U.S. patent application number 09/752723 was filed with the patent office on 2002-02-07 for engineered protein kinases which can utilize modified nucleotide triphosphate substrates.
This patent application is currently assigned to Princeton University. Invention is credited to Shokat, Kevan M..
Application Number | 20020016976 09/752723 |
Document ID | / |
Family ID | 26724237 |
Filed Date | 2002-02-07 |
United States Patent
Application |
20020016976 |
Kind Code |
A1 |
Shokat, Kevan M. |
February 7, 2002 |
Engineered protein kinases which can utilize modified nucleotide
triphosphate substrates
Abstract
Engineered protein kinases which can utilize modified nucleotide
triphosphate substrates that are not as readily utilized by the
wild-type forms of those enzymes, and methods of making and using
them. Modified nucleotide triphosphate substrates and methods of
making and using them. Methods for using such engineered kinases
and such modified substrates to identify which protein substrates
the kinases act upon, to measure the extent of such action, and to
determine if test compounds can modulate such action. Also
Engineered forms of multi-substrate enzymes which covalently attach
part or all of at least one (donor) substrate to at least one other
(recipient) substrate, which engineered forms will accept modified
substrates that are not as readily utilized by the wild-type forms
of those enzymes. Methods for making and using such engineered
enzymes. Modified substrates and methods of making and using them.
Methods for using such engineered enzymes and such modified
substrates to identify the recipient substrates the enzymes act
upon, to measure the extent of such action, and to measure whether
test compounds modulate such action.
Inventors: |
Shokat, Kevan M.; (San
Francisco, CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS
1800 M STREET NW
WASHINGTON
DC
20036-5869
US
|
Assignee: |
Princeton University
|
Family ID: |
26724237 |
Appl. No.: |
09/752723 |
Filed: |
January 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09752723 |
Jan 3, 2001 |
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09367065 |
Nov 17, 1999 |
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09367065 |
Nov 17, 1999 |
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PCT/US98/02522 |
Feb 9, 1998 |
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PCT/US98/02522 |
Feb 9, 1998 |
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08797522 |
Feb 7, 1997 |
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60046727 |
May 16, 1997 |
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Current U.S.
Class: |
800/8 ; 424/94.5;
435/15; 435/194; 536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
G01N 33/5041 20130101; G01N 33/5008 20130101; G01N 33/5011
20130101; A61P 43/00 20180101; A01K 2217/075 20130101; G01N 33/5091
20130101; C07K 2319/00 20130101; C07K 2319/23 20130101; G01N
33/5026 20130101; A61P 31/18 20180101; A61K 38/00 20130101; A61P
35/00 20180101; G01N 33/502 20130101; A01K 2217/05 20130101; A61P
25/28 20180101 |
Class at
Publication: |
800/8 ; 424/94.5;
536/23.2; 435/15; 435/194 |
International
Class: |
A61K 038/51; A61K
038/52; A61K 038/53; C12Q 001/48; C07H 021/04; C12N 009/12 |
Goverment Interests
[0001] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others, as provided for by the terms of NSF Grant No.
MCB9506929 and DHHS NCI Grant No. R01CA70331-01.
Claims
1. A mutant multi-substrate enzyme which accepts at least one
orthogonal substrate analog, whereby catalytic activity results in
the combination of all or part of said orthogonal substrate with at
least one other substrate of said enzyme.
2. The mutant enzyme of claim 1 wherein said multi-substrate enzyme
is a transferase.
3. The mutant enzyme of claim 1 wherein said multi-substrate enzyme
is a signal transduction mediator.
4. A mutant protein kinase which accepts an orthogonal nucleotide
triphosphate analog as a phosphate donor substrate.
5. The mutant protein kinase of claim 4 wherein said mutant protein
kinase binds to the orthogonal nucleotide triphosphate with an
affinity which is higher than its affinity for the nucleotide
triphosphate which is the primary intracellular phosphate donor
substrate for the wild-type protein kinase.
6. The mutant protein kinase of claim 4 wherein said orthogonal
nucleotide triphosphate analog is an orthogonal analog of ATP.
7. The mutant protein kinase of claim 4 wherein said orthogonal
nucleotide triphosphate analog is a derivative of ATP having a
substituent comprising at least three carbon atoms covalently
attached to the N.sup.6 position of said ATP.
8. The mutant protein kinase of claim 7 wherein said orthogonal
nucleotide triphosphate analog is selected from the group
consisting of N.sup.6-(cyclopentyl)ATP,
N.sup.6-(cyclopentyloxy)ATP, N.sup.6-(cyclohexyl)ATP,
N.sup.6-(cyclohexyloxy)ATP, N.sup.6-(benzyl)ATP,
N.sup.6-(benzyloxy)ATP, N.sup.6-(pyrolidino)ATP, and
N.sup.6-(pipperidino)ATP.
9. The mutant protein kinase of claim 4 wherein said orthogonal
nucleotide triphosphate analog is N.sup.6-(cyclopentyl)ATP.
10. The mutant protein kinase of claim 4 which is a mutant protein
tyrosine kinase.
11. The mutant protein kinase of claim 10 which is a mutant of a
Src protein tyrosine kinase.
12. The mutant protein kinase of claim 10 which is a mutant of a
Rous sarcoma virus Src protein tyrosine kinase.
13. The mutant protein kinase of claim 4 wherein the amino acid
sequence differs from that of the wild type protein kinase in that
at least one amino acid at a position homologous to the position
selected from the group consisting of v-Src position 323 and v-Src
position 338 has been replaced with an amino acid selected from the
group consisting of alanine and glycine.
14. The mutant protein kinase of claim 4 wherein the amino acid at
a position homologous to v-Src position 338 has been replaced with
glycine.
15. The mutant protein kinase of claim 4 wherein the amino acid at
a position homologous to v-Src position 323 and the amino acid at a
position homologous to v-Src position 338 have been replaced with
alanine.
16. The mutant protein kinase of claim 4 wherein said mutant
protein kinase has been expressed as a fusion protein.
17. The mutant protein kinase of claim 16 which has been expressed
as fusion protein selected from the group consisting of a
glutathione-S-transferase fusion protein and a G-Histidine fusion
protein.
18. A nucleotide sequence which encodes a mutant multi-substrate
enzyme which accepts at least one orthogonal substrate analog,
whereby catalytic activity of said enzyme results in the
combination of all or part of said orthogonal substrate with at
least one other substrate of said enzyme.
19. A nucleotide sequence which encodes a mutant protein kinase
which accepts an orthogonal nucleotide triphosphate analog as a
phosphate donor substrate.
20. The nucleotide sequence of claim 19 wherein said nucleotide
sequence is selected from the group consisting of mRNA, cDNA, gDNA,
mitochondrial DNA, chloroplast DNA, satellite DNA, plasmid DNA,
viral RNA, and viral DNA.
21. A method for producing a nucleic acid sequence encoding a
mutant protein kinase which accepts an orthogonal nucleotide
triphosphate analog as a phosphate donor substrate, comprising the
steps of: (a) identifying, from the crystal structure of an
identical or homologous enzyme bound to its phosphate donor
substrate, one or more amino acids other than glycine which are
close enough to an atom of said bound phosphate donor substrate
that they would sterically exclude an orthogonal substituent
attached to the corresponding atom in said orthogonal nucleotide
triphosphate analog; and (b) mutating a nucleotide sequence which
encodes the wild-type protein kinase such that the nucleotide
triplets encoding one or more of the identified amino acids are
converted to nucleotide triplets that encode amino acids having
side chains that are sterically less bulky than the identified
amino acids.
22. The method of claim 21 wherein said amino acids of step (a) are
within about five angstroms of said atom of said bound phosphate
donor substrate.
23. The method of claim 21 wherein said phosphate donor substrate
is ATP.
24. The method of claim 23 wherein said atom is the N.sup.6 amino
group of ATP.
25. A method for producing a mutant protein kinase which accepts an
orthogonal nucleotide triphosphate analog as a phosphate donor
substrate, comprising expressing the mutant sequence of claim 21,
whereby said mutant protein kinase is produced.
26. A method for producing a nucleic acid sequence encoding a
mutant protein kinase which accepts an orthogonal nucleotide
triphosphate analog as a phosphate donor substrate, comprising the
steps of: (a) identifying, from the crystal structure of an
identical or homologous enzyme bound to its phosphate donor
substrate, one or more amino acids other than glycine which are
close enough to an atom of said bound phosphate donor substrate
that they would sterically exclude the orthogonal substituent
attached to the corresponding atom in said orthogonal nucleotide
triphosphate analog; (b) preparing a plurality of mutant protein
kinase-encoding nucleotide sequences having one or more mutations
in one or more nucleotide triplets encoding amino acids within ten
amino acids of said one or more amino acids, in both the amino
terminal and carboxy terminal directions; (c) expressing said
plurality of mutant kinase-encoding nucleotide sequences to produce
a plurality of mutant kinases; and d. testing said plurality of
mutant kinases to select one or more which have the ability to
utilize said orthogonal nucleotide triphosphate analog as phosphate
donor substrate.
27. A method for producing a mutant protein kinase which accepts an
orthogonal nucleotide triphosphate analog as a phosphate donor
substrate, comprising expressing one or more mutant sequence of
claim 26 found to express such a mutant protein kinase, whereby
said mutant protein kinase is produced.
28. A method for producing a nucleic acid sequence encoding a
mutant multi-substrate enzyme which accepts at least one orthogonal
donor substrate analog, whereby catalytic activity results in the
combination of all or part of said orthogonal donor substrate with
at least one other, recipient substrate of said enzyme, comprising
the steps of: (a) identifying, from the crystal structure of an
identical or homologous enzyme bound to its donor substrate, one or
more amino acids other than glycine which are close enough to an
atom of said bound donor substrate that they would sterically
exclude an orthogonal substituent attached to the corresponding
atom in said orthogonal donor substrate analog; and (b) mutating a
nucleotide sequence which encodes the wild-type form of said
multi-substrate enzyme such that the nucleotide triplets encoding
one or more of the identified amino acids are converted to
nucleotide triplets that encode amino acids having side chains that
are sterically less bulky than the identified amino acids.
29. The method of claim 28 wherein said amino acids of step (a) are
within about five angstroms of said atom of said bound donor
substrate.
30. A method for producing a multi-substrate enzyme which accepts
at least one orthogonal donor substrate analog, comprising
expressing the mutant sequence of claim 28, whereby said mutant
multi-substrate enzyme is produced.
31. A method for producing a nucleic acid sequence encoding a
mutant multi-substrate enzyme which accepts at least one orthogonal
donor substrate analog, whereby catalytic activity results in the
combination of all or part of said orthogonal donor substrate with
at least one other, recipient substrate of said enzyme, comprising
the steps of: (a) identifying, from the crystal structure of an
identical or homologous enzyme bound to its donor substrate, one or
more amino acids other than glycine which are close enough to an
atom of said bound phosphate donor substrate that they would
sterically exclude the orthogonal substituent attached to the
corresponding atom in said orthogonal donor substrate analog; (b)
preparing a plurality of mutant multi-substrate enzyme-encoding
nucleotide sequences having one or more mutations in one or more
nucleotide triplets encoding amino acids within ten amino acids of
said one or more amino acids, in both the amino terminal and
carboxy terminal directions; (c) expressing said plurality of
mutant multi-substrate enzyme-encoding nucleotide sequences to
produce a plurality of mutant multi-substrate enzymes; and d.
testing said plurality of mutant multi-substrate enzymes to select
one or more which have the ability to utilize said orthogonal donor
substrate analog as donor substrate.
32. A method for producing a mutant multi-substrate enzyme which
accepts at least one orthogonal donor substrate analog as a donor
substrate, comprising expressing one or more mutant sequence of
claim 31 found to express such a mutant, whereby said mutant
multi-substrate enzyme is produced.
33. A method of detecting the one or more intracellular components
that are recipient substrates for a multi-substrate enzyme that
covalently transfers part or all of a donor substrate to a
recipient substrate, comprising: I. combining: (a) cells, selected
from the group consisting of permiablized cells, lysed cells, and
cells which are naturally permeable to the orthogonal donor
substrate analog, which cells express a mutant of said
multi-substrate enzyme, which mutant accepts said orthogonal donor
substrate analog as a donor substrate; and (b) said orthogonal
substrate analog, having a detectable moiety on the portion thereof
that is catalytically transferred to a recipient substrate by said
multi-substrate enzyme; II. incubating said cells under conditions
sufficient to allow the mutant multi-substrate enzyme to transfer
part or all of the labeled orthogonal donor substrate to the
recipient substrate; and III. detecting the presence or absence of
said detectable label on cellular components, where by the presence
of said label on a cellular component indicates that said component
is a recipient substrate for said multi-substrate enzyme, and the
absence of said label on a cellular component indicates that said
component is not a recipient substrate for said multi-substrate
enzyme.
34. A method of detecting the one or more intracellular protein
substrates for a protein kinase, comprising: I. combining: (a)
cells, selected from the group consisting of permiablised cells,
lysed cells, and cells which are naturally permeable to the
orthogonal nucleotide triphosphate substrate analog, which cells
express a mutant of said protein kinase, which mutant accepts said
orthogonal nucleotide triphosphate analog as a phosphate donor
substrate; and (b) said orthogonal nucleotide triphosphate analog,
having a detectably labeled terminal phosphate; II. incubating said
cells under conditions sufficient to allow the mutant protein
kinase to phosphorylate its one or more protein substrates using
said orthogonal nucleotide triphosphate as phosphate donor; and
III. detecting the presence or absence of said detectably labeled
phosphate on cellular proteins, whereby the presence of said label
on a cellular protein indicates that said protein is a substrate
for said protein kinase, and the absence of said label on a
cellular protein indicates that said protein is not a substrate for
said protein kinase.
35. The method of claim 34 wherein said mutant binds to said
substrate with an affinity that is higher than its affinity for the
primary intracellular phosphate donor substrate for the wild-type
protein kinase.
36. A method for determining whether a test compound modulates the
activity of a multi-substrate enzyme, comprising the steps of: I.
combining: (a) cells, selected from the group consisting of
permiablized cells, lysed cells, and cells which are naturally
permeable to the orthogonal donor substrate analog, which cells
express a mutant of said multi-substrate enzyme, which mutant
accepts said orthogonal donor substrate analog as a donor
substrate; and (b) said orthogonal substrate analog, having a
detectable moiety on the portion thereof that is catalytically
transferred to a recipient substrate by said multi-substrate
enzyme; and (c) said test compound; II. incubating said cells under
conditions sufficient to allow the mutant multi-substrate enzyme to
transfer part or all of the labeled orthogonal donor substrate to
the recipient substrate; and III. detecting whether there has been
an increase or decrease in the presence or absence of said
detectable label on cellular components relative to that observed
in one or more control experiments where said test compound was
omitted, whereby a relative increase in the presence of said label
on a cellular component indicates that said test compound has
positively modulated the action of said multi-substrate enzyme on
that component, and a relative decrease in the presence of said
label on a cellular component indicates that said test compound has
negatively modulated the action of said multi-substrate enzyme on
that component.
37. A method for determining whether a test compound modulates the
activity of a protein kinase, comprising the steps of: I.
combining: (a) cells, selected from the group consisting of
permiablised cells, lysed cells, and cells which are naturally
permeable to the orthogonal nucleotide triphosphate substrate
analog, which cells express a mutant of said protein kinase, which
mutant accepts said orthogonal nucleotide triphosphate analog as a
phosphate donor substrate; (b) said orthogonal nucleotide
triphosphate analog, having a detectably labeled terminal
phosphate; and (c) said test compound; II. incubating said cells
under conditions sufficient to allow the mutant protein kinase to
phosphorylate its one or more protein substrates using said
orthogonal nucleotide triphosphate as phosphate donor; and III.
detecting whether there has been an increase or decrease in the
presence or absence of said detectable label on cellular proteins
relative to that observed in one or more control experiments where
said test compound was omitted, whereby a relative increase in the
presence of said label on a cellular protein indicates that said
test compound has positively modulated the action of said protein
kinase on that component, and a relative decrease in the presence
of said label on a cellular protein indicates that said test
compound has negatively modulated the action of said protein kinase
on that component.
38. An inhibitable engineered protein kinase or multi-substrate
enzyme selected from kinases prepared in accordance herewith,
synthetic analogs thereof, active fragments thereof, congeners
thereof, and combinations thereof, for use both diagnostic and
therapeutic procedures selected from drug assays, methods of
treatment or intervention in disease states such as cancer, HIV or
the like.
39. A transgenic animal that may function as a "knock out" model
for drug screening, wherein the wild-type gene corresponding to a
particular kinase associated with a particular disease state is
replaced with a gene encoding a mutant kinase, and said screen is
used by the interaction of said model with a kinase inhibitor
hereof.
40. A method for the transformation of a target cell in an animal
by the preparation of a vector containing DNA molecules that code
on expression for a material selected from the group consisting of
mutant kinases of claim 1, kinase inhibitors, agonists and
antagonists thereto, active fragments thereof, analogs thereof,
degenerate variants thereof, muteins thereof, and combinations
thereof.
41. A drug screen and associate screening method that utilizes an
agent selected from the mutant kinase of claim 1, variants thereof,
inhibitors thereof, active fragments thereof, analogs thereof, and
combinations thereof.
42. A pharmaceutical composition comprising an active agent
selected from a mutant multi-substrate enzyme in accordance with
claim 1, inhibitors thereof, agonists thereof, active fragments
thereof, alleles thereof, analogs thereof, conserved variants
thereof, and a pharmaceutically acceptable carrier.
43. Use of the pharmaceutical composition of claim 41 for the
treatment of a disease selected from cancer, HIV, Alzheimer's
Disease.
Description
FIELD OF THE INVENTION
[0002] The present invention is in the field of biotechnology. More
specifically, the invention is in a field often referred to as
enzyme engineering, in which through genetic alterations or other
means, the amino acid sequences of enzymes of interest are changed
in order to alter or improve their catalytic properties. The
embodiments of the invention which are described below involve
methods in the fields of genetic engineering and enzymology, and
more particularly, to the design of protein kinases and other
multi-substrate enzymes, including inhibitable such enzymes, and to
related materials, techniques and uses.
BACKGROUND OF THE INVENTION
[0003] It is only logical that cell-to-cell communications in a
multicellular organism must be fast, and that they must be able to
allow cells to respond to one another in diverse and complex ways.
Typically, the intracellular signals used are molecules called
"ligands" and a given ligand can bind to a particular type of
receptor on the surface of those cells that are to receive that
signal. But this simple ligand binding alone is not enough to
provide for the complex responses that the receiving cells may need
to make. Cells therefore amplify and add complexity to this signal
through complex, often cascading mechanisms leading to the rapid
modulation of catalytic activities inside the cell, which in turn
can produce complex, and sometimes dramatic, intracellular
responses. This process as a whole, from initial ligand binding to
completion of the intracellular response, is called "signal
transduction." Signal transduction is often accomplished by the
activation of intracellular enzymes that can act upon other enzymes
and change their catalytic activity. This may lead to increases or
decreases in the activity certain metabolic pathways, or may lead
to even large intracellular changes, for example, the initiation of
specific patterns of gene expression. The ability of one enzyme to
alter the activity of other enzymes generally indicates that the
enzyme is involved in cellular signal transduction.
