis, 50mM Potassium phosphate, SmM DTT, 2mM EDTA, ImM
PMSF, pH 7.0
2) 40um WP Carboxy-sulfon column --~633mgs
3) Phosphotyrosine Agarose column-->512mgs
4) Dialysis against 50mM Potassium phosphate, 10% Glycerol, 500mM NaCl,
5mM EDTA, 0.02% NaN3, pH 7.0
B. Saturation Experiments
As in a previous example(s), saturation experiments were performed with fixed
concentrations of probe and increasing concentrations of Src-SH2-SH3 protein.
The following Src SH3 specific probe ("FPSI 13 ") was synthesized using the
techniques described in Exarnple 1:
Fluor-PLARRPLPPLP-NH2
in which "Fluor" represents 5-carboxyfluorescein conjugated through an arnide bond to
the peptide containing the sequence PLARRPLPPLP, which is specific for Src SH3
domain binding. This probe had appropriate characteristics and an appropriate
saturation curve with the Src protein to (e.g., appropriate Kd and total mP difference on
protein binding) to serve as a probe for a competition assay.
~ Exarnple 8- A FLUORESCENCE-POLARIZATION BASED Src SH2-SH3 DOUBLE
BINDING ASSAY
A. Assay- General
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Proteins such as Src contain both an SH2 and an SH3 domain. Certain proteins
can bind to such SH2-SH3 proteins not through SH2 or SH3 domains alone, but through
both domains at once. One example is the protein, pl30CAS, which is thought to bind
Src via both SH2 and SH3 domains. The SH3 and SH2 binding sequences of p 130CAS
have been identified by deletional and site-specific mutagenesis (Nakamoto et. al.. JBC
271, 8959-8965, 1996). Residues 733-738 (RPLPSP) have been shown to be involved in
binding (presumably to the SH3 domain), as has residue Tyr-762 (presumably
phosphorylated and responsible for binding Src SH2). Fluorescent probes for FP assays
were designed based on these sequences. Such probes can be used in assays that allow
simultaneous screening for both SH2-specific and SH3-specific inhibitors. One such
probe ("FPC130 ") is the following:
Fluor-RPLPSPPKFTSQDSPDGQYENSEGGWMEDpYDWHL
This is the native sequence from pl30CAS (733-767). Derivative probes based
on the foregoing but using different component SH2 and/or SH3 sequences may be used
to vary the affinity or protein specificity, and especially to increase the overall affinity of
probe for the desired target protein.
All assay methods, including buffers and instrumental usage, were performed
generally as in the case of Src SH2 assay described in Example 4, but substituting a
protein containing peptide sequence spanning the Src SH2 and SH3 domains (for Src
SH2) and the SH2-SH3 probe described above (in place of the Src-specific SH2 probe).
B. Saturation Experiments
As in a previous example(s), saturation experiments were performed with fixed
concentrations of probe and increasing concentrations of protein (Src-SH2-SH3 in this
case).
The Src SH2-SH3 specific probe, FPC130, was synthesized using the techniques
described in Example 1:
Fluor-RPLPSPPKFTSQDSPDGQYENSEGGWMEDpYDYVHL
in which "Fluor" represents S-carboxyfluorescein conjugated through an amide bond to
the peptide containing the sequence PLARRPLPPLP, which is specific for Src Sh3
domain binding (any other fluorescent probe might be substituted). This probe had
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appropriate characteristics and an appropriate saturation curve with the Src protein to
(e.g., ~p,opl,ate Kd and total mP difference on protein binding) to serve as a probe for a
competition assay.
Example 9 - A FL UORESCENCE-POLA~IZATION BASED HUMAN
Src-SH2 BINDING ASSAYUSING AN ALTERNATE PROBE:
A. Assay- General
A competition assay was developed for Src-SH2 (applicable to any fragment of Srccontaining the SH2 domain) with a non-fluorescein fluorophore. The fluorophore is one
of a family of fluorescent molecules cont~ining the core fluorophore, 4,4-difluoro-4
bora-3a,4a-diaza-s-indacene (commercially available from Molecular Probes Inc.). The
structure of the fluorophore-peptide probe is:
OH O
~N ~I'HN~ JlH ~ NHZ
~3~
The fluorophore is BODIPY-TRX, and has spectral characteristics very similar to the
Texas Red family of fluorophores. The use of a red fluorophore widens the utility of the
assay, and permits one to screen test compounds that might have fluorescence
characteristics similar to fluorescein itself.