[0004] The most common covalent modification used in signal
transduction process is phosphorylation, which results in the
alteration of the activity of those enzymes which become
phosphorylated. This phosphorylation is catalyzed by enzymes known
as protein kinases, which are often simply referred to as
"kinases."
[0005] Several key features of the kinases make them ideally suited
as signaling proteins. One is that they often have overlapping
target substrate specificities, which allows "cross-talk" among
different signaling pathways, thus allowing for the integration of
different signals (Mustelin, (1994) Immunity 1:351-356). This is
thought to be a result of the need for each kinase to phosphorylate
several substrates before a response is elicited, which in turn
provides for many types of diverse signaling outcomes. For example,
a given kinase may in one instance transmit a growth inhibitory
signal and in another instance transmit a growth promoting signal,
depending on the structure of the extracellular ligand that has
bound to the cell surface (Renshaw et al., (1992) EMBO. J.
11:3941-3951).
[0006] A second key feature is that the kinases are organized into
several modular functional regions, or "domains" (Cohen et al.,
(1995) Cell 80:237-248). One domain known as "SH3" is a
proline-rich region of 55-70 amino acids in length, and another,
known as "SH2" is a phosphotyrosine binding region of about 100
amino acids in length. These two domains are believed to be
involved in recognizing and binding to the protein substrates. The
third domain, "SH1" is comprised of about 270 amino acids, and is
the domain which is responsible for catalysis. It also contains the
binding site for the nucleoside triphosphate which is used as
energy source and phosphate donor (Cohen et al., (1995) Cell
80:237-248). Other domains, including myristylation and
palmitylation sites, along with SH2 and SH3, are responsible for
assembling multiprotein complexes which guide the catalytic domain
to the correct targets (Cohen et al., (1995) Cell 80:237-248; Mayer
et al., (1994) Mol. Cell. Bio. 14:2883; Mayer et al., (1992) Mol.
Cell. Bio. 12:609-618). Molecular recognition by the various
domains has been studied using by x-ray diffraction and by using
NMR methods (Koyama et al., (1993) Cell 72:945-952; Yu et al.,
(1992) Science 258:1665-1668; Kohda et al., (1993) Cell 72:953-960;
Waksman et al., (1993) Cell 72:779-790; Eck et al., (1993) Nature
362:87 (1993).
[0007] These domains appear to have been mixed and matched through
evolution to produce the large protein kinase "family." As many as
1000 kinases are thought to be encoded in the mammalian genome
(Hunter, (1987) Cell 50:823-829), and over 250 kinases have already
been identified. The large number of kinases and the large number
of phosphorylation-modulated enzymes that are known to exist inside
cells allow for rapid signal amplification and multiple points of
regulation.
[0008] A third key feature of the kinases is their speed. The
kinetics of phosphorylation and dephosphorylation is extremely
rapid in many cells (on a millisecond time scale), providing for
rapid responses and short recovery times, which in turn makes
repeated signal transmission possible (Eiseman et al., (1992)
Nature 355).
[0009] These features of the kinases have apparently led them to be
used in a vast array of different intracellular signal transduction
mechanisms. For example, growth factors, transcription factors,
hormones, cell cycle regulatory proteins, and many other classes of
cellular regulators utilize tyrosine kinases in their signaling
cascades (Ullrich et al., (1990) Cell 61:203-212; Bolen et al.,
(1992) FASEB J. 6:3403-3409). Tyrosine kinases catalytically attach
a phosphate to one or more tyrosine residues on their protein
substrates. The tyrosine kinases include proteins with many diverse
functions including the cell cycle control element c-abl (Cicchetti
et al., (1992) Science 257:803-806; Sawyers et al., (1994) Cell
77:121-131; Kipreos et al., (1992) Science 256:382-385), epidermal
growth factor receptor which contains a cytoplasmic tyrosine kinase
domain (Ullrich et al., (1990) Cell 61:203-212), c-src, a
nonreceptor tyrosine kinase involved in many immune cell functions
(Bolen et al., (1992) FASEB J. 6:3403-3409), and Tyk2, a
cytoplasmic tyrosine kinase which is involved in phosphorylation of
the p91 protein which is translocated to the nucleus upon receptor
stimulation and functions as a transcription factor (Velazquez et
al., (1992) Cell 70:313-320). The serine/threonine kinases make up
much if not all of the remainder of the kinase family; these
catalytically phosphorylate serine and threonine residues in their
protein substrates, and they have similarly diverse roles. They
share homology in the 270 amino acid catalytic domain with tyrosine
kinases. As such, although the discussion which follows focuses
more particularly on the tyrosine kinases, that discussion is
generally applicable to the serine/threonine kinases as well.
[0010] Unfortunately, the very features which make kinases so
useful in signal transduction, and which has made them evolve to
become central to almost every cellular function, also makes them
extremely difficult, if not impossible, to study and understand.
Their overlapping protein specificities, their structural and
catalytic similarities, their large number, and their great speed
make the specific identification of their in vivo protein
substrates extremely difficult, if not impossible, using current
genetic and biochemical techniques. This is today the main obstacle
to deciphering the signaling cascades involved in tyrosine
kinase-mediated signal transduction (Hunter, (1987) Cell
50:823-829; Murray, (1994) Chem. Biol. 1:191-195; White, (1991) J.
Bioenerg. Biomembr. 23:63-83; Hunter, (1995) Cell 80:225-236).
[0011] Efforts to dissect the involvement of specific tyrosine
kinases in signal transduction cascades have been frustrated by
their apparent lack of protein substrate specificity in vitro and
in vivo (Hunter, (1987) Cell 50:823-829; Hunter, (1995) Cell
80:225-236). The catalytic domains of tyrosine kinases possess
little or no inherent protein substrate specificity, as
demonstrated by domain swapping experiments (Duyster et al., (1995)
Proc. Natl. Acad. Sci. USA 92:1555-1559; Mayer et al., (1991) Proc.
Natl. Acad. Sci. USA 88:627-631; Kamps et al., (1986) Cell
45:105-112; Muller et al, (1993) Proc. Natl. Acad. Sci. USA
90:3457-3461; Mayer et al., (1994) Mol. Cell. Biol. 14:2883-2894;
Mayer et al., (1992) Mol. Cell. Biol. 12:609-618). The catalytic
domain from one tyrosine kinase can be substituted into a different
tyrosine kinase with little change in the protein substrate
specificity of the latter (Mayer et al., (1994) Mol. Cell. Biol.
14:2883-2894).
[0012] The poor in vitro specificity of kinases also makes it
difficult, if not impossible, to extrapolate what the in vivo
function of given kinases might be. An isolated tyrosine kinase of
interest will often phosphorylate many test protein substrates with
equal efficiency (Wang et al., (1982) J. Biol. Chem.
257:13181-13184). This apparently poor substrate specificity is
also found in vivo; for example, many genetic approaches, such as
gene knock out experiments, give no interpretable phenotype due to
compensation by other cellular tyrosine kinases (Schwartzerg et
al., (1991) Cell 65:1165-1175; Tybulewicz et al., (1991) Cell
65:1153-1163).
[0013] Another complication is that many tyrosine kinases have been
proposed to phosphorylate downstream and upstream proteins which
are themselves tyrosine kinases; although this appears to make
complex positive feedback loops possible, it also makes dissecting
the cascade even more difficult (Mustelin, (1994) Immunity
1:351-356).
[0014] One important avenue for deciphering the role and
understanding the function of enzymes, both in vitro and in vivo,
is the use of specific enzyme inhibitors. If one or more compound
can be found that will inhibit the enzyme, the inhibitor can be
used to modulate the enzyme's activity, and the effects of that
decrease can be observed. Such approaches have been instrumental in
deciphering many of the pathways of intermediary metabolism, and
have also been important in learning about enzyme kinetics and
determining catalytic mechanisms.
[0015] In addition, such inhibitors are among the most important
pharmaceutical compounds known. For example, aspirin
(acetylsalicylic acid) is such an inhibitor. It inhibits an enzyme
that catalyzes the first step in prostaglandin synthesis, thus
inhibiting the formation of prostaglandins, which are involved in
producing pain (Lehninger et al., (1993) Principles of
Biochemistry, Worth Publishers). Traditional drug discovery can be
characterized as the design and modification of compounds designed
specifically to bind to and inactivate a disease-causing protein;
the relative success of such an effort depends upon the selectivity
of the drug for the target protein and its lack of inhibition of
non-disease associated enzymes with similar enzyme activities.
[0016] Such approaches would appear to be promising ways to develop
treatments for cancer, since many human cancers are caused by
disregulation of a normal protein (e.g., when a proto-oncogene is
converted to an oncogene through a gene translocation). And since
kinases are key regulators, they have turned out to be very common
proto-oncogenes, and thus ideal drug design targets.
[0017] The process of designing selective inhibitors is relatively
simple in cases where few similar enzymes are present in the target
organism, for example in cases where inhibitors of a protein unique
to bacteria can be targeted. But unfortunately, the similarities
between the kinases and their large number has almost completely
frustrated the discovery and design of specific inhibitors, and has
blocked most hopes of developing specific pharmaceutical treatments
aimed at the proto-oncogene level. It is expected that the vast
majority of candidate inhibitors will inhibit multiple kinases,
even though they may have initially been identified as inhibiting a
particular, purified kinase.
[0018] This is not to say, however, that inhibitors with at least
some degree of kinase specificity cannot be found. Several natural
products have been identified which are relatively specific for
particular kinase families, but attempts to derive general rules
about kinase inhibition based on these has failed. Furthermore, as
the following examples show, specificity in most cases is quite
limited. For example, the compound Damnacanthal was reported to be
a "highly potent, selective inhibitor" of the kinase p561ck
(Faltynek et al., (1995) Biochemistry 34:12404-12410); as shown in
FIG. 2A, this compound has an inhibition constant (IC.sub.50) for
that kinase which is almost seven times lower than for the kinase
src (the IC.sub.50 is the concentration of inhibitor which must be
added to reduce catalytic activity by 50%). The compound PPI (FIG.
2B) has a binding affinity for the kinase Ick which is very strong
(IC.sub.50 0.005 .mu.M); but unfortunately, the inhibition of other
kinases of the src family is very similar. It inhibits the kinase
fyn with an almost identical IC.sub.50, 0.006 .mu.M, and has only
about a 4-fold higher IC.sub.50 for the kinase hck (IC.sub.50=0.020
.mu.M). The compound CGP 57148 (FIG. 2C) has been reported to be
"semi-selective" for the kinases abl (IC.sub.50=0.025 .mu.M) and
PDGFR (IC.sub.50=0.030 .mu.M) (Hanke et al., (1996) J. Biol. Chem.
271:695-701). Nevertheless, considering the vast number of kinases
and their relative cellular importance, and also considering that
the above-described inhibitors have only been reported in the last
two years, it appears that success in discovering or designing
selective kinase inhibitors has been remarkably limited. These
difficulties described above have implications well beyond the mere
frustration of scientists; they have frustrated efforts to decipher
the kinase cascades and the function of individual kinases in those
cascades and other cellular mechanisms. Such an understanding of
kinase activity and function may be essential before certain human
diseases can be effectively treated, prevented or cured. For
example, it has been known for over thirty years that the oncogene
bcr-abl is a protein kinase that is responsible for chronic
myelogenous leukemia; but the physiological substrates that it acts
upon to cause oncogenesis, which may be important drug design
targets, have yet to be definitively identified (Kurzrock et al.,
(1988) New Engl. J. Med. 319:990-998). On the bright side, despite
this shortcoming, the above-described inhibitor CGP 57148 is
reportedly now undergoing clinical trials for use in treating
myelogenous leukemia, even though the substrates it may block
phosphorylation of in vivo are not known.
[0019] The medical significance of these difficulties is further
illustrated by the Rous sarcoma virus (RSV), which has become an
important model system for studying the role of kinases in
oncogenesis. RSV transformation of fibroblasts is controlled by a
single viral gene product, the protein tyrosine kinase v-Src
(Brugge et al., (1977) Nature 269:346-348). It is the rapid time
course and the dramatic morphological changes during RSV fibroblast
transformation that have made RSV a paradigm for studies of
oncogene activity in all cells. The origin (Jove et al., (1987)
Ann. Rev. Cell Biol. 3:31-56), regulation (Cohen et al., (1995)
Cell 80:237-248; Hunter, (1995) Cell 80:225-236; Erpel et al.,
(1995) Curr. Opin. in Cell Biol. 7:176-182; Pawson, (1995) Nature
373:573-580), and structure (Yu et al., (1992) Science
258:1665-1668; Waksman et al., (1993) Cell 72:779-790; Waksman et
al., (1992) Nature 358:646-653) of v-Src have been extensively
studied and are well understood (Hunter, (1995) Cell 80:225-235;
Taylor et al., (1993) Curr. Opin. Genet. Dev. 3:26-34; Brown et
al., (1996) Biochim. Biophys. Acta 1287:121-149). But central
questions about this intensely studied kinase remains unanswered:
what are its direct cellular substrates? Does inhibition of its
catalytic activity effectively inhibit, or even reverse,
transformation? Would such inhibition be an effective therapy for
or prophylactic against RSV transformation? Unfortunately, as
discussed above, the answers to these questions are not
forthcoming, largely because the number of cellular kinases is
enormous (it is estimated that 2% of the mammalian genome encodes
protein kinases (Hunter, (1987) Cell 50:823-829) and because
tyrosine kinases display overlapping substrate specificities
(Hunter, (1995) Cell 80:225-236; Songyang et al. (1995) Nature
373:536-539) and share catalytic domains, making the design of
specific inhibitors enormously difficult.
[0020] The expression of v-Src in fibroblasts results in the
tyrosine phosphorylation of over fifty cellular proteins (Taylor et
al., (1993) Curr. Opin. Genet. Dev. 3:26-334). These same
substrates are also phosphorylated by other kinases in
untransformed fibroblasts (Kamps et al., (1988) Oncogene Res.
3:105-115). Even the most sophisticated biochemical and genetic
techniques, including anti-phosphotyrosine protein blots of
transformed fibroblasts, transfection of fibroblasts with
transformation-defective v-Src mutants, temperature-sensitive v-Src
mutants, gene knock-out studies of cellular Src host-range
dependent Src mutants, anti-v-Src immunoprecipitation, and use of
kinase specific inhibitors, have not led to the unambiguous
identification of v-Src's direct substrates (see reference to Brown
et al., (1996) Biochim. Biophys. Acta 1287:121-149) for a
comprehensive review). But this situation is not unique; in fact,
the direct substrates for the majority of cellular kinases remain
unidentified (Hunter, (1995) Cell 80:225-236). Furthermore, as
discussed above, there also are remarkably few compounds known to
selectively inhibit individual kinases, or even groups of related
kinases.
[0021] Although the forgoing difficulties are daunting, new methods
of rational drug design and combinatorial organic synthesis make
the design or discovery of kinase-specific inhibitors feasible
given sufficient resources. However, because the kinase networks
are highly degenerate and interconnected in unknown ways, there is
considerable uncertainty with regard to many diseases which kinases
should be targeted for inhibition. Moreover, it is by no means
clear that a specific inhibitor of a given kinase will have any
effect on the disease, either in vitro or in vivo. Because kinases
can be highly promiscuous, there is a significant chance that
inhibiting one kinase will simply force another kinase to "take its
place." Therefore, there is a need for a simple and direct way to
determine the biochemical and cellular effects of inhibiting a
given kinase, before herculean efforts are undertaken to design or
discover specific inhibitors.
[0022] From the forgoing, it is clear that there has been a long
felt but unsatisfied need for ways to identify which cellular
proteins are acted upon by individual protein kinases. Such a
method would ideally also allow for the quantitative measurement of
relative activity of a given kinase on its protein substrates,
which could be used, for example, to detect how or whether actual
or potential drug compounds might modulate kinase activity. In
addition, there has also been a need for specific inhibitors of
individual kinases or kinase families, which could be used to
identify protein substrates (by looking for which proteins are not
phosphorylated or are more weakly phospohorylated in the presence
of the inhibitor), to study the biochemical and phenotypic effects
of rapidly down-regulating a given kinase's activity, for use as
drugs to treat kinase-mediated diseases, and to confirm that
tedious efforts to design or develop more traditional inhibitor
drugs would be worthwhile. As is described in considerable detail
below, the present invention for the first time provides a method
for the highly specific inhibition of individual kinases, which
have been engineered to bind the inhibitor more readily than the
wild-type form of that kinase or other, non-engineered kinases. The
invention also provides for the engineered kinases and the
inhibitors to which they are adapted.
[0023] Moreover, as will become apparent, this method is even more
broadly applicable, as it would provide similar advantages for the
study of other enzymes which, like the kinases, covalently attach
part of at least one substrate to at least one other substrate.
[0024] The present invention involves the engineering of kinases
and other multi-substrate enzymes such that they can become bound
by inhibitors which are not as readily bound by their wild-type
forms. Modified substrates and mutant enzymes that can bind them
have been used to study an elongation factor (Weijland et al.,
(1993) Science 259:1311-1314) and a receptor for cyclophilin A
(Belshaw et al., (1995) Angew. Chem. Int. Ed. Engl. 34:2129-2132).
However, prior to the present invention, it was not known how, or
even if, multi-substrates enzymes which covalently attach part or
all of a donor substrate onto a recipient substrate could be
engineered to bind to an inhibitor, yet still retain at least some
catalytic activity and at least some specificity for the recipient
substrate in the absence of the inhibitor. The present invention is
that this can be done, as explained below; and this invention for
the first time opens the door to the selective inhibition of
individual kinases, which are not only important tools for
understanding of the kinase cascades and other complex catalytic
cellular mechanisms, but also may provide avenues for therapeutic
intervention in diseases where those mechanisms come into play.
SUMMARY OF THE INVENTION
[0025] The present invention provides a solution to the
above-described problems by providing materials and methods by
which a single protein kinase can be specifically inhibited,
without the simultaneous inhibition of other protein kinases.
[0026] In a first aspect, the present invention involves the
engineering of kinases and other multi-substrate enzymes such that
they can utilize modified substrates which are not as readily used
by their wild-type forms. The invention further provides such
chemically modified nucleotide triphosphate substrates, methods of
making them, and methods of using them. The methods of the present
invention include methods for using the modified substrates along
with the engineered kinases to identify which protein substrates
the kinases act upon, to measure the extent of such action, and to
determine if test compounds can modulate such action.
[0027] In a further aspect, the invention provides engineered
protein kinases which can bind inhibitors that are not as readily
bound by the wild-type forms of those enzymes. Methods of making
and using all such engineered kinases are also provided. The
invention further provides such inhibitors, methods of making them,
and methods of using them. The methods of the present invention
include methods for using the inhibitors along with the engineered
kinases to identify which protein substrates the kinases act upon,
to measure the kinetics of such action, and to determine the
biochemical and cellular effects of such inhibition. They also
relate to the use of such inhibitors and engineered kinases to
elucidate which kinases may be involved in disease; these kinases
can then become the subject of efforts to design or discover more
traditional specific inhibitors of their wild-type forms, which may
prove to be valuable in treating the kinase-related disease or
disorder.
[0028] Furthermore, methods are provided for inserting the
engineered kinase into cells or whole animals, preferably in place
of the corresponding wild-type kinase, and then using the inhibitor
to which it has been adapted as a tool for study of the disease
kinase relationship, and ultimately, as a drug for the treatment of
the disease.
[0029] The present invention also more generally relates to
engineered forms of multi-substrate enzymes which covalently attach
part or all of at least one (donor) substrate to at least one other
(recipient) substrate. These engineered forms will accept modified
substrates and inhibitors that are not as readily bound by the
wild-type forms of those enzymes.
[0030] The invention also relates to methods for making and using
such engineered enzymes, as well as the modified donor substrates.
The methods of the present invention include methods for using the
modified substrates and inhibitors along with the engineered
enzymes to identify which substrates the enzymes act upon, to
measure the kinetics of such action, and in the instance of the
modified substrates, to determine the recipient substrates to which
part or all of the donor substrate becomes attached, to measure the
extent of such action, and to identify and measure the extent of
modulation thereof by test compounds.
[0031] In the instance of inhibitors, the methods seek to determine
the biochemical and cellular effects of such inhibition. The
methods also extend to the use of such inhibitors and engineered
enzymes to elucidate which enzymes may be involved in disease;
these enzymes can then become the subject of efforts to design or
discover specific inhibitors of their wild-type forms, which may
prove to be valuable in treating the enzyme-related disease or
disorder. Furthermore, methods are provided for inserting the
engineered enzyme into cells or whole animals, preferably in place
of the corresponding wild-type enzyme, and then using the inhibitor
to which it has been adapted as a tool for study of the
disease-enzyme relationship, and ultimately, as a drug for the
treatment of the disease.
[0032] According to the present invention, through enzyme
engineering a structural distinction can be made between the
nucleotide binding site of a protein kinase of interest, and the
nucleotide binding sites of other kinases. This distinction allows
the engineered kinase to use a nucleotide triphosphate or an
inhibitor that is not as readily bound by the wild-type form of
that kinase, or by other kinases. In a preferred embodiment with
respect to the inhibitor, the inhibitor used is one that is
"orthogonal" to the "natural" nucleotide triphosphate substrate for
that kinase, or is orthogonal to a less specific inhibitor (e.g.,
one which is readily bound by the wild-type form of that kinase).
The term "orthogonal" as further discussed below, means that the
substrate or inhibitor is similar in structure (including those
that are geometrically similar but not chemically similar, as
described below), but differs in a way that limits its ability to
bind to the wild-type form.
[0033] An engineered kinase made according to the present invention
will be able to use an orthogonal nucleotide triphosphate substrate
that is not as readily used by other, nonengineered kinases present
in cells. Preferably, it will be able to use an orthogonal
nucleotide triphosphate that is not substantially used by other
kinases; and most preferably, it will be able to use an orthogonal
nucleotide triphosphate substrate that can not be used at all by
other kinases. By labeling the phosphate on the orthogonal
substrate, e.g., by using radioactive phosphorous (.sup.32P), and
then adding that labeled substrate to permiabilized cells or cell
extracts, the protein substrates of the engineered kinase will
become labeled, whereas the protein substrates of other kinases
will be at least labeled to a lesser degree; preferably, the
protein substrates of the other kinases will not be substantially
labeled, and most preferably, they will not be labeled at all.
[0034] The detailed description and examples provided below
describe the use of this strategy to uniquely tag the direct
substrates of the prototypical tyrosine kinase v-Src. Through
protein engineering a chemical difference has been made in the
amino acid sequence which imparts a new structural distinction
between the nucleotide binding site of the modified v-Src and that
of all other kinases. The v-Src kinase Applicant has engineered
recognizes an ATP analog (A*TP), N.sup.6-(cyclopentyl)ATP, which is
orthogonal to the nucleotide substrate of wild-type kinases. The
generation of a v-Src mutant with specificity for an orthogonal
A*TP substrate allows for the direct substrates of v-Src to be
uniquely radiolabeled using [.gamma.-.sup.32P]N.sup.6-(cyclop-
entyl)ATP, because it is able to serve as substrate to the
engineered v-Src kinase, but is not substantially able to serve as
substrate for other cellular kinases.
[0035] The detailed description and examples provided below
describe the use of this strategy to uniquely identify the direct
substrates of the prototypical tyrosine kinase, v-Src. Through
protein engineering a chemical difference has been made in the
amino acid sequence which imparts a new structural distinction
between the nucleotide binding site of the modified v-Src and that
of all other kinases. The engineered v-Src kinases that have been
made and presented herein bind to an orthogonal analog of the more
general kinase inhibitor PP3: the compound N-4 cyclopentoyl PP3.
The generation of a v-Src mutant with specificity for such an
inhibitor allows for the mutant to be inhibited, whereas other
kinases in the same test system are not substantially inhibited,
not even the wild-type form of that same kinase.
[0036] As is apparent from the forgoing, it is one object of the
present invention to provide a mutant protein kinase which accepts
an orthogonal nucleotide triphosphate analog as a phosphate donor
substrate.
[0037] Another object of the present invention to provide a
nucleotide sequence which encodes such a mutant protein kinase; and
it is a further object to provide a method for producing such a
nucleic acid sequence.
[0038] It is also an object of the invention to provide methods for
producing such a mutant protein kinase, for example, by expressing
such a nucleic acid sequence.
[0039] It is also an object of the present invention to provide
such orthogonal nucleotide triphosphates and methods for their
synthesis, including N.sup.6-(cyclopentyl)ATP,
N.sup.6-(cyclopentyloxy)ATP, N.sup.6-(cyclohexyl)ATP,
N.sup.6-(cyclohexyloxy)ATP, N.sup.6-(benzyl)ATP,
N.sup.6-(benzyloxy)ATP, N.sup.6-(pyrolidino)ATP and
N.sup.6-(piperidino)ATP (Waksman et al., (1993) Cell
72:779-790).
[0040] It is yet another object of the invention to provide a
method for determining whether a test compound positively or
negatively modulates the activity of a protein kinase with respect
to one or more protein substrates.
[0041] More particularly, and in accordance with the further aspect
of the invention, it is a primary object provide a mutant protein
kinase which binds to and is inhibited by an inhibitor, which
inhibitor less readily binds to or inhibits the corresponding
wild-type kinase.
[0042] A further object of the present invention is to provide a
nucleotide sequence which encodes such a mutant protein kinase; and
it is a further object to provide a method for producing such a
nucleic acid sequence.
[0043] It is also an object of the invention to provide methods for
producing such a mutant protein kinase, for example, by expressing
such a nucleic acid sequence.
[0044] It is another object of the present invention to provide
such inhibitors, such as the compound N-4 cyclopentoyl PP3, and
methods for their synthesis.
[0045] Another object is to provide a method for determining what
are the substrates for a given protein kinase.
[0046] It is yet another object of the invention to provide a
method for determining whether specific inhibition of a particular
kinase produces a biochemical or phenotypic effect in a test system
such as a cell-free extracts, cell cultures, or living
multicellular organisms.
[0047] It is a further object of the invention to provide a method
to determine whether inhibition of a particular kinase might have
therapeutic value in treating disease.
[0048] It is yet another object to provide methods for the study of
the activity, kinetics, and catalytic mechanisms of a kinase by
studying the inhibition of the corresponding mutant of the present
invention.
[0049] A further object is to provide a methods of preventing and
treating kinase-mediated diseases by introducing an
inhibitor-adapted mutant kinase of the present invention into a
diseased organism, and preferably diminishing or, most preferably,
depleting the organism of the wild-type enzyme; and then
administering the inhibitor to regulate the activity of the now
disease-mediating mutant kinase so as to diminish or eliminate the
cause or symptoms of the disease.
[0050] Based upon the forgoing and the detailed description of the
present invention provided below, one of ordinary skill in the art
will readily recognize that the present invention can be used more
generally to study multi-substrate enzymes which covalently
transfer a donor substrate or portion thereof to a recipient
substrate, as do the kinases. Such applications of the present
invention are also further described in the detailed description
which follows.
[0051] Accordingly, it is yet a further object of the present
invention to provide a mutant multi-substrate enzyme which binds to
an inhibitor, which inhibitor is less readily bound to the
wild-type enzyme or to other enzymes with similar activity.
[0052] It is another object of the invention to provide a
nucleotide sequence which encodes such a mutant multi-substrate
enzyme; and it is a further object to provide a method for
producing such a nucleic acid sequence.
[0053] It is also an object of the invention to provide methods for
producing such a mutant multi-substrate enzyme, for example, by
expressing such a nucleic acid sequence.
[0054] It is also an object of the present invention to provide
such inhibitors and methods for their synthesis.
[0055] Another object is to provide a method for determining what
are the substrates for a given multi-substrate enzyme.
[0056] It is yet another object of the invention to provide a
method for determining whether specific inhibition of a particular
multi-substrate enzyme produces a biochemical or phenotypic effect
in a test system such as a cell-free extract, cell culture, or
living multicellular organism.
[0057] It is a further object of the invention to provide a method
to determine whether inhibition of a particular multi-substrate
enzyme might have therapeutic value in treating disease.
[0058] It is yet another object to provide methods for the study of
the activity, kinetics, and catalytic mechanisms of a
multi-substrate enzyme by studying the inhibition of the
corresponding mutant of the present invention.
[0059] A further object is to provide a methods of preventing and
treating multi-substrate enzyme-mediated diseases by introducing an
inhibitor-adapted multi-substrate enzyme of the present invention
into a diseased organism, and preferably diminishing or, most
preferably, depleting the organism of the wild-type enzyme; and
then administering the inhibitor to regulate the now
disease-mediating mutant enzyme so as to diminish or eliminate the
cause or symptoms of the disease.
[0060] These and other objects of the present invention will, from
the detailed description, examples and claims set forth below,
become apparent to those of ordinary skill in the art.
BRIEF DESCRIPTION OF THE FIGURES
[0061] FIG. 1 is a schematic representation of the protein domain
structures of v-Src, of XD4 (which has a deletion of residues
77-225), of the glutathione S-transferase (GST)-XD4 fusion protein,
and of the GST-XD4 fusion protein double mutant (V323A, I338A);
[0062] FIG. 2 is a schematic representation of adenosine
triphosphate (ATP), with an "X" bound to the N.sup.6 position; and
in the box below, schematic representations are provided for the
twelve side chains that take the place of "X" in each of the
orthogonal ATP analogs described in the examples (which are always
referred to by the numbers 1-12 set forth in bold typeface);
[0063] FIG. 3 is an anti-phosphotyrosine immunoblot showing the
level of protein tyrosine phosphorylation following treatment of a
murine lymphocyte cell lysate with ATP or one of the ATP analogs
(A*TPs);
[0064] FIG. 4 provides a close-up view of the X-ray model showing
the ATP binding domain in cAMP dependent protein kinase (1ATP);
[0065] FIG. 5 shows (a) an anti-phosphotyrosine blot of cell
lysates expressing XD4 and GST-XD4 (V323A, I338A), (b) an
autoradiogram showing levels of phosphorylation when cell lysates
are provided only radiolabeled ATP or only radiolabeled
N.sup.6(cyclopentyl)ATP, and (c) an autoradiogram showing
autophosphorylation of GST XD4 and GST-XD4 (V323A, I338A) by
radiolabeled ATP and radiolabeled N.sup.6(cyclopentyl)ATP(A*TP(-
8));
[0066] FIG. 6 is a bar chart showing the relative degree to which
ATP and each of the twelve ATP analogs inhibits GST-XD4 and GST-XD4
(V323A, I338A) catalyzed phosphorylation by radiolabeled ATP;
[0067] FIG. 7 shows autoradiograms indicating the levels of
autophosphorylation by several v-Src position 338 single mutants
when provided with either radiolabeled ATP and radiolabeled
N.sup.6(cyclopentyl)ATP as phosphate donor substrate;
[0068] FIG. 8 is a schematic diagram of a method of the present
invention for determining which phosphorylated substrates in cells
were phosphorylated by a particular kinase, here v-Src;
[0069] FIG. 9 is a schematic diagram of how an engineered kinase of
the present invention can be inhibited by an inhibitor of the
present invention, even in the presence of other kinases, and can
be used to reveal the kinase's protein substrates;
[0070] FIG. 10 shows the chemical structures for three known kinase
inhibitors, Damnacanthal, PPI and CGP 57148, along with summaries
of their inhibition constants (IC.sub.50) for several kinases;
[0071] FIG. 11A shows the core structure of adenosine and PP3;
and
[0072] FIG. 11B shows the structures of several bulky substitutents
which can be added to N-4 nitrogen of PP3 to produce the inhibitor
candidate compounds whose IC.sub.50 values are listed in Table
1;
[0073] FIG. 12 shows the chemical structure of N-4 cyclopentoyl
PP3, and autogradiograms of electrophoresed proteins which have
become radiolabeled in the presence of N-4 cyclopentoyl PP3 in the
presence of either wild-type v-Src or the mutant (I338G);
[0074] FIGS. 13A-C is a chart presenting additional inhibitor
analogs prepared and tested in accordance with the present
invention;
[0075] FIG. 14A is a schematic representation of the specificity
problems associated with using small molecule protein kinase
inhibitors to deconvolute cell signaling. Kinase catalytic domains
(red ovals) are highly conserved. Thus, the majority of potent
inhibitors block the activity of closely related kinases and
broadly down regulate pathways mediated by kinase activity.
[0076] FIG. 14B is a schematic representation of the approach
toward selective protein kinase inhibition described here. A space
creating mutation is introduced into the ATP binding site of the
kinase of choice (Src). This mutation creates an active site pocket
(notch) in Src which can be uniquely recognized by a rationally
designed small molecule inhibitor. This inhibitor contains a bulky
chemical group (bump) which makes it orthogonal to wild type
protein kinases. Design of the complementary kinase/inhibitor pair
allows for highly selective inhibition of the target kinase in the
context of a whole cell.
[0077] FIG. 15A--Structure of N-6 cyclopentyloxyadenosine (1). B.
Synthesis of pyrazolo[3,4-d]pyrimidine inhibitor analogues. (2) was
synthesized according to Hanefeld et al., (1996) J. Chem. Soc.
Perkin Trans. 1:1545-1552, (I) RCOC1 (10 equiv.), pyridine,
5.degree. C., one hour; then warm to 22.degree. C., eleven hours;
(ii) LiAlH.sub.4 (3.0 equiv.), dry THF under argon, 0.degree. C.,
thirty minutes; then heat to reflux for thirty minutes. All
compounds were characterized by .sup.1H NMR (300 MHz) and high
resolution mass spectrometry (EI).
[0078] FIG. 16A--Chemical structures of quercetin (5) and AMP PNP
(6). B. Predicted binding orientation of (2) in src family kinase
active sites. The crystal structures of Hck bound to AMP PNP (red)
and Hck bound to quercetin (blue) were superimposed according to
the Hck protein backbone (white). The structure of (2) (yellow) was
subsequently docked into the kinase active site by superimposing
the pyrazolo[3,4-d]pyrimidine ring system of (2) onto the adenine
ring of AMP PNP. C. Predicted close contact between N-4 of (2) and
the side chain of residue 338 in src family kinases. Molecule (2)
has been docked into the ATP binding site of the Src family kinase,
Hck, as in FIG. 3B. The atoms of the threonine 338 side chain and
(2) are colored according to their elemental makeup (green carbon,
blue =nitrogen, red=oxygen, white=hydrogen) and the Hck backbone is
shown in purple. The methyl hydrogens of the threonine side chain
are not shown. Images were generated using the program
InsightII.
[0079] FIG. 17--Inhibitor analogue (3g) does not inhibit B cell
receptor mediated tyrosine phosphorylation. Murine spleen cells
were incubated with 1.1% DMSO (lanes 1-2), 100 mM (3g) in 1.1% DMSO
(lane 3), or 100 mM (2) in 1.1% DMSO (lane 4). B cell stimulation
(lanes 2-4) was initiated by the addition of 10 mg/ml goat
anti-mouse IgM. Cellular proteins were resolved by 10% PAGE,
transferred to nitrocellulose, and immunoblotted with a monoclonal
antibody for phosphotyrosine (4G10).
[0080] FIG. 18--Inhibitor (3g) blocks p36 phosphorylation in I338G
v-Src but not WT v-Src transformed NIH3T3 fibroblasts.
Non-transformed NIH3T3 cells (lane 1), WT v-Src transformed NIH3T3
cells (lanes 2-3), and I338G v-Src transformed NIH-3T3 cells (lanes
4-5) were incubated with 1.1% DMSO (lanes 1, 2 and 4) or 100 mM
(3g) in 1.1% DMSO (lanes 3 and 5). After 12 hours, the cells were
lysed. Phosphorylation levels were determined as in FIG. 4.
[0081] FIG. 19--I338G v-Src transformed fibroblasts selectively
acquire a flattened morphology and selectively regain actin stress
fibers upon incubation with (3g). Non-transformed (a-b), WT v-Src
transformed (c, d, g, h) and I338G v-Src transformed (e, f, i, j)
NIH-3T3 fibroblasts were treated with either 1.1% DMSO (a-c, e, g,
i) or 100 mM (3g) in 1.1% DMSO (d, f, h, j). After 48 hours cells
were photographed (a, c-f), stained with phalloidin-FITC, and
visualized (b, g-j) by fluorescence microscopy.
DETAILED DESCRIPTION OF THE INVENTION
[0082] FIG. 9
[0083] This figure shows a schematic representation of an
experiment to Identify Kinase Substrates below which uses the
invention for discovery of the substrates of a Src protein kinase.
The ovals at the top of the figure represent protein kinase
substrates which become phosphorylated by the protein kinases
adjacent to the arrow. The protein kinases containing several ovals
connected by lines are members of the "Src-Family" of protein
kinases (Src, Fyn, Lck). One kinase (Src) contains a notch cut out
which represents the I338G mutation which creates an extra space in
the adenine binding pocket of this kinase. The symbol above this
kinase represents the orthogonal inhibitor which contains a
protrusion which complements the mutation in the Src I338G kinase,
resulting in its unique inhibition. The kinase with a large round
oval and two protruding stings is the F-Actin Dependent protein
kinase (FAK). The protein kinases with only an oval are members of
the serine or threonine specific protein kinase family. The ovals
below the arrow containing small P's represent the phosphorylated
(P) substrates after action by the protein kinases. The simulated
gels at the bottom of the figure represent the expected results if
cells expressing either all wild-type kinases (on left) or one
mutant kinase (Src-I338G) in place of wild-type Src are treated
with the orthogonal inhibitor. The inhibitor should have no effect
on the phosphoproteins present in the cells which do not express
the mutant Src kinase (identical pattern in the gel on the left)
and several phosphoproteins should be absent following treatment of
the mutant expressing cells with the inhibitor (gel on the
right).