All assay methods, including buffers and instrumental usage, were performed
generally as in the case of Src SH2 assay described in Example 4, but substituting the
new probe, shown above, and using different filters on the fluorimeter, to match the
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spectral characteristics of this alternate probe. Specifically, the excitation/emission
filters used with the above are 591/635, compared to 485/530 for fluorescein.
Synthesis of the peptide portion of the probe was as described in Example 1, with
production of the peptide NH2-pYEEI. The probe was further synthesized by coupling
the free amine peptide, NH2-pYEEI, with the succinimidyl ester of the BODIPY-TRXprobe in a 66% DMSO buffer (34% O.lM NaBicarbonate). Completion of reaction was
monitored by silica gel TLC (4:1:1, butanol, H20, Acetic Acid solvent system), and was
done by 24 hrs. The resultant peptide was quite hydrophobic, and was purified by the
same TLC system as just described.
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B. Saturation Experiments
As in a previous example(s), saturation experiments were performed with fixed
concentrations of probe and increasing concentrations of Src-SH2 protein. The only
difference was the use of an alternate set of filters for the red probe, as described in A
above.
This probe showed an adequate Kd and total mP difference on protein binding to be
a probe for a competition assay.
This invention is not to be limited in scope by the specific embodiments
described herein. Indeed, various modifications of the invention in addition to those
described herein wil} become apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the scope of the appended
claims.
The disclosures of the patents, patent applications and publications cited herein
are incorporated by reference in their entireties.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Ariad Pharmaceuticals, Inc.
(ii) TITLE OF INVENTION: In Vitro Fluorescence Polarization Assay
(iii) NUM8ER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD
(B) STREET: 28 State Street, 24th Floor
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02109-1875
(v) COMPUTER READA8LE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US97/06746
(8) FILING DATE: April 18, 1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/029,870
(B) FILING DATE: 06-NOV-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Elizabeth A. Hanley
(B) REGISTRATION NUMBER: 33,505
(C) REFERENCE/DOCKET NUMBER: AFI-006PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617)227-7400
(B) TELEFAX: (617)742-4214
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~ CA 022~0067 1998-09-21
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= Tyr is Phosphorylated Tyrosine
(xi) S~Uu~N~ DESCRIPTION: SEQ ID NO:l:
Tyr Glu Glu Ile
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1,2
(D) OTHER INFORMATION: /note= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Tyr Tyr Glu Glu Ile
1 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Tyr Gly Gly Leu
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
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,
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= Tyr is Phosphorylated Tyrosine
(xi) S~u~ DESCRIPTION: SEQ ID NO:4:
Tyr Glu Asp Leu
(2) INFORMATION FOR SEQ ID NO:5:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v~ FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Mofified-site
(B) LOCATION: 4
(D~ OTHER INFORMATION: /note= Tyr is Phosphorylated Tyrosine
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:5:
Asp Gly Val Tyr Thr Gly Leu
1 5
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
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-
Phe Leu Val Arg Glu Ser
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Tyr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Asn Xaa Pro Tyr
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 022~0067 1998-09-21
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Asn Pro Xaa Tyr
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gly Tyr Glu Glu Ile
1 5
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1-3
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Tyr Tyr Tyr Ile Glu
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(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Tyr Thr Gly Leu
(2) INFORMATION FOR SEQ ID NO:13:
( i ) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2,14
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Gly Tyr Asn Glu Leu Asn Leu Gly Arg Arg Gly Glu Glu Tyr Glu Val
1 5 10 15
Leu
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 022~0067 1998-09-21
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2,13
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ala Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Gly Tyr Val Asn Val
1 5
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
CA 022~0067 l998-09-2l
(xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:16:
Gly Tyr Val Asn Val
1 5
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Pro Leu Ala Arg Arg Pro Leu Pro Pro Leu Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Pro Leu Ala Arg Arg Pro Leu Pro Pro Leu Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 30
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
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.
t
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Arg Pro Leu Pro Ser Pro Pro Lys Phe Thr Ser Gln Asp Ser Pro Asp
1 5 10 15
Gly Gln Tyr Glu Asn Ser Glu Gly Gly Trp Met Glu Asp Tyr Asp Tyr
Val His Leu
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2-4
(D) OTHER INFORMATION: note/= Tyr is Phosphorylated Tyrosine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gly Tyr Tyr Tyr Ile
1 5
AFI\006PC\SEQLIST DOC