[0084] The Inhibitors
[0085] FIGS. 11A and 11B show the structures of a variety of bulky
substituents which, when added to either N-4 of PP3 or to N.sup.6
of adenosine diphosphate, or to N.sup.6 of adenosine monophosphate,
or to N.sup.6 of adenosine (specifically N.sup.6 cyclopentyloxy
adenosine) to produce inhibitors of the mutant kinase v-Src
(T120G), which is an engineered kinase of the present invention;
the synthesis and inhibition constants for these inhibitors are
discussed in Example 12 below.
[0086] Such inhibitors may be useful in studies directed towards
developing other useful mutants of this and other kinases, and for
the several methods described elsewhere herein. However, the scope
of the present invention is not limited to the use of these
particular inhibitors, and those of ordinary skill in the art will
recognize that many other possible structures could be substituted
for or supplement those described herein.
[0087] For example, different, simpler, and even more complex
aliphatic or aromatic groups could be added to the N.sup.6 position
of ADP or to the N.sup.4 position of PP3. In addition, the
inhibitors of the present invention are not limited to
modifications of nucleotides at the N.sup.6 position or
modifications of PP3 at the N.sup.4 position. Chemical means to
modify various positions on such compounds are known, and any of
the resulting derivatives would be within the scope of the present
invention; it is even possible to make changes or substitutions in
their ring structures. Exemplary variants are presented herein, and
particular reference is made to FIG. 13 where both analogs and data
relating to their activity is set forth. Of course, the use of such
inhibitors may require that different positions in the protein
sequence of the kinase be modified in order to make an engineered
kinase that will bind to them, but such different modifications are
well within the scope of the present invention.
[0088] In addition, it is important to note that the inhibitors of
the present invention are not limited to ADP and PP3 derivatives.
For example, it should be possible to utilize derivatives of other
natural nucleotide phosphate donor substrate as such inhibitors.
For studying some kinases, different analog bases may in fact be
preferred. For example, it is known that some kinases utilize GTP
as phosphate donor substrate and energy source; to make inhibitors
for engineered forms of such kinases, analogs of guano sine
diphosphate would be suitable.
[0089] Furthermore, it is well known that related compounds (e.g.,
other bases) and compounds chemically unrelated to the natural
substrate can sometimes nevertheless bind to an active site, and
can (but for the purposes of this invention need not), be acted
upon or act upon other substrates through chemical catalysis by the
enzyme. Sometimes they participate in the catalyzed reaction in the
same way as the natural substrate, sometimes in different ways.
Such compounds and their derivatives would be suitable starting
points for the design of inhibitors that are orthogonal to them,
and which would be within the scope of the present invention.
Similarly, other known kinase inhibitors can be used as a starting
point for synthesis of inhibitors of the present invention, such as
those whose structures appear in FIG. 10. Of course, even
derivatives of inhibitors that are currently unknown would, once
identified, be suitable core structures for the design of
inhibitors of the present invention, as illustrated herein and made
a part hereof.
[0090] Furthermore, the inhibitors of the present invention are not
limited to those made by chemical synthetic means, but also include
compounds which may be found in nature, and which can serve that
role, some of which are discussed above. In addition, those of
ordinary skill in the art will appreciate that there are other
variations besides those set forth here, and that these are all
within the scope of the present invention.
[0091] The inhibitors that are candidates for use in accordance
with the present invention can conveniently be screened to
determine the extent to which they are accepted by wild-type
kinases, using a screening procedure such as that set forth in
Example 13 below, or by a screening procedure involving the use of
a cell or cells which are rich in protein kinase activity as set
forth in Example 9 herein. By such an assay, one can determine
whether each inhibitor is bound by wild-type kinases to a lesser
degree than the engineered kinases, or preferably, if the wild-type
kinases do not substantially bind to that inhibitor, or most
preferably, do not bind the inhibitor at all. For those substrates
that are least less readily bound, it may be worthwhile to try to
engineer the kinase of interest so that it will more readily bind
to them. Of course, one could make the engineered kinase first and
then assay it along side the wild-type enzyme to determine whether
it uses a given orthogonal substrate better than the wild-type
kinase; this was the approach used in Example 13. However, under
most circumstances, pre-screening as described above will be
preferred. Of course, other assay approaches will be apparent to
those in the field, and the use of such assays would be within the
scope of the present invention.
[0092] The Engineered Kinases
[0093] There are several criteria that should be satisfied in
reengineering a kinase in order to uniquely tag its authentic
substrates in the presence of wild type tyrosine and
serine/threonine kinases. The engineered kinase should: (1) accept
an ATP analog (A*TP) that is utilized less readily by wild-type
protein kinases; preferably, accept an A*TP that is not
substantially utilized by wild-type kinases; and most preferably,
accept an A*TP that is not utilized by wild-type kinases at all;
(2) preferably, use the A*TP analog with high catalytic efficiency;
and (3) preferably, have reduced catalytic efficiency for the
natural nucleotide substrate (ATP) so that in the presence of
cellular levels of ATP (1-2 mM) the mutated kinase would
preferentially utilize A*TP as the phosphodonor. If such engineered
kinases are to be used to study the protein substrate specificity
of the wild-type kinase, then these criteria must be met without
substantially altering the protein target specificity of the
kinase.
[0094] Likewise several criteria should be satisfied in
reengineering a kinase in order that it will be inhibited by the
inhibitors of the present invention. The engineered kinase should:
(1) bind to an inhibitor which is bound less readily by wild-type
protein kinases; preferably, the inhibitor will not substantially
bind to wild-type kinases; and most preferably, will not bind at
all to wild type kinases; (2) preferably, the engineered kinase
will bind the inhibitor with high affinity (i.e., low IC.sub.50).
It is not generally of particular importance whether the inhibitor
binds to the wild-type form of the kinase that corresponds to the
engineered kinase, as such binding and the resulting inhibition
would augment that of the engineered kinase. However, it is most
likely that the wild-type form of that kinase will not bind the
inhibitor any better than other wild-type kinases. If an
inhabitable engineered kinase is to be used to study the protein
substrate specificity of the wild-type kinase, or to replace the
wild-type form of that kinase through gene therapy or other means,
as further discussed below, then a further concern is that the
above-described criteria must preferably be met without
substantially altering the protein target specificity of the
engineered kinase when compared with the corresponding wild-type
form.
[0095] When viewed from the perspective of the state of the art
when the present invention was made, it was not predictable whether
it would be possible to satisfy all of these criteria
simultaneously; in fact, it was doubtful, because the ATP binding
site that is engineered is very close to the second substrate
binding site, i.e., the peptide binding site. However, as shown by
the examples below, all of these criteria, including the preferred
criteria, were in fact met simultaneously when Applicant made the
described v-Src mutants, provided them with
N.sup.6-(cyclopentyl)ATP and inhibited them using
N.sup.4-(cyclopentyl) PP3.
[0096] Example 1 describes the twelve ATP analogs which were used
in the studies on mutant v-Src, which are described in the further
examples which follow. These orthogonal ATP analogs may be useful
in studies directed towards developing other useful mutants of this
and other kinases, and for the several methods described elsewhere
herein. However, the scope of the present invention is not limited
to the use of these particular ATP analogs, and those of ordinary
skill in the art will recognize that many other possible orthogonal
substrates could be substituted for or supplement those described
herein. For example, different and even more complex aliphatic or
aromatic groups could be added to the N.sup.6 position of ATP. In
addition, the orthogonal substrates of the present invention are
not limited to modifications of nucleotides at the N.sup.6
position. Chemical means to modify various positions on adenosine
are known, and any of these would be within the scope of the
present invention; and it is even possible to make changes or
substitutions in the ring structures of nucleotides. Of course, the
use of such orthogonal substrates may require that different
positions in the protein sequence of the kinase be modified in
order to make an engineered kinase that will bind to them, but such
different modifications are well within the scope of the present
invention.
[0097] In addition, it is important to note that the orthogonal
substrates of the present invention are not limited to ATP
derivatives. For studying different kinases different analog bases
may in fact be preferred. For example, it is known that some
kinases utilize GTP as phosphate donor substrate and energy source;
for studies of such kinases, analogs of guanosine triphosphate
would be preferred. It is well known that compounds chemically
unrelated to the natural substrate can sometimes nevertheless bind
to an active site, and can even be acted upon or act upon other
substrates through chemical catalysis by the enzyme. Sometimes they
participate in the catalyzed reaction in the same way as the
natural substrate, sometimes in different ways. Such compounds and
their derivatives would also be within the scope of the terms
"natural substrate" and "orthogonal substrate" as used herein.
[0098] Furthermore, the orthogonal substrates of the present
invention are not limited to those made by chemical synthetic
means, but also include compounds which may be found in nature, and
which can serve that role. Those of ordinary skill in the art will
appreciate that there are other variations besides those set forth
here, and that these are all within the scope of the present
invention.
[0099] The orthogonal nucleotides that are candidates for use in
accordance with the present invention can conveniently be screened
to determine the extent to which they are accepted by wild-type
kinases, using a screening procedure such as that set forth in
Example 2 below. By such an assay, one can determine whether each
orthogonal substrate is accepted by wild-type kinases to a lesser
degree than the normal substrate for such kinases, or preferably,
do not substantially accept that substrate, or most preferably, do
not accept it at all. For those substrates that are least less
readily accepted, it may be worthwhile to try to engineer the
kinase of interest so that it will more readily accept them. Of
course, one could make the engineered kinase first and then assay
it along side the wild-type enzyme to determine whether it uses a
given orthogonal substrate better than the wild-type kinase.
However, under most circumstances, pre-screening such as is
described in Example 2 will be preferred. Of course, other assay
approaches will be apparent to those in the field, and the use of
such assays would be within the scope of the present invention.
[0100] The design of an engineered v-Src is described in Example 3
below. As is described, the engineered form was designed by
reference to the crystal structures of other kinases which have
domains that are homologous to those found in most if not all
kinases. As will be seen, the example mutant kinases described
herein have been constructed as fragments of protein kinases,
rather than as containing the entire sequences; but it was found
there is no substantial difference in performance when the entire
sequence is used. Of course, the concepts and the practicalities
are the same whether fragments or whole kinases are used, and both
are within the scope of the present invention. As such, the term
"kinase" should be viewed as including the whole enzyme or a
fragment of one, including when interpreting the claims.
[0101] Using this approach, it is possible to design similar
mutants of virtually any other kinase. The method for doing this
comprises the steps of: (a) identifying, from the crystal structure
of an identical or homologous enzyme bound to its phosphate donor
substrate or to a known kinase inhibitor (which may be non-specific
for kinases, specific for kinases generally but not for that
kinase, or specific for that kinase), one or more amino acids other
than glycine which are close enough to a substituent on the bound
phosphate donor substrate or inhibitor that they would sterically
restrict entry of a bulky substituent attached to that substituent
in a putative orthagonal inhibitor; and (b) mutating a nucleotide
sequence which encodes the wild-type protein kinase such that the
nucleotide triplets encoding one or more of the identified amino
acids, are converted to nucleotide triplets that encode amino acids
having side chains that are sterically less bulky than the
identified amino acids.
[0102] The above-described method uses steric restriction of entry
or exclusion as the criteria for deciding which amino acid(s) to
change, and how to change them. However, the present invention is
not so limited. It is also possible to engineer a kinase to change
its ability to bind to an orthogonal substrate by considering other
factors, such as hydrophobicity, hydrophilicity, ionic binding or
repulsion, hydrogen bonding, forming covalent bonds between the
enzyme and electrophilic groups on orthogonal substrates, etc.
[0103] The study of protein kinases using the present invention
will be greatly facilitated by the vast knowledge regarding the
domain structure of many different kinases, and their generally
homologous sequences. The Protein Kinase Fact Book (Hardie &
Hanks, (1995) Academic Press, San Diego) provides protein sequence
data for the three functional domains in literally hundreds of
protein kinases, and this along with sequence information available
in the primary literature, should greatly facilitate the further
application of the present invention to the kinases. Similar
information is available regarding other multi-substrate enzymes,
which should facilitate their study and use according to the
present invention.
[0104] Although the preferred method of the present invention
involves the rational design of substrate analogs and mutant
protein kinases, both could alternatively be made by use of methods
known as combinatorial methods. There are many combinatorial
methods of synthesizing organic compounds. Using one such method,
one could synthesize nucleoside analogs on resin beads using
sequential chemical steps, and then release them from the resin
prior to phosphorylation to make the nucleotide triphosphates.
After using such a method to make a collection or library of
putative orthogonal substrates for mutants of v-Src kinase, other
protein kinase, or other multi-substrate enzymes, the collection or
library could be screened for particularly favorable binding or
catalytic properties. This may allow for the more thorough search
of structural, conformational, and electronic features of such
putative orthogonal substrates. Moreover, it is often found that
when larger numbers of analogs of a given substrate are
investigated, and unexpectedly efficient substrate or inhibitor can
be found. Furthermore, sometimes the compounds which are the most
desirable would not have been chosen if only well understood
parameters were used to specifically design the best compound.
[0105] There are also many combinatorial methods known in the art
for making protein mutants. These include "error prone" polymerase
chain reaction (PCR), "sexual" PCR or PCR using primers with random
nucleotides at fixed positions in the protein sequence. Other
sequence randomization methods might include using chemical
mutagens of cDNA or plasmid DNA, or MutD type strains of bacteria,
which are known to introduce mutations randomly in proteins that
they express. It would be possible to carry out the present
invention by exploiting such methods for making randomly mutated
protein kinases or other multi-substrate enzymes, and then
screening for one with particularly high activity with a particular
orthogonal substrate, or with some or all of the putative
orthogonal substrates made using combinatorial synthesis, as
described in the paragraph above. The assay methods described in
the examples below would be suitable for this purpose, and those in
the art would be readily able to design alternative approaches.
[0106] These methods and other methods which are or may be
developed to explore protein sequence space and the structural
space of small organic molecules might be particularly useful for
the technological application described here, where one is changing
or altering both the protein and the putative inhibitor in order to
find the best possible non-natural (i.e., orthogonal) fit. The use
of any of these or any of the other methods described herein would
be within the scope of the present invention.
[0107] The synthesis of one engineered kinase is described in
Example 4. The focus of this effort was on amino acid side chains
that were within about 4 .ANG. of the N.sup.6 of ATP; but there is
nothing magical about that distance. Residues with side chains that
are within about 1 .ANG., 2 .ANG., 3 .ANG., 4 .ANG., 5 .ANG., 6
.ANG., 7 .ANG., 8 .ANG., 9 .ANG., 10 .ANG. or lesser, greater or
intermediate distances should also be considered as targets for
modification. Amino acids with side chains that are within about 3
.ANG. to about 6 .ANG. would be preferred targets. Generally those
amino acids with the closer side chains will be preferred over
those with more distant side chains, as they would be expected to
cause the greatest steric or other interference with the orthogonal
substituent on the inhibitor; and those with the very closest side
chains would be the most preferred.
[0108] Of course, there are many other ways to modify and express
genetic sequences today than those used in the examples, such as
site-directed mutagenesis, and one can expect that other methods
will be developed in the future. The use of any or all of these
would be within the scope of the present invention. In addition,
although the use of genetic engineering is today probably the
preferred method to prepare such mutants, it is not the only way.
For example, one could design an engineered kinase and then
synthesize that protein by known methods of chemical peptide
synthesis. Or, it may be possible to chemically modify a given
enzyme in a specific location such that one or more side chain
changes in size, hydrophobicity, or other characteristic, such that
it can more readily utilize an orthogonal substrate. The use of all
such methods are within the scope of the invention.
[0109] Example 7 describes testing which could be done to determine
whether the engineered kinase had retained its protein substrate
specificity. It is preferred that the wild-type protein substrate
specificity be substantially retained if, as in the examples, the
goal is to use the engineered kinase to study what substrates the
kinase acts upon and to what degree it does so, or it is to be used
to replace or supplement the corresponding wild-type kinase in
vivo, e.g., through genetic engineering. However, although for such
purposes it is important that the kinase still recognize the same
substrates as the wild type, it is not critical that it do so with
the same kinetics; i.e., if it does so slower or faster, or to a
greater or lesser degree, the engineered kinase may still have
substantial value for such purposes. If the engineered kinase does
not recognize the same protein substrates as the wild-type enzyme,
it may have less value in studying the wild-type enzyme, but may
still have substantial value in studying protein phosphorylation
and kinases in general, and would still be within the scope of the
invention.
[0110] Of course, the particular assays used in Example 7, although
useful, need not be used. Those of skill in the art will readily be
able to develop or adopt other assays that can provide comparable
information.
[0111] Once a mutant kinase has been made which accepts a given
orthogonal substrate analog, or which is inhibited by a given
inhibitor, it can be characterized using classical enzyme kinetic
analysis, as illustrated in Examples 5 and 6. Also, as shown in
Example 8, one can study the degree to which the mutant can utilize
or be inhibited by the analog, and whether the analog is a "dead"
(i. e., wholly ineffective) inhibitor for the wild-type enzyme. Of
course, the methods used in the examples are not the only ways
these studies can be done, and those of skill in the art can easily
design alternate approaches.
[0112] As illustrated in Example 10, it is not necessary to make
multiple amino acid substitutions to provide a mutant that will be
inhibited by an inhibitor of the present invention. It may only be
necessary to make a single amino acid change, as is the case with
the mutants GST-XD4 (I338A) and GST-XD4 (I338G).
[0113] Assay to Identify Kinase Substrates
[0114] A very simple embodiment of the present invention would be
as follows. First, the orthogonal inhibitor is added to two samples
of the cell of interest which either express an added gene for the
engineered kinase or express the normal copy of the kinase of
interest. The inhibitor can be added before, after, or during the
activation of a signaling cascade (such as permeabilized cells,
cell extracts, or cells that are naturally permeable to them). Then
a method which allows detection of all phosphorylated proteins in a
cell or cell fraction, e.g., by using radioactive phosphorous
[.gamma.-.sup.32P]ATP or by using monoclonal antibodies specific
for phosphorylated amino acids is used to reveal the result of
specifically inhibition of the kinase of interest. In the cells
expressing the normal copy of the kinase of interest, the protein
substrates of the native kinase will become labeled, even in the
presence of the inhibitor, whereas the protein substrates of the
engineered kinase will at least be labeled to a lesser degree;
preferably, the protein substrates of the engineered kinases will
not be substantially labeled, and most preferably, they will not be
labeled at all.
[0115] It is also preferable if the wild-type kinase corresponding
to the mutant has been removed from the cells, e.g., by "knock-out"
of the cellular gene(s) for it. If the labeled proteins of such an
assay are examined in tandem with control samples containing the
wild-type kinase but not the mutant kinase, certain bands will be
diminished in intensity in the mutant-treated sample relative to
the control. Preferably, the difference in intensity will be high;
most preferably, there will be bands which are missing in the
mutant-containing samples treated with the inhibitor. This would
indicate that the wild-type form of that kinase phosphorylates
those differentially labeled proteins; when the kinase is
inhibited, those bands do not get labeled.
[0116] Example 10 provides one example of a method of using a
mutant kinase of the present invention, along with its orthogonal
substrate analog or its inhibitor, as the case may be, to detect
which are the intracellular protein substrates for that protein
kinase. Developing such a test was a primary goal of the research
that led to the present invention.
[0117] Generally, the method described in Example 10 and in FIG. 8
would appear to be generally applicable; however, there are many
other possible approaches that could be used, once a mutant that
accepts an orthogonal substrate analog or inhibitor has been
prepared. The natural phosphate donor substrate is first prepared
to contain a labeled moiety on the terminal phosphate, for example,
by replacing the phosphate with [.gamma.-.sup.32P] phosphate. This
substrate, along with the analog or inhibitor, is then added to a
sample of lysed cells, cell extracts, permiabilized cells, or cells
which are naturally permeable to the orthogonal nucleotide
triphosphate substrate analog or to the inhibitor, and which
express the mutant kinase, or to which the mutant kinase has been
exogenously added (e.g., by microinjection). After incubation under
conditions that will allow the mutant kinase to become inhibited,
and/or to phosphorylate its protein substrates to the extent not
inhibited, the labeled products are then extracted and analyzed in
comparison with those produced by a control sample, which was
treated substantially the same way, but without the addition of the
analog or inhibitor, respectively. Methods for the detection of
labeled proteins are well known, and include both quantitative and
qualitative methods. In addition, all methods for characterizing
and identifying proteins can be used to determine with specificity
what the protein substrates are, and what their functions are.
Ultimately, it should be possible to develop an understanding of
what protein substrates each of the various protein kinases act
upon, and reveal in great detail the mysteries of cellular signal
transduction.
[0118] Once one or more cellular protein substrate has been
identified, similar assays can be used to identify drugs or other
compounds that can modulate the activity of a given protein kinase
on one or more substrates. For example, one could add small amounts
of solutions of a variety of such compounds to test samples
containing cell-free extract, mutant kinase, along with a labeled
orthogonal substrate analog and/or inhibitor. The labeled proteins
can then be identified, e.g., by gel electrophoresis followed by
autoradiography, and compared with a duplicate test sample treated
the same way, but to which no drug or other compound was added.
[0119] If a protein is not labeled in a sample having an added
compound plus substrate analog and/or inhibitor that does get
labeled in a sample treated with the analog and/or the inhibitor,
this indicates that the added compound has caused the kinase to
phosphorylate a protein that it does not act on in the absence of
the compound, i e., the compound upwardly modulates the activity of
the kinase for that protein. Alternatively, if a labeled protein
appears in a test sample to which the compound or drug was added,
but does not appear in a test sample not having the compound or
drug added, this indicates that the added compound has prevented
the kinase from phosphorylating a protein that it does act on in
the absence of the compound, i.e., the compound downwardly
modulates the activity of the kinase for that protein
substrate.
[0120] Furthermore, if quantitative measurements are made for each
labeled protein, e.g., by scanning autoradiograms and integrating
the data, more subtle effect on kinase activity can be detected.
For example, it may be found that a protein is more fully or less
fully phosphorylated in the presence or absence of a given compound
(i.e., has been less dramatically modulated). It can also be
expected that some compounds will upwardly modulate kinase activity
for some proteins and downwardly modulate activity for others at
the same time.
[0121] Use in Screening for Drug Design Target Kinases
[0122] As mentioned above, because kinases play key roles in
various diseases, it is of great interest to develop inhibitors
which can specifically inhibit a single wild-type kinase or group
of wild-type kinases. By down-modulating the activity of these
diseases involved kinases, it should be possible to reduce the
disease symptoms, or even cure the disease.
[0123] However, the great difficulty which has been experienced in
making such inhibitors of wild-type kinases, as briefly described
above, limits the potential of that approach. The primary
difficulty is finding inhibitors which are specific, and do not
inhibit other kinases than the intended target. The reasons for
such non-specificity are (i) the nucleotide triphosphate binding
sites of kinases are highly conserved in evolution, and (ii) many
kinases are "degenerate" that is, they have sufficiently similar
activities and specificities that they can substitute for other
kinases that because of gene deletion or other reason are absent or
diminished in concentration in the cells. The problem of binding
site similarities can in many instances be overcome, e.g., by
careful rational inhibitor design, or by selection of inhibitors
from combinatorial libraries on the basis of specificity. However,
efforts to do so with a kinase that is truly degenerate with
another kinase will likely be unfruitful; either all of the
co-degenerate kinases will be inhibited by even the best candidate
compounds, or even if the target is inhibited, it will be
impossible to tell, because a degenerate kinase will "take over"
the activity of the inhibited one.
[0124] Because of this, there is a need for a way to screen kinases
to determine which wild-type kinases are degenerate, and thus
probably poor candidates for specific inhibition, and which are not
degenerate, and therefore preferred candidates for specific
inhibition. The present invention provides such a method. The
present invention provides a means to generate a specific, unique
kinase inhibitor for any kinase of interest, by making a mutant of
the kinase that is specifically designed to be inhibited by
candidate inhibitors selected, and then studying the effects of
that inhibition.
[0125] One way to accomplish this is to test cells or cell extracts
in vitro. For example, one could add ATP to such a sample which has
one kind of label (the "first label") on the terminal phosphate,
and add the specific inhibitor which is differently labeled (the
"second label") at the terminal phosphate. The decrease in
appearance of the second label on a given protein substrate (e.g.,
as viewed by gel electrophoresis) indicates specific inhibition of
the mutant kinase; and appearance of the first label on that same
substrate indicates that the other kinases have taken over that
phosphorylation role, the degree of which is shown by the relative
degree of such labeling. If it turns out that the engineered kinase
is specifically inhibited, and other kinases do not take over
phosphorylation of the substrates of the engineered kinase when it
is inhibited, or at least do not completely take over, then that
kinase is not degenerate, or at least not completely so; it is thus
probably not a good candidate for development of a specific
inhibitor of the wild-type for use as a drug to treat the disease
it relates to. However, if inhibition of the mutant kinase with an
inhibitor of the present invention is not compensated for by the
other kinases, then it is a preferred candidate for the development
of an inhibitor of the wild-type kinase.
[0126] Another, preferred method of such screening would be to
produce animal models for the disease of interest, and then "knock
out" the wild-type gene, and then, by genetic engineering, insert
into the genome a gene encoding a mutant kinase of the present
invention "knock-in" Then, an inhibitor of the present invention,
preferably one which has been shown in vitro to inhibit the mutant,
can be used to down-regulate the mutant kinase. If down regulation
leads to a decrease in the symptoms or morbidity of the disease in
the model animal, or eliminates the disease, then that kinase is a
preferred candidate for the development of a specific inhibitor of
the wild-type form.
[0127] Gene Therapy Applications
[0128] The mutant kinases and inhibitors of the present invention
can also be used directly to treat diseases in humans and animals.
Just as described above for the animal model systems, gene
substitution could be used on patients with diseases which are
mediated by those kinases. The wild-type gene for one or more such
wild-type kinase would be deleted, e.g., by "knock-out" methods
known in the art, and then specifically inhibitable mutants of
those one or more kinases would be added to the animal's genome,
e.g., by "knock-in" or gene therapy methods which are known in the
art. Then, the inhibitor could be used as a drug to down-modulate
those one or more mutant kinases, such that the disease is
ameliorated to at least some degree, but the degree of activity of
those kinases which may be found to be necessary for normal
cellular function could be maintained. Of course, the kinases could
also be essentially "turned off" strong inhibition, if that proved
to be therapeutically effective. Furthermore, if it is found that
the disease is greatly improved or cured by a period of
down-regulation or being turned off, then administration of the
inhibitor could be discontinued, and the disease well might not
return or exacerbate. If not, then inhibition could be discontinued
on a long term or even permanent basis, and the mutants could be
left to function in the place of the wild-type kinase for the
remainder of that patient's life. Since the specific inhibitors of
the present invention are not present in the environment, the
mutant kinases should behave just like the wild-type (except to the
extent that the engineering may have changed their activity or
kinetics). And if the disease should recur or flare up again in the
future, the patient could again be treated with the inhibitor,
without the need to repeat the gene exchange.
[0129] Other Multi-substrate Enzymes
[0130] As mentioned above, the present invention is not limited to
mutant kinases, orthogonal inhibitors, and their synthesis and use.
The present invention will work just as well for other
multi-substrate enzymes which covalently transfer part or all of
one substrate, here called the donor, to another substrate, here
called the recipient; and there are surely more such enzymes yet to
be discovered. In any such instance, one of skill in the art who
has studied the present specification will well appreciate the
applicability of the present invention to such enzymes. The tasks
at hand in such an instance are quite similar to those described in
detail here for the kinases. First, it is necessary to identify
what the donor substrate is, and/or to identify compounds which can
inhibit that kinase, even if it is not specific for that
kinase.
[0131] Second, it is necessary to consider where a bulky
substitutent might be added to the substrate or the inhibitor such
that it will not bind as readily to the wild-type kinase, or
preferably will not bind substantially to the wild-type kinase, and
preferably, will not bind at all. Of course, it is not really
necessary, in the case of kinases or in other multi-substrate
enzymes as described above, to be restrictive with respect to which
analogs of these to make; one can make a variety of them, even
including some that seem unlikely to be ideal, and determine by
screening which one or ones are the best. Further guidance
regarding how to do this can be gained from the examples below. The
inhibition assay, the results of which are shown in FIG. 6, is a
non-limiting example of an assay particularly well suited to such
screening.
[0132] The third step is to engineer the kinase such that one or
more amino acid in the three-dimensional location where the bulky
group would be expected to be if the analog did bind are replaced
with amino acids having less bulky side chains, thus "making room"
for the bulky moiety of the inhibitor. Steps two and three can, of
course, be carried out in the reverse order.
[0133] For example, transferase enzymes would be most interesting
candidates for study using the present invention. One could,
following the teachings provided herein, prepare mutant
transferases which will accept orthogonal inhibitors, and these
could be used together in order to identify the direct substrates
of one particular transferase in a large family of homologous
transferases, by the methods described above for the kinases. The
family of methyl-transferases would be of clear interest, and could
quite easily be studied using the methods provided herein. These
enzymes all use the same nucleotide based cofactor,
S-adenosylmethionine (AdoMet), as a methyl (CH.sub.3) group donor.
The different members of the family can transfer the methyl group
of AdoMet to a wide variety of cellular components such as proteins
(in which case the methyl group is added to arginine, aspartate,
and glutamate side chains), DNA (in which case the methyl group is
added to the C-5 position of cytosine, or the N-7 of guanine), to
components of cell membrane components such as phospholipids, and
also to a number of small amine containing hormones. Many new
targets are also being identified for this diverse family of
enzymes. The present invention provides the opportunity to decipher
the tremendously complex cellular mechanisms that these enzymes are
carrying out.
[0134] For example, one could synthesize a set of AdoMet analogs
that contain additional bulky hydrophobic groups at the N-6
position, or at other ring positions, which would make the analogs
orthogonal, and thus not be accepted as readily by wild-type
methyltransferases as is the natural substrate; and the structure
in the region of the transferred methyl group might be altered such
that the methyl group is more chemically resistant to transfer; or,
for example, S-adenosylcysteine might be used as the starting
compound instead. Using the crystal structures of DNA
methyltransferase M.HhaI and the catechol methyltransferase
catechol O-methyl-transferase (COMT), one can identify those amino
acids in the adenine binding pocket which are candidates for
mutation as Applicant has done for the protein kinases; and one of
ordinary skill in the art should readily be able to identify a set
of residues to mutate in order to accommodate the bulky hydrophobic
groups of one or more of the orthogonal substrates.
[0135] For example, one might mutate large hydrophobic groups to
smaller alanine or glycine residues, or replace hydrogen bonding
amino acids with others that compliment the orthogonal purine
analogs of AdoMet. Of course, a myriad of other possible mutations
may work as well, and all would be within the scope of the present
invention. In addition, from sequence alignments and crystal
structures of methyltransferases, it is known that they have a
common catalytic domain structure (Schluckebeir et al., (1995) Mol.
Biol. 247:16-20); so this approach is not limited to M.HhaI and
COMT, but should be equally applicable to other methyl
transferases.
[0136] After a methyltransferase mutant is identified which accepts
an orthogonal inhibitor, radiolabeled AdoMet can then be
synthesized which contains a C-14 labeled methyl group attached to
the sulfur atom of AdoMet. When this radiolabeled analog is added
to cells expressing one mutant methyltransferase, the direct
substrates (e.g., protein or DNA, or polyamines) of all
methyltransferases in the sample will be specifically radiolabeled
with the C-14 methyl group. But when this is done in the presence
of the orthogonal inhibitor, the specific substrates for the
methyltransferase of interest will be less labeled in comparison to
the sample not containing the inhibitor; preferably, they will not
be substantially labeled, and most preferably, will not be labeled
at all. In this way, or through the use of other methods described
herein for the study of the kinases, direct substrates of
methyltransferases can be identified which are important in cancer,
embryonic development, chemotaxis of polymorphonuclear leukocytes,
or in neurological disorders. In addition, the methods of the
present invention can then be used to determining whether compounds
can be identified that modulate the activity of the enzyme. The
several other aspects of the present invention, although perhaps
not described here, could also be applied to the methyl
transferases, and also to other multi-substrate enzymes.
[0137] The forgoing discussion of the application of the present
invention to the methyl transferases is not intended to limit the
scope of the present invention, but to illustrate of the
applicability of the present invention to multi-substrate enzymes
other than the protein kinases. As will be appreciated by those in
the art, the present invention could be applied similarly to other
multi-substrate enzymes using similar approaches.
[0138] Terms
[0139] As is generally the case in biotechnology, the description
of the present invention herein has required the use of a
substantial number of terms of art. Although it is not practical to
do so exhaustively, definitions for some of these terms are
provided here for ease of reference. Definitions for other terms
also appear elsewhere herein, and those are not repeated here. It
is important to note that it is not intended that the terms defined
here or elsewhere herein be given a meaning other than that which
those skilled in the art would understand them to have when used in
the field, and it is therefore urged that other sources also be
consulted in interpreting the meaning of these terms and those
defined elsewhere herein. However, the definitions provided here
and elsewhere herein should always be considered in determining the
intended scope and meaning of the defined terms.
[0140] The term "orthogonal" is used here to mean a compound that
is similar, structurally and/or geometrically, to the natural
substrate for a given enzyme, or to an inhibitor of the wild-type
form of the enzyme, but has differences in chemical structure which
make that compound less able to bind to the wild-type form of the
enzyme than is the natural substrate. By "natural" substrate
Applicant means that substrate which is utilized by the wild-type
form of that enzyme. The orthogonal inhibitors of the present
invention may be referred to in different ways herein; for example,
sometimes they are referred to as "modified substrates" or
"modified inhibitors" or "analogs" or "derivatives" just as
"substrates" or "inhibitors" and perhaps by other terms as well.
However, in each instance, the same meaning is intended. Of course,
the meaning of "orthogonal" and its synonyms are further explained
in the descriptions of the invention provided above.
[0141] The putative orthogonal substrates and inhibitors of the
embodiments of the invention described herein were made by adding
bulky substituents to an atom on the natural substrate or known
kinase inhibitor, respectively. However, the present invention is
not so limited. For example, it is possible to make an orthogonal
substrate that is smaller than a known inhibitor or the natural
substrate, e.g., by preparing an analog that is missing one or more
atoms or substituents that are present in the natural substrate.
With such putative orthogonal substrates or inhibitors, one could
mutate the enzyme to contain one or more amino acids having more
bulky side chains than those found in the wild-type amino acid
sequence, so that when the orthogonal substrate or inhibitor binds,
those more bulky amino acid side chains fill or partially fill the
extra space created by the missing atoms or substituents. In this
way, it would be expected that the mutant would bind to and/or be
inhibited by the orthogonal substrate or inhibitor, but would not
substantially utilize the normal substrate, because the added bulky
amino acids present a steric hindrance to its binding. Such an
approach would allow for highly selective control of the resulting
mutant.
[0142] It is important to keep in mind that even though the
substrates and inhibitors of the examples herein are of the
non-competitive type, this should not be viewed as a limitation of
the scope of the present invention. Many different types of enzyme
substrates and inhibitors are known, e.g., competitive,
non-competitive, uncompetitive, "suicide" inhibitors, etc.
Competitive inhibitors compete with a substrate for its binding
site; but since the inhibitor cannot participate in the catalytic
reaction which that enzyme carries out, it slows down catalysis.
Non-competitive inhibitors bind to the active site, but then become
covalently or ionically bound to the protein structure of the
enzyme, such that they cannot come off. Thus, they inhibit
catalysis by taking molecules of enzyme out of the reaction
altogether. More detailed descriptions of these and other
competitive mechanisms can be found in a variety of sources (e.g.,
Lehninger et al., (1993) Worth Publishers). By applying the
understanding of the art regarding such mechanisms to the design of
inhibitors of the present invention, all such types of inhibitors
could be made.
[0143] For example, an analog which can bind, but not react, would
provide a competitive inhibition, and an analog which becomes
covalently attached to the enzyme upon binding, would be a
non-competitive inhibitor, i. e., a poison. All such types of
inhibitors are within the scope of the present invention.
[0144] The term "homologous to" has been used to describe how
information about how to modify one enzyme can be deduced from
information regarding the three-dimensional structure of other,
related enzymes. As those in the field well know, a part of one
enzyme which is "homologous" to part of a second enzyme has a
protein sequence which is related to that of the second enzyme.
This relationship is that they have a number of amino acids in the
same relative location to one another. For example, the imaginary
sequence Asp-Met-Phe-Arg-Asp-Lys-Glu and the imaginary sequence Asp
Met-Ile-Arg-Glu-Lys-Asp have four amino acids in the same relative
location, and three which are different, and they would be said to
have homologous sequences. Note that the three amino acids that are
different between the chains are "conservative" differences, in
that the substitutions in the second sequence relative to the first
are with amino acids that have similar functionalities on their
side chains. For example, Glu and Arg both have aliphatic side
chains terminated in carboxylic acid groups, and both Phe and Ile
are hydrophobic. Although this is often the case with homologous
protein sequences, it need not be the case, and these two imaginary
sequences would still be considered homologous even if the
differences were not conservative.
[0145] Whether a particular sequence or domain is homologous to
another cannot be stated with any particularity, e.g., by using
percentages, as there is no such absolute yardstick; one must leave
it to the art to define which sequences are and are not considered
"homologous." Hardie & Hanks, (1995) Protein Kinase Facts Book,
Academic Press, gives a good overview of which domains of the known
kinases are considered by the art to be "homologous" In addition,
although the art may not generally agree, it is intended here that
sequences that are identical to one another also be considered to
be "homologous" to one another.
[0146] The term "domain" is also one well known in the art, and it
refers to a region in a protein which has been identified as having
a particular functionality. For example, the three domains in
protein kinases have been discussed elsewhere herein, and their
functional roles have been discussed. Often, as is the case with
the kinases, different enzymes of the same family will have the
same number of domains with each serving the same function, and
they are often (but probably not always) arranged in the same order
along the protein sequence. Interestingly, as is the case for the
kinases, one enzyme may have a different length of protein sequence
between its domains than does another. However, since the domains
of two related enzymes are generally (but probably not always)
homologous to one another, this does not generally hamper the
identification of corresponding domains.
[0147] In describing the broader aspects of the present invention,
the term "multi-substrate" is used. This is intended to mean
enzymes which bind two or more substrates. Those multi-substrate
enzymes of most interest here are those which catalytically attach
at least part of one substrate to at least one other substrate. The
kinases and the transferases are but two families of such
multi-substrate enzymes, and those of skill in the art will readily
recognize that there are other such enzymes and enzyme
families.
[0148] The term "recognize" is sometimes used here to describe the
ability of a substrate to specifically bind to the active site on
an enzyme. This simply refers to the fact that an enzyme's
substrate (or sometimes substrate derivatives or even completely
different compounds that mimic the substrate) can contact and bind
to the enzyme's active site, but other compounds will not. This
concept is well known in the art. Enzymologists often say that the
enzyme has an affinity for its substrate, or that the substrate has
an affinity for the enzyme. They also say that an enzyme has
"substrate specificity" These all really describe the same
phenomenon.
[0149] A related term is the term "bind." An inhibitor generally
binds, or sticks to, an active site through one or more
hydrophobic, hydrophilic, hydrogen, and/or ionic bonds, or, in the
case of non-competitive inhibitors, through covalent bonds.
[0150] Although the complex understanding in the art regarding
inhibitor binding and the reasons for inhibition may be of
interest, such an understanding is not essential to understanding
the present invention. It is sufficient to simply note that binding
by an inhibitor causes inhibition of the catalytic reaction.
[0151] The terms "mutant" and "engineered form" when used to
describe the enzymes of the present invention, simply mean that
they have sequences that have a different amino acid at one or more
position when compared to the sequence of the wild-type enzyme.
[0152] In describing such mutants, two letters separated by a
number indicate the amino acid mutations made. The letters are
single-letter amino acid codes, and the numbers are the amino acid
residue positions in the intact, wild-type enzyme. For example, GST
XD4 is a fusion protein containing a fragment, XD4, that has the
same sequence as a specific part of the wild-type v-Src. In the
designation GST-XD4 (V323A, I338A), the valine in the sequence of
v-Src fragment XD4 that represents position 323 in the complete
wild type v-Src sequence has been replaced by alanine, and the
ioleucine in the XD4 fragment that represents position 338 in the
complete wild type v-Src sequence has also been replaced with
alanine.
[0153] As described in the examples below, using the present
invention Applicant has designed, made and demonstrated the utility
of a v-Src kinase which shows high specificity for a synthetic
inhibitor while maintaining its wild-type specificity for tyrosine
containing peptides and proteins, thus satisfying Applicant's
initial research goals. By exploiting the highly conserved nature
of the ATP binding site across the kinase superfamily and the
availability of structural information from other protein kinases,
Applicant was able to engineer novel inhibition specificity for
v-Src without any detailed structural information about v-Src
itself. That Applicant used an unrelated kinase as a blueprint for
designing orthogonal ATP analogs to tag the direct cellular
substrates of v-src and have prepared inhibitors from like origins
demonstrates that this approach should work for other kinases as
well.
EXAMPLES
[0154] The following examples are provided to describe and
illustrate the present invention. As such, they should not be
construed to limit the scope of the invention. Those in the art
will well appreciate that many other embodiments also fall within
the scope of the invention, as it is described hereinabove and in
the claims.
Example 1
[0155] Synthesis of ATP Analogs
[0156] Twelve different orthogonal ATP analogs were synthesized.
FIG. 2 is a schematic representation of their structure. The figure
shows adenosine triphosphate (ATP), with an "X" bound to the 6
position; and in the box below, schematic representations are
provided for the twelve side chains that take the place of "X" in
each of the orthogonal ATP analogs described in the examples (which
are always referred to by the numbers 1-12 set forth in bold
typeface). Those analogs are:
[0157] 1. N.sup.6(methoxy)ATP
[0158] 2. N.sup.6 (ethoxy)ATP
[0159] 3. N.sup.6 (acetyl)ATP
[0160] 4. N.sup.6 (i-propoxy)ATP
[0161] 5. N.sup.6-(benzyl)ATP
[0162] 6. N.sup.6-(benzyloxy)ATP
[0163] 7. N.sup.6-(pyrolidino)ATP
[0164] 8. N.sup.6-(cyclopentyl)ATP
[0165] 9. N.sup.6-(cyclopentyloxy)ATP
[0166] 10. N.sup.6-(pipperidino)ATP
[0167] 11. N.sup.6-(cyclohexyl)ATP
[0168] 12. N.sup.6-(cyclohexyloxy)ATP
[0169] Analogs 1, 2, 4, 6, 9, and 12 were synthesized via Dimroth
rearrangement of the corresponding N.sup.1 alkoxy adenine
derivatives in four steps starting from adenosine, according to the
procedure of Fuji et al. (Fuji et al., (1973) Chem. Pharm. Bull.
21:1676-168243). Analog 5 was synthesized similarly via Dimroth
rearrangement of N.sup.1 benzyladenosine (Robins et al., (1976)
Biochem. 2:2179-2187). Analog 3 was prepared via in situ protection
of the adenosine hydroxyl groups as trimethylsilyl ethers and
subsequent treatment with acetyl chloride, according to McLaughlin
et al. (McLaughlin et al., (1985) Synthesis 00:322-323). Analogs 7,
8, 10 and 11 were synthesized via treatment of 6-chloropurine
riboside (Aldrich) with pyrrolidine, cyclopentylamine, piperidine
& cyclohexylamine, respectively (Kikugawa et al., (1973) J.
Med. Chem. 16:358-364).
[0170] Triphosphate synthesis was carried out according to the
method of Ludwig (Ludwig et al., (1981) Acta Biochim. Biophys.
Acad. Sci. Hung. 16:131-133) with the exception of the preparation
of pyrophosphate. Accordingly, bis-tri-N-butyl ammonium
pyrophosphate was prepared by mixing 1 equivalent of pyrophosphoric
acid with 2 equivalents of tributyl amine in a (1:1) water:ethanol
mixture until a homogenous solution was obtained. Solvent was
removed under vacuum to dryness and the pyrophosphate was stored
over P.sub.2O.sub.5 overnight.
[0171] All non-radioactive nucleotides were characterized by
.sup.1H-NMR, mass spectral analysis and strong anion exchange (SAX)
HPLC (Rainin #83-E03-ETI).
[0172] [.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP was synthesized
according the method of Hecht & Kozarich, (1973) Biochim.
Biophy. Acta 331:307-309. The radiolabeled analog was purified by
DEAE (A-25) Sephadex (Pharmacia) column chromatography and the
triphosphate was identified by co-injection of the radiolabeled
material with an authentic sample of N.sup.6-(cyclopentyl) ATP on
an SAX-anion exchange HPLC column (Rainin) (linear gradient of
5-750 mM ammonium phosphate pH 3.9 in 10 minutes at 0.5 ml/min).
The chemical yield of the reaction varied from 70% to 80%.
Example 2
[0173] Screening of Nucleotide Analogs
[0174] To identify compounds that would not be accepted as
substrates by any existing cellular kinases (Kwiatkowski et al.,
(1987) Biochemistry 26:7636-7640), Applicant screened a panel of
synthetic A*TP analogs in a murine lymphocyte lysate (CF) rich in
protein tyrosine kinases (Bolen et al., (1992) FASEB J.
6:3403-3409).
[0175] The assays were performed using spleenocytes (8-30 week old
male and female C57/B6 mice from the Princeton University Animal
Facility) which were isolated and washed in RPMI-1640 medium
containing 5% Bovine Calf Serum (BCS), 1% Hepes and DNAseI (1
.mu.g/ml). Red cells were lysed at 4.degree. C. by treatment with
17 mM Tris ammonium chloride (pH 7.2). The cells were hypotonically
lysed on ice for ten minutes in 1 mM Hepes (pH 7.4), 5 mM
MgCl.sub.2, leupeptin (10 .mu.g/ml), aprotinin (10 .mu.g/ml) and
100 .mu.M PMSF according to the method of Fukazawa et al., (1993)
Anal. Biochem. 212:106-110. After vortexing and centrifugation at
500.times.g, the supernatant was collected. Cells were stored at
4.degree. C. for twenty minutes to attenuate the basal protein
phosphorylation level, after which the buffer was adjusted to 20 mM
Hepes (pH 7.4), 10 mM MgCl.sub.2 and 1 mM NaF. Sodium vanadate (100
.mu.M) was then added to inhibit the activity of phosphotyrosine
phosphatases.
[0176] Each nucleotide triphosphate was added to a final
concentration of 100 .mu.M to 10.times.10.sup.6 cell equivalents
and incubated at 37.degree. C. for five minutes after which
4.times. Laenunli gel loading buffer was added to the cell lysate
to quench the reaction. Proteins were separated by 12.5% SDS-PAGE
and transferred to Protran BA85 (Schleicher-Schuell). The blot was
probed with the anti-phosphotyrosine monoclonal antibody 4G10
(Upstate Biotechnology) and the bound antibody was detected via
enhanced chemiluminescence (Pierce) following treatment with
HRP-coupled goat anti-mouse antibody (VWR catalog # 710133)
according to the manufacturer's instructions.
[0177] The results are shown in FIG. 3, which is an
anti-phosphotyrosine protein immunoblot showing the level of
protein tyrosine phosphorylation following treatment of a murine
lymphocyte cell lysate (CF) with 100 .mu.M of ATP or A*TPs (1-12).
The cell lysate used includes the tyrosine kinases Src, Fyn, Lck,
Lyn, Yes, Fgr, Hck, Zap, Syk, Btk, Blk, and other tyrosine kinases
present in B and T lymphocytes, macrophages, and follicular
dendritic cells (Bolen et al., (1992) FASEB J. 6:3403-3409).
Molecular size standards (in kilodaltons) are indicated. The A*TPs
containing the smallest N.sup.6 substituents, (1) (methoxy), (2)
(ethoxy), and (3) (acetyl) showed some ability to serve as cellular
tyrosine kinase substrates (FIG. 3, lanes 3-5). The A*TPs with
sterically demanding N.sup.6 substituents, (4) (i-propoxy), (5)
(benzyl), and (6) (benzyloxy), and all analogs containing cyclic
aliphatic substituents (7-12) showed little or no protein
phosphorylation (FIG. 3, lanes 6-8, 11-16).
[0178] To test for possible metathesis of orthogonal A*TPs (7-12)
with cellular ADP to give A*DP and ATP, Applicant added 1 mM ADP to
cell lysate kinase reactions identical to those shown in FIG. 3;
(data not shown); the pattern of phosphoproteins was the same,
indicating that no significant metathesis of A*TP occurs in a
complete cell lysate system.
[0179] Based upon these results, it appears that analogs (7-12) are
"dead substrates" for wild type tyrosine kinases, i.e., the
wild-type substrates do not substantially, or at all, accept these
as phosphate donor substrate. These analogs thus were chosen as the
most preferred targets for reengineering the nucleotide binding
site of v-Src.
Example 3
[0180] Designing the Mutant v-Src
[0181] No crystal structures of any tyrosine kinases in an active
conformation have been solved to date although several structures
of inactive kinases have been solved (Hubbard et al., (1994) Nature
372:746-754; Mohammadi et al., (1996) Cell 86:577-587). However,
two crystal structures of catalytically active ser/thr kinases have
been solved (Zheng et al., (1993) Biochemistry 32:2154-2161;
Jeffrey et al., (1995)Nature 376:131-320). There is a high degree
of functional homology between the ser/thr and the tyrosine kinase
catalytic domains as shown by affinity labeling of the identical
catalytically active lysine residue in both kinase families (K72 in
cAMP dependent kinase (PKA), K295 in v-Src) (Kamps et al., (1984)
Nature 312:589-592; Zoller et al., (1981) J. Biol. Chem.
256:10837-10842). Inspection of the PKA (Zheng et al., (1993)
Biochemistry 32:2154-2161) and cyclin dependent kinase-2
(CDK2)-cyclin A (Jeffrey et al. (1995) Nature 376:313-320) crystal
structures revealed two amino acid side chains within a 4 A sphere
of the N.sup.6 amino group of bound ATP: V104/M120 (PKA) and
V64/F80 (CDK2) (Taylor et al. (1995) Structure 2:345-355).
[0182] FIG. 4 shows a close-up view of the ATP binding site in cAMP
dependent protein kinase (PKA), which is bound to ATP. Three
residues within a 4 A sphere of the N.sup.6 amine of ATP (Val104,
Met120 and Glu121) and the catalytically essential lysine residue
(Lys72) are shown in ball-and-stick representation. The remainder
of the protein is shown in ribbon format. This figure was created
by feeding the output of Molscript into the Raster3D rendering
program (Merritt et al., (1994) Acta Cryst. D50:869-873; Bacon et
al., (1988) J. Molec. Graphics 6:219-220). Note that in the model,
the side chain of Glu121 is pointed away from the adenine ring
binding region, and therefore Glu121 was not a candidate for
alteration.
[0183] The sequence alignment of the ATP binding regions of PKA
(SEQ ID NO: 1), CDK2 (SEQ ID NO: 2), and v-Src (SEQ ID NO: 3) are
shown below. The residues shown in bold correspond to the amino
acids with side chains in a 5 A sphere of the N.sup.6 amino group
of kinase bound ATP.
1 Subdomain PKA (SEQ ID NO: 1) (99)NFPFLVKLEFSFKDNSNLYMVMEYVPG(125)
CDK2 (SEQ ID NO: 2) (59)NHPNIVKLLDVIHTENKLYLVFEFLHQ(85) v-Src (SEQ
ID NO: 3) (318)RHEKLVQLYAVVSE-EPIYIVIEYMSK(343)
[0184] Based on the functional similarity between the
above-described kinases, Applicant decided to mutate positions V323
and I338 in the v-Src catalytic domain, which correspond to
V104/M120 in PKA & V64/F80 in CDK2. By mutating these residues
to alanine, Applicant hoped to create an additional "pocket" in the
nucleotide binding site of v-Src to allow binding of one of the
preferred orthogonal A*TPs (4-12).
Example 4
[0185] Mutant Synthesis, Expression and Purification
[0186] The mutant (V323A, I338A) was made as described below. Both
the wild-type and the double alanine mutant of the v-Src catalytic
domain, (the XD4 fragment) were made as glutathione S-transferase
(GST) fusion proteins (GST-XD4) (DeClue et al., (1989) J. Virol.
63:542-554; Seidel-Dugan et al., (1992) Mol. Cell Biol.
12:1835-1845). These were made in E. coli, which is a good
expression host because it lacks any endogenous tyrosine kinases,
as described in the following Example. Applicant used the XD4
fragment of v-Src because it contains an intact SH1 catalytic
domain but lacks the non-catalytic regulatory SH3 and SH2 domains,
and exhibits higher specific activity than full-length v-Src.
[0187] Overlap extension PCR was used to make GST-XD4 (V323A,
I338A) (Reikofski et al., (1992) Biotech. Adv. 10:535-554). Pfu
polymerase (Stratagene) was used in the PCR reactions according to
the manufacturer's protocol. Six synthetic oligonucleotides were
used:
2 SEQ ID NO:4 (5'-TTTGGATCCATGGGGAGTAGCAAGAGCAAG) SEQ ID NO:5
(5'-TTTGAATTCCTACTCAGCGACCTCCAACAC) SEQ ID NO:6
(5'-TGAGAAGCTGGCTCAACTGTACGCAG) SEQ ID NO:7
(5'-CTGCGTACAGTTGAGCCAGCTTCTCA) SEQ ID NO:8
(5'-CTACATCGTCGCTGAGTACATGAG) SEQ ID NO:9
(5'-CTCATGTACTCAGCGACGATGTAG)
[0188] Primer SEQ ID NO: 4 contains a BamHI site and primer SEQ ID
NO: 5 contains an EcoRI site (shown in italics). Primers SEQ ID NO:
6 and SEQ ID NO: 7 contain the nucleotide sequence changes to
introduce the V323A mutation (nucleotides encoding mutations are
shown in bold). Primers SEQ ID NO: 8 and SEQ ID NO: 9 contain the
I338A mismatch. The XD4 gene from YEp51-XD4 plasmid (a gift of B.
Cochran at Tufts Medical School) was amplified with primers SEQ ID
NO: 4 and SEQ ID NO: 5. The PCR product was digested with BamHI and
EcoRI and ligated into BamHI and EcoRI digested pGEX-KT and then
transformed into the E. coil strain DH5.alpha..
[0189] The GST-XD4 (V323A) was constructed using primer SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 with the GST-XD4
plasmid as the template. The PCR product from the two step
procedure was digested with BamHI and EcoRI, ligated into BamHI and
EcoRI-digested pGEX-KT, and transformed into DH5.alpha. E. coli
cells. GST-XD4 (V323A, I338A) was made in the same manner using
primers SEQ ID NO: 8 & SEQ ID NO: 9 with GST-XD4 (V323A) as the
template.
[0190] Expression and purification of the GST fusion kinases were
carried out in E. coli strain DH5.alpha. as described by Xu et al.,
(1995) J. Biol. Chem. 270:29825-29830, with the exception that the
cells were stored at 4.degree. C. overnight prior to centrifugation
and lysis by French press (overnight storage is essential for
producing highly active kinases).
[0191] Expression of 6-His-XD4 and 6-His-XD4 (V323A, I338A) in Sf9
insect cells was accomplished using the Life Technologies
BAC-to-BAC system. Briefly, the 6-His-XD4 and 6-His-XD4 (V323A,
I338A) genes were generated by PCR using the corresponding pGEX
vectors as templates with primers SEQ ID NO: 4 and SEQ ID NO: 5
followed by digestion with BamHI and EcoRI. The resulting PCR
fragment was cloned into pFASTBAC which had been digested with
BamHI and EcoRI. Transformation of HB10BAC cells and subsequent
transfection of Sf9 cells with the Bacmid containing XD4 or XD4
(V323A, I338A) were carried out as suggested by the
manufacturer.
[0192] In an alternate procedure performed herein, transfection of
v-src or v-src (I338G) mutant kinase was performed by cloning the
v-src gene from the pGEX-v-Src vector (Hunter, (1995) Cell
80:225-236) into the pBabe vector (Eiseman et al., (1992) Nature
355:78-80) which contains the LTR promotor for high level of
expression in NIH3T3 cells. The pBabe v-Src (I338G) plasmid was
transfected into viral packaging cell line BOSC23 (Murray, (1994)
Chem. Bio. 1:191-195) and viral particles harvested after two days
as described (Murray, (1994) Chem. & Biol. 1:191-195). NIH3T3
cells were infected as described (White, (1991) J. Bioenerg.
Biomembr. 23:63-83) with these viral particles and stable
transfectants were selected in puromycin containing media as
described (Eiseman et al., (1992) Nature 355:78-80). Stable
transfectants were maintained in media containing puromycin to
ensure no loss of expression of v-Src.
[0193] The final results are shown in FIG. 1, which is a diagram
showing the domain structure of v-Src including the Src-homology 3,
2, and 1 (SH3, SH2 & SH1) domains, with the domain boundaries
indicated by the amino acid residue numbers listed above each boxed
domain. The domain structure of XD4 is also represented, which
contains a deletion of residues 77-225 (A77-225). Domain
organizations of the glutathione S-transferase (GST) fusion with
XD4 (numbering from v-Src), and the doubly mutated GST-XD4
(representing both V323A, I338A and I338G) are also shown
schematically.
Example 5
[0194] Testing the Mutant v-Src for Ability to Bind Orthogonal ATP
Analogs
[0195] Applicant next evaluated the ability of the N.sup.6
substituted ATP analogs (1-12) to differentially inhibit wild-type
and mutant kinase phosphorylation of RR-Src with
[.gamma.-.sup.32P]ATP, which is a measure of their ability to bind
to the respective ATP binding sites. Assays were carried out in
triplicate at 37.degree. C. in a final volume of 30 .mu.L buffered
at pH 8.0 containing 50 mM Tris, 10 mM MgCl.sub.2, 1.6 mM
glutathione, 1 mg/ml BSA, 1 mM RR-Src peptide with either GST-XD4
(100 nM) or GST XD4 (V323A, I338A) (100 nM) and 10 .mu.M
[.gamma.-.sup.32P]ATP (1000 cpm/pmol) (Dupont-NEN). Cold ATP or
A*TP analogs (100 .mu.M) (1-12) were added prior to addition of the
kinase. After thirty minutes the reactions were quenched by
spotting 25 .mu.l of the reaction volume onto p81 phosphocellulose
disks (Whattman) and these were immersed in 250 ml of 10% acetic
acid for at least thirty minutes followed by washing and
scintillation counting according to standard methods (Lee et al.,
(1995) J. Biol. Chem. 270:5375-5380).
[0196] The results are shown in FIG. 1. Relative inhibition of
GST-XD4 is shown by solid bars, and relative inhibition by GST-XD4
(V323A, I338A) is represented by the diagonal filled bars. Percent
inhibition (1-v.sub.l/v.sub.o) is reported as a ratio of v.sub.l
(cpm in the presence of 100 .mu.M of the indicated triphosphate and
10 .mu.M [.gamma.-.sup.32P]ATP (1000 cpm/pmol)/v.sub.o (cpm in the
presence of 10 .mu.M [.gamma.-.sup.32P]ATP (1000 cpm/pmol) alone
background cpm due to non-specific 10 .mu.M [.gamma.-.sup.32P]ATP
binding to the phosphocellulose disks (<0.1% of total input
counts). Error bars represent the S.D. determined from four
separate experiments with three replicates.
[0197] The wild-type kinase GST-XD4 displays poor binding affinity
for most A*TP analogs (FIG. 6, solid bars) as expected from the
lymphocyte kinase assay (FIG. 3). In contrast, the doubly mutated
GST-XD4 (V323A, I338A) shows excellent inhibition by more
sterically demanding N.sup.6 substituted ATP analogs (FIG. 6,
shaded bars). Most significantly, the GST-XD4 (V323A, I338A) mutant
is inhibited by ATP analogs (5), (8), (9) and (11) almost as well
as the wild-type kinase, GST-XD4, is inhibited by its natural
substrate ATP. Applicant has confirmed that GST-XD4 (V323A, I338A)
and the full length GST-v-Src (V323A, I338A) display the same
inhibition pattern with A*TPs (1-12) (data not shown).
[0198] Four of the nine "dead" substrates identified in the screen
of wild-type kinase specificity (FIG. 3) bind well to the mutant
kinase. This high success rate in identifying new substrates for a
mutant v-Src which are not accepted by wild-type kinases suggests
that Applicant has identified a key feature of the v-Src nucleotide
binding site, namely the residues which make a close fit around the
N.sup.6 amino group of ATP. It is worth noting that Applicant is
not aware of any wild-type protein kinases which contain an alanine
at the position corresponding to I338 in v-Src (position 120 in
PKA). If a sterically demanding amino acid side chain at this
position also plays a critical role in determining the specificity
of other kinases, it should well be possible to engineer them to
accept orthogonal substrates using an approach very similar to the
one described here, and such engineered kinases would be well
within the scope of the present invention.
Example 6
[0199] Determining Catalytic Efficiency of Mutant v-Src with the
Most Preferred Orthogonal ATP Analog
[0200] Applicant chose to test the ability of N.sup.6-(cyclopentyl)
ATP, (8), to serve as a catalytically competent substrate of both
wild-type GST-XD4 and the GST-XD4 (V323A, I338A) mutant over the
other three ATP analogs (5), (9) and (11) because analog (8)
exhibited a slightly lower level of phosphorylation with wild-type
kinases (FIG. 3, lane 12).
[0201] ATP and N.sup.6-(cyclopentyl)ATP dependent RR-Src
phosphorylation (1 mM) by GST-XD4 (V323A, I338A) and GST-XD4 were
carried out at low substrate conversion (<5%) in triplicate.
Kinetic constants were determined by analysis of Lineweaver-Burk
plots of the rate data (Fersht, (1985) Enzyme structure and
mechanism, W. H. Freeman). Assays were carried out in triplicate at
37.degree. C. in a final volume of 30 .mu.l buffered at pH 8.0
containing 50 mM Tris, 10 mM MgCl.sub.2, 1.6 mM glutathione, 1
mg/ml BSA, 1 mM RR-Src peptide with either GST-XD4 (100 nM) or
GST-XD4 (V323A, I338A) (100 nM) and 10 .mu.M [.gamma.-.sup.32P]ATP
(1000 cpm/pmol) or [.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP (5000
cpm/pmol) as indicated.
3TABLE 1 Kinetics for Phosphate Donor Substrates GST-XD4 GST-XD4
(V323A, I338A) Nucleotide K.sub.cat (min.sup.-1) K.sub.M (.mu.M)
K.sub.cat /K.sub.M (min.sup.-1M.sup.-1) K.sub.cat (min.sup.-1)
K.sub.M (.mu.M) K.sub.cat/K.sub.M (min.sup.-1M.sup.-1) ATP 2 .+-.
0.5 12 .+-. 3 1.6 .times. 10.sup.5 0.8 .+-. 0.2 150 .+-. 20 5.3
.times. 10.sup.3 N.sup.6-(cp)ATP 2000 (K.sub.i) (5 .+-. 2) .times.
10.sup.-2 15 .+-. 3 3.3 .times. 10.sup.3
[0202] As shown in Table 1 above, the wild-type kinase GST-XD4 did
not substantially phosphorylate the RR-Src peptide with
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP, confirming Applicant's
previous observations that this analog is not a significant
substrate for the wild-type kinase. In contrast, GST-XD4 (V323A,
I338A) displayed Michaelis-Menten kinetics with the orthogonal
A*TP, [.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP. The K.sub.M of
the mutant for the orthogonal substrate is quite close to the
K.sub.M of GST-XD4 for ATP. On the other hand, the mutant has a
K.sub.M for ATP which is more than 10-fold higher than the K.sub.M
of GST-XD4 for ATP.
[0203] The parameter used to rank catalysts for competing
substrates is the ratio of the turnover number to the
Michaelis-Menten constant, K.sub.cat/K.sub.M (the "specificity
constant") (Fersht, (1985) Enzyme structure and mechanism, W. H.
Freeman). The K.sub.cat/K.sub.M of the engineered mutant GST-XD4
(V323A, I338A) with the orthogonal substrate
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP is only 50-fold lower
than the K.sub.catK.sub.M value of the wild-type kinase with its
natural substrate, ATP. This catalytic efficiency with the
orthogonal A*TP substrate, coupled with the mutant kinase's lower
catalytic efficiency with ATP when compared to the wild-type,
satisfy two of the design criteria discussed above.
[0204] It is even more significant that the new substrate,
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP, is not substantially
utilized by wild-type GST-XD4, as demonstrated by the apparent
complete inability of GST-XD4 to use this analog as a phosphodonor
for autophosphorylation; this is illustrated in FIG. 5C, lane 3.
FIG. 5C is an autoradiogram showing [.gamma.-.sup.32P]ATP dependent
autophosphorylation of GST-XD4, lane 1, or GST-XD4 (V323A, I338A),
lane 2; and [.gamma.-.sup.32P]N.sup.6-- (cyclopentyl)ATP dependent
phosphorylation of GST-XD4, lane 3, or GST-XD4 (V323A, I338A)
phosphorylation, lane 4. Note that in contrast to GST-XD4, the
engineered kinase is efficiently autophosphorylated with
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP (FIG. 5C, lane 4).
Example 7
[0205] Confirming Retention of Protein Substrate Specificity
[0206] As shown in Table 2 below, Applicant has found that the
wild-type GST-XD4 kinase phosphorylated a well characterized
peptide substrate of v-Src, RR-Src, with kinetics consistent with
literature reports (Czemilofsky et al., (1980) Nature 287:198-200).
This indicates that the sequence engineering had not substantially
affected the catalytic activity of the enzyme with respect to its
protein substrates.
4TABLE 2 Kinetics for Protein Substrate RR-Src GST-XD4
GST-XD4(V323A, 1338A) Nucleotide K.sub.M (.mu.M) K.sub.M (.mu.M)
ATP 2.6 .+-. 0.9 3.1 .+-. 0.9 N.sup.6-(cp)ATP 2.1 .+-. 0.9
[0207] Assays of GST-XD4 and GST-XD4 (V323A, I338A) phosphorylation
of RR-Src were carried out in triplicate at 37.degree. C. in a
final volume of 30 .mu.l buffered at pH 8. 0 containing 50 mM Tris,
10 mM MgCl.sub.2, 1.6 mM glutathione, 1 mg/ml BSA, 1 mM RR-Src
peptide with either GST-XD4 (100 nM) or GST-XD4 (V323A, I338A) (100
nM) and 10 .mu.M [.gamma.-.sup.32P]ATP (1000 cpm/pmol).
[0208] To determine whether the alanine mutations have any effect
on the protein substrate specificity, Applicant measured the
K.sub.M of both the wild-type and the mutant fusion proteins for
the RR-Src peptide. At saturating concentrations of
[.gamma.-.sup.32P]ATP the wild-type and the mutant display
essentially the same K.sub.M for RR-Src, 2.6.+-.0.9 mM and
3.1.+-.0.9 mM, respectively (Czernilofsky et al., (1980) Nature
287:198-200). In addition, the K.sub.M of the mutant for the
protein substrate in the presence of saturating amounts of the
orthogonal substrate was also essentially the same, 2.1.+-.0.9 mM.
These findings suggest that the alanine mutations in the ATP
binding pocket, which is proximal to the adjacent phospho-acceptor
binding site, do not affect the protein target specificity.
[0209] In support of this, the engineered kinase phosphorylates the
same broad set of proteins that are phosphorylated by wild-type XD4
when each is expressed in Sf9 insect cells. This is shown in the
FIG. 5A, which shows an anti-phosphotyrosine protein blot of cell
lysates (108 cell equivalents/lane) from Sf9 insect cells
expressing 6-His-XD4, lane 2 or 6-His-XD4 (V323A, I338A), lane 3.
These blots were carried out following lysis of 10.sup.6 cells in a
buffer containing 0.1% Triton X-100, 50 mM Tris (pH 8.0) using a
procedure similar to that of the blots of Example 2.
[0210] The Sf9 insect cell system is a good host for expressing
small amounts of tyrosine kinases because these cells contain most
of the same machinery necessary to carry out post-translational
modifications to proteins resulting in kinases which are more
similar in activity to those found in mammalian cells. Furthermore,
uninfected Sf9 cells lack endogenous tyrosine kinase activity, as
shown in FIG. 5A, lane 1, and thus the phosphotyrosine containing
proteins in lanes 2 and 3 of FIG. 5A are substrates of the
expressed 6-His-XD4 or mutant 6-His-XD4 kinases. Applicant
attributes the small differences in phosphorylation level of
particular proteins to the lower catalytic activity of the mutant
XD4 (V323A, I338A) compared to the wild-type kinase.
[0211] Taken together, these data show that the peptide specificity
of the engineered kinase is virtually identical to that of
wild-type v-Src.
Example 8
[0212] Confirmation that the Engineered Kinase Accepts the
Preferred Orthogonal Substrate, but the Wild-type Kinase does not
Substantially Accept it
[0213] The ultimate goal of this work is to use mutant kinases
specific for synthetic substrate analogs to tag the direct protein
substrates in whole cells or cell lysates. For this it is
preferable that no wild-type kinase, including ser/thr specific
kinases (which carry out the bulk of cellular phosphorylation, as
only 0.03% of all phosphoamino acids are tyrosine) (Hunter et al.,
(1980) Proc. Natl. Acad. Sci. USA 77:1311-1315), substantially
accept the synthetic substrate. To establish that
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP is essentially a "dead
substrate" for all wild-type cellular kinases, in vitro kinase
reactions with [.gamma.-.sup.32P]ATP or
[.gamma.-.sub.32P]N.sup.6-(cyclopentyl)ATP were performed with
murine lymphocyte lysates.
[0214] These assays were performed in a manner similar to the
procedure set forth in Example 2, with the exception of the use of
radiolabeled [.gamma.-.sup.32P]ATP or
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP (5000 cpm/pmole) added
to a final concentration of 100 .mu.M with 5.times.10.sup.6 cell
equivalents and incubated at 37.degree. C. for ten minutes, after
which 4.times. Laemmli gel loading buffer was added to the cell
lysate to quench the reaction. Proteins were separated by 12.5%
SDS-PAGE. The gel was soaked in 10% acetic acid, 10% isopropanol
for one hour after which it was dried in a gel dryer and exposed to
Biomax MS film (Kodak) for one hour.
[0215] The results are shown in FIG. 5B, which is an autoradiogram
showing the level of phosphorylation in hypotonically lysed murine
lymphocytes with [.gamma.-.sup.32P]ATP, lane 1 or
[.gamma.-.sup.32P]N.sup.6-(cyclopen- tyl)ATP, lane 2. There are no
radiolabeled phosphoproteins in the cell lysate following addition
of [.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP, confirming the true
orthogonal nature of N.sup.6-(cyclopentyl)ATP with respect to all
wild type protein kinases. The same result was found when in vitro
kinase reactions with [.gamma.-.sup.32P]ATP or
[.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP and NIH3T3 cell lysates
were used instead of freshly isolated murine lymphocytes (not
shown). In principle, the ability to follow one protein kinase's
activity in the presence of all other cellular kinases would allow
for the identification of the direct kinase targets in a particular
cell type. To accomplish this Applicant is currently using membrane
permeabilization (Ozawa et al., (1993) J. Biol. Chem.
268:1749-1756) and a cell permeable form of A*TP to introduce
[.gamma.-.sup.32P]A*TP into cells (Schultz et al., (1994) Mol.
Pharmacol. 46:702-708).
Example 9
[0216] Construction and Analysis of Single Mutation v-Src
Mutants
[0217] In order to determine whether a single mutation might be
sufficient to allow N.sup.6-(cyclopentyl)ATP to be efficiently used
as a substrate, three additional v-Src derived mutants were
prepared, using methods comparable to those of Example 4. However,
these had only single mutations, at position 338. These were again
expressed as GST-XD4 fusion proteins. These mutants, GGST-XD4
(I338A), GST-XD4 (I338S) and GST-XD4 (I338G), were then tested as
described in Example 8.
[0218] The results are shown in FIG. 7. The gel lanes shown on the
top left of FIG. 7 show that the mutant with alanine at the 338
position was able to utilize the natural substrate, ATP, more
readily than the mutant with serine at that same position. The gel
lanes shown on the bottom left of FIG. 7 show that the mutant with
alanine in position 338 is also better able to use ATP as a
substrate than is the mutant with glycine at that position.
[0219] The panels on the right side of FIG. 7 tell an even more
interesting story. From the top right panel, it is clear that the
mutant with serine at position 338 is not able to utilize
N.sup.6-(cyclopentyl)ATP nearly as well as is the mutant with
alanine at that position. However, the bottom panel shows that the
mutant with glycine at position 338 is better able to use
N.sup.6-(cyclopentyl)ATP as substrate than is the mutant with
alanine at that position.
[0220] These results are most promising. It appears that a single
mutation is enough to allow the use of this orthogonal substrate.
Notably, the mutant with glycine at position 338 appears to be the
best engineered v-Src mutant that Applicant has produced to date.
Moreover, it is quite surprising that a glycine substitution would
work here.
[0221] Generally, glycine substitution is usually not expected to
work in such situations, because it introduces too much flexibility
into the enzyme structure, and thus detrimentally affects the
desired outcome.
Example 11
[0222] Identifying the Substrates of v-Src
[0223] A schematic representation of an experimental approach to
identifying v-Src substrates is shown in FIG. 8. The engineered
v-Src, such as GST-XD4 (V323A, I338A), is added to cell extracts or
permiablized cells, along with a radiolabeled orthogonal substrate,
such as [.gamma.-.sup.32P]N.sup.6-(cyclopentyl)ATP. Typically, this
would be done in triplicate. After incubation, the cells would be
lysed (if not already lysed), and the resulting samples would be
separated by polyacrylamide gel electrophoresis.
[0224] A western blot taken from the gel and labeled with
anti-phosphotyrosine would show all phosphorylated proteins in the
sample; and an autoradiogram of the gel would reveal which of those
were phosphorylated by v-Src.
Example 12
[0225] Synthesis of Inhibitors
[0226] The pyrazolopyrimadine backbone for the first six inhibitors
is shown in FIG. 11A. Synthesis of
4-amino-1-tert-butyl-3-phenylpyrazolo [3,4-d]pyrimidine, having a
phenyl group in the "R" position, compound 1 (which is the same
structure as PP1, shown on FIG. 10, but without the para-methyl
group on the phenyl ring) was carried out according to the method
of Hanefeld et al., (1996) J. Chem. Soc. Perkin Trans. 1:
1545-1552. Compounds (2-6) (FIG. 11), having cyclobutoyl,
cyclopentoyl, cyclohexoyl, benzoyl, and 2-furoyl substituents at
the "R" position, respectively, were synthesized by treatment of
(1) with cyclobutoyl chloride, cyclopentoyl chloride, cyclohexoyl
chloride, benzoyl chloride, or furoyl chloride, respectively in dry
pyridine for one hour at room temperature. The structures of each
of the substituents are shown in FIG. 11B. Purification by silica
gel chromatography afforded pure products in 6-84% yield. Compounds
(1-6) were characterized by .sup.1H-NMR and mass spectral
methods.
Example 13
[0227] Screening of Inhibitors Which are Orthogonal to Wild-type
Kinases
[0228] To identify compounds that would not inhibit any existing
cellular kinases, Applicant screened the panel of synthetic
pyrazolo pyramidine analogs (1-6) against two closely related
purified tyrosine kinases, v-Src and Fyn, in a peptide
phosphorylation assay using [.gamma.-.sup.32P]ATP as the radiolabel
tracer of kinase activity, as described in Shah et al., (1997)
Proc. Natl. Acad. Sci. USA 94:3565-3570.
[0229] The results showed that each of the compounds (2-6) had
IC.sub.50 values of over 400 .mu.M for inhibition of Src and
compounds (3) and (5) showed at over 400 .mu.M IC.sub.50 values for
inhibition of wild-type Fyn, indicating that these analogs (2) and
(5) are orthogonal to (do not inhibit) these representative
wild-type kinases.
Examples 14-16
[0230] Deconvoluting protein kinase signaling pathways using
conventional genetic and biochemical approaches has been difficult
due to the overwhelming number of closely related kinases. If cell
permeable inhibitors of each individual kinase could be designed,
the role of each protein kinase could be systematically
assessed.
[0231] Results:
[0232] Applicant has devised an approach combining chemistry and
genetics to develop the first uniquely specific cell permeable
inhibitor of the oncogenic protein tyrosine kinase, v-Src. A
functionally silent active site mutation was made in v-Src in order
to distinguish it from all other cellular kinases. A tight binding
(IC.sub.50=430 nM) cell permeable inhibitor of this mutant kinase
was designed and synthesized which does not inhibit wild-type
kinases. In vitro and whole cell assays established the unique
specificity of the mutant v-Src/inhibitor pair. This inhibitor
reverses the transforming effects of cellular expression of the
engineered v-Src, but does not disrupt wild type v-Src mediated
cellular transformation. These cell lines differ only by a single
amino acid in a single protein kinase, establishing that dramatic
changes in cellular signaling can be directly attributed to
specific inhibition of the engineered kinase. The generality of
this method was tested by engineering another tyrosine kinase, Fyn,
to contain the corresponding silent mutation. The same compound was
found to be a potent inhibitor (IC.sub.50=830 nM) of this mutant
kinase as well, confirming the generality of the strategy toward
making allele specific inhibitors of multiple tyrosine kinases.
[0233] Conclusions:
[0234] Allele specific cell permeable inhibitors of individual Src
family kinases can be rapidly developed using a combined chemical
and genetic approach.
[0235] Treatment of mutant v-Src transformed NIH3T3 fibroblasts
with a uniquely specific v-Src reverts the morphological hallmarks
of transformation. The inhibitor exhibits no effect on cells
transformed by the wild-type v-Src allele strongly suggesting that
the phenotype induced by inhibitor treatment is a result of a
single inhibitory event.
[0236] The ability to rapidly generate kinase specific inhibitors
in a generalizable way will be useful for deconvolution of kinase
mediated cellular pathways and for validating novel kinases as good
targets for drug discovery both in vitro and in vivo.
[0237] As stated earlier, a combined chemical and genetic strategy
has been devised which allows for the generation of "chemical
sensitive" mutant kinases which are uniquely inhibited by a
rationally designed small molecule inhibitor. Applicant's approach
involves engineering a unique pocket in the active site of the
kinase of interest with a functionally silent mutation. A specific
inhibitor of the engineered kinase is then synthesized by
derivatizing a known kinase inhibitor with a bulky group designed
to fit the novel active site pocket. The bulky group kills the
potency of the inhibitor for wild type kinases. Successful
complementary design, therefore, leads to favorable binding
interactions that are only possible in the engineered
kinase/inhibitor complex. Transfection of cells with the gene
encoding the engineered kinase generates a cell in which only one
kinase can be blocked by the designed inhibitor (see FIG. 14).
[0238] Importantly, since the mutant kinase serves the same
function as the wild-type kinase, an inhibitor of the mutant will
affect cell signaling in the same manner as a selective inhibitor
of the wild-type kinase in non-transfected cells. The ability to
observe the phenotype of cells after selective inhibition of any
protein kinase provides a rapid method for determining the unique
roles of individual kinases in signal transduction cascades.
[0239] Applicant has targeted the src family protein tyrosine
kinases for specific inhibitor design because of their ubiquitous
importance in mediating cell function. Despite intense
investigation, the roles of individual src family members have been
difficult to assess because of cellular co-localization and their
high sequence identities. Although some potent inhibitors of Src
family kinases are known, no molecules which can effectively
discriminate (twenty-fold selectivity for one src family member)
between these closely related enzymes have been identified.
[0240] Two functionally important src kinases, v-Src and Fyn, were
chosen as the primary targets of Applicant's mutant
kinase/inhibitor pair design. Src kinase has emerged as a leading
drug target because of its implication in the oncogenesis of
breast, lung, and colon cancers. Although v-Src is the prototype
for oncogenic tyrosine kinases, no small molecule inhibitors which
are highly selective for this kinase have been discovered. Fyn is
an Src family tyrosine kinase which is important in T cell receptor
mediated lymphocyte activation. Src and Fyn share a similar domain
structure and have approximately 85% amino acid identity in their
catalytic domains. The close structural relationship of the Src
family members provides the ideal test of Applicant's ability to
engineer enzyme/inhibitor specificity between highly homologous
kinases. If one can discriminate between these closely related Src
members using a cell permeable inhibitor, it is likely that
specificity for members of other protein kinase families can also
be achieved using a similar approach.
[0241] Results and Discussion
[0242] Enzyme Engineering
[0243] From Applicant's previous efforts to engineer kinases with
novel ATP specificity, Applicant identified a functionally
conserved residue in the ATP binding pocket of v-Src (Ile338) which
could be mutated to glycine without altering the phosphoacceptor
specificity or biological function of the kinase. The space
creating mutation causes only a modest drop in K.sub.cat, a modest
increase in the K.sub.M for ATP and no quantitative change in the
level of fibroblast transformation (Shah, unpublished results). The
biological substrates of the mutant v-Src are unchanged and I338G
v-Src carries out the same biological functions as wild type v-Src.
All crystal structures of ATP bound protein kinases have revealed a
close contact interaction between the residue corresponding to 338
(Src numbering) and ATP. Analysis of protein kinase sequence
alignments confirmed that residue 338 contains a bulky side chain
(usually Thr, Ile, Leu, Met, or Phe) in all known eukaryotic
protein kinases. Thus, a glycine mutation at the 338 position
should create a novel pocket that is not present in any wild type
kinase. Due to the expanded ATP binding site, the glycine mutant
kinases should accept bulky inhibitors that could not bind wild
type kinases. Using standard methods Applicant cloned, expressed
and purified the glutathione-S-transferase (GST) fusion protein of
the WT and I338G v-Src catalytic domains as described previously.
WT Fyn, T339G Fyn (Src numbering), and WT Abl were also expressed
and purified as GST fusion proteins.
[0244] Inhibitor Design and Synthesis
[0245] To test Applicant's basic design strategy Applicant screened
the WT and I338G v-Src SHI domains against a previously synthesized
panel of N-6 substituted adenosine molecules for selective
inhibition of I338G v-Src over WT v-Src. Because adenosine is only
a moderate inhibitor of src family tyrosine kinases, Applicant did
not expect to discover a potent inhibitor of the engineered kinase.
As expected, all of the N-6 adenosine analogues inhibited I338G
v-Src more potently than WT v-Src (data not shown). The most potent
inhibitor found in this screen was N-6 cyclopentyloxyadenosine
((1), FIG. 15A) with an IC.sub.50 of 1 mM for I338G v-Src.
Subsequent experiments to test for selectivity demonstrated that
N-6 cyclopentyloxyadenosine showed no detectable in vitro
inhibition of WT v-Src or Fyn at concentrations up to 400 mM. This
first screen encouraged Applicant to pursue the strategy of
developing novel inhibitors of I338G v-Src since Applicant's design
had allowed Applicant to readily over come selectivity barriers
which are major problems in conventional inhibitor discovery.
[0246] As inhibitors, adenosine analogues are not ideal because of
the many cellular functions performed by adenosine as well as the
large number of cellular proteins which bind adenosine. N-6
adenosine analogues have been shown to act as adenosine receptor
agonists and antagonists, and one can imagine N-6 adenosine
analogues acting as substrates for nucleoside kinases. For these
reasons Applicant turned to a class of known tyrosine kinase
inhibitors that are not direct analogues of biologically known
molecules. Applicant's design strategy called for a core structure
which exhibits potent inhibition of multiple wild type kinases and
is easily synthesized. Also, the binding orientation of the
molecule in the enzyme active site must be known or readily
predictable. In addition, the molecule must bind in a manner in
which the site pointing toward Ile338 can be easily modified. As
Applicant's core inhibitor structure
4-amino-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidin- e was chosen
((2), FIG. 15B). This molecule is a derivative of 4-amino-l
-tert-butyl-3-(p-methylphenyl)pyrazolo [3,4-d]pyrimidine (PP 1)
which was reported by Hanke and co-workers as a potent src family
kinase inhibitor. Based on the co-crystal structure of the src
family kinase, Hck, bound to the general kinase inhibitor,
quercetin ((5), FIG. 16), Applicant postulated that (2) binds to
src family kinases in a conformation similar to that of ATP. The
predicted binding orientation of (2) in Hck is shown in an overlay
with the known Hck co-crystal structures of AMP PNP (2) and
quercetin (FIG. 16B). In this conformation the easily derivatizable
N-4 position of (2) corresponds to the N-6 of ATP (close contact
with residue 338, FIG. 16C) and the tert-butyl moiety roughly
corresponds to the ribose ring of ATP. Applicant further
hypothesized that in this orientation, the C-3 phenyl ring of (2)
could bind in a pocket that surrounds the N-7 of ATP as seen in the
Hck/quercetin co-crystal structure. This analysis lead Applicant to
synthesize a small panel of N-4 derivatized analogues of (2) (FIG.
2).
[0247] Identification of a Uniquely Selective Inhibitor
[0248] The panel of pyrazolo[3,4-d]pyrimidines was screened against
WT and I338G v-Src kinases (see FIG. 13). All of the analogues are
better inhibitors of the engineered v-Src as compared to wild type,
confirming Applicant's prediction of the binding orientation of (2)
in the kinase active site. Any derivatization of (2) at the N-4
position destroys the inhibitory activity against WT v-Src (no
detectable inhibition at the limit of solubility, 300 mM). All ten
analogues demonstrated measurable inhibition of I338G v-Src and
several of the compounds have IC.sub.50 in the low mM range. The
N-4(p-tert-butyl)benzoyl analogue (3g) is the most potent inhibitor
of I338G v-Src in the panel (IC.sub.50=430 nm). This molecule shows
no inhibition of WT v-Src at 300 mM suggesting that (3g) is at
least a thousand-fold better inhibitor of the mutant v-Src as
compared to wild type. The large size of the derivatization needed
to achieve sub-micromolar potency for the I338G v-Src active site
was rather unexpected. Applicant removed only four carbon atoms
from the ATP binding site and derivatized the parent molecule with
eleven carbon atoms. This discrepancy may be due to an imperfection
in Applicant's binding prediction. Also the Ile to Gly mutation may
confer greater flexibility to the enzyme active site allowing the
mutant kinase to accept a larger inhibitor analogue than predicted.
To confirm that (3g) does inhibit I338G v-src at the ATP binding
site Applicant investigated its kinetics of inhibition at various
ATP concentrations. Lineweaver-Burk analysis confirmed that (3g)
does inhibit I338G v-Src competitively with respect to ATP with an
inhibitory constant (K.sub.i) of approximately 400 nM (data not
shown).
[0249] The panel of inhibitor analogues was next screened against
WT Fyn to investigate their potential to cross react with this
kinase. WT Fyn was chosen as the "worst case" control of wild type
kinases because the published parent molecule, PP1 and (2) are
highly potent (low nM) Fyn inhibitors. Many of the ten synthetic
analogues did not display high selectivity for the target kinase
(see FIG. 13). The N-acyl analogues with saturated ring systems
(3a-3c) effectively inhibit wild type Fyn. The N-methylene
compounds (4b, 4d, 4e) are sufficiently orthogonal to WT Fyn but
show only poor to moderate inhibition of the engineered v-Src.
Importantly, (3g), the most potent inhibitor of the mutant v-Src
inhibited WT Fyn very weakly (IC.sub.50=300 mM). Thus, (3g)
inhibits the engineered v-Src over 700 times more effectively than
WT Fyn, which is likely to be the wild type cellular kinase which
is most capable of binding the molecule.
[0250] Applicant also tested whether other non-src family kinases
were fortuitously inhibited by (3g) in vitro. The serine/threonine
kinases, PKCd and PKA, were not detectably inhibited at
concentrations up to 300 mM. Likewise, (3g) exhibited only weak
inhibition (IC.sub.50>300 mM) of the Abl tyrosine kinase.
Therefore (3g) satisfied all of Applicant's initial design
requirements for potent selective inhibition of one engineered
kinase.
[0251] Selectivity in Whole Cells
[0252] To further demonstrate that (3g) does not inhibit wild type
tyrosine kinases Applicant investigated the effects of (3g)
treatment on the B cell receptor (BCR) mediated phosphorylation
cascade. Src family (Fyn, Lyn, Lck, Blk) and non-src family
tyrosine kinases (Btk, Syk) are known to be activated upon BCR
cross-linking. Due to the amplifying nature of the BCR mediated
cascade, inhibition of any of these kinases would dramatically
alter the distribution and intensity of post-activation cellular
phosphotyrosine. Because (3g) was designed to be sterically
incompatible with the active sites of wild type kinases, it should
not disrupt tyrosine phosphorylation dependent signaling in wild
type B cells. FIG. 17 (lane 3) demonstrates that 100 mM (3g)
treatment of antigen receptor cross linked murine B cells has no
effect on the phosphotyrosine pattern of B cell stimulation
(compare to lane 2). The signal intensities of all the major bands
are unchanged and only slight depletion of some minor bands is
detectable, confirming that (3g) does not appreciably inhibit the
panel of tyrosine kinases that are activated by BCR cross linking.
Treatment of B cells with 100 mM (2), however, causes a significant
reduction in tyrosine phosphorylation (FIG. 4, lane 4) that is
consistent with its potent inhibition of wild type src family
kinases.
[0253] Selective Inhibition of I338G v-Src in NIH3T3 Cells
[0254] In order to use Applicant's selective inhibitor to study a
Src mediated pathway Applicant retrovirally introduced both WT and
I338G v-Src into NIH3T3 fibroblasts. These cells acquire a
transformed phenotype which is dependent on v-Src expression.
Applicant sought to show that (3g) could selectively disturb the
Src dependent signal transduction pathway of I338G v-Src
transformed cells while not affecting WT transformed cells.
Treatment of WT v-Src infected cells (100 mM (3g)) causes no loss
of tyrosine phosphorylation compared to control DMSO treated lanes
(FIG. 18), demonstrating that the designed inhibitor does not
inhibit WT v-Src or any of the other tyrosine kinases that are
activated by v-Src mediated cellular transformation. Equivalent
treatment of I338G v-Src transformed cells gives rise to a dramatic
diminution in the tyrosine phosphorylation of the putative v-Src
substrate, p36, as well as a moderate overall decrease in the
cellular level of phosphotyrosine. Previously, it has been shown
that treatment of v-Src transformed cells with general tyrosine
kinase inhibitors causes a reduction in the tyrosine
phosphorylation of a 36 kDa protein. It is thought that p36 is
associated with a specific phosphotyrosine phosphatase, possibly
explaining its rapid dephosphorylation in inhibitor treated cells.
The (3g) IC.sub.50 for p36 phosphotyrosine signal in I338G v-Src
expressing cells (50 mM) is roughly one-hundred times the in vitro
value (data not shown). This is presumably due to the fact that the
inhibitor must compete with millimolar concentrations of ATP for
the kinase active site in the cellular experiments.
[0255] Selective Inhibition of I338G Mutant v-Src Reverses
Transformed Cell Morphology
[0256] V-Src activity is required for Rous sarcoma virus
transformation of mammalian cells. Treatment of the I338G v-Src
expressing NIH3T3 cells with 100 mM (3g) caused dramatic changes in
cell morphology which are consistent with the reversal of
transformation (FIG. 19). The mutant cells that were treated with
inhibitor (3g) appeared flat and did not exhibit growth
characteristics of transformed cells (i.e., the ability to grow on
top of one another). Under identical conditions, WT v-Src infected
cells demonstrated the prototypical rounded morphology and
overlapping growth patterns of transformed cells.
[0257] To further demonstrate the selective reversal of cell
morphology Applicant used fluorescence microscopy to view (3g)
treated cells after staining the cellular polymerized actin with
phalloidin-FITC (FIG. 19). Non-transformed NIH-3T3 cells show long
actin spindles that form across the cells. v-Src transformed cells
(both WT and I338G) appear rounded with no discernible pattern of
actin formation. In agreement with the light microscopy data,
inhibitor treated WT v-Src expressing cells appear
indistinguishable from untreated WT cells. However, (3g) treated
I338G v-Src expressing cells have defined polymerized actin
strings, strongly resembling the actin formations of
non-transformed NIH-3T3 fibroblasts. These inhibitor treated cells
have an exaggerated flattened morphology and show peripheral
actinstaining that is not present in the non-transformed NIH3T3
cells. This data shows that (3g) can uniquely induce morphological
changes in cells which are engineered to contain a single amino
acid change in the kinase of interest. This is the first
demonstration that a small molecule inhibitor selective for a
tyrosine kinase oncogene product can revert the morphological
changes associated with cellular transformation. Previous examples
of morphological reversion of transformation by herbimycin A (and
other benzoquinone ansamycins) have recently been shown to operate
via a mechanism unrelated to kinase inhibition consisting of heat
shock protein (hsp90) mediated targeting of the oncogenic tyrosine
kinase to the proteasome.
[0258] Generalization to Other Kinases
[0259] The advantage of using mutagenesis to provide a unique
molecular difference between the enzyme of interest and all others
is that, due to the conserved kinase fold, the approach should be
extendible across the kinase superfamily. Almost all known protein
kinases contain a bulky side chain at the position corresponding to
residue 338 of v-Src. Therefore a space creating mutation at this
position should render multiple kinases susceptible to selective
inhibition. To test this Applicant measured the inhibition of the
analogues against T339G Fyn (Table 1). There exists a striking
similarity in the structure activity relationships of the analogues
for I338G v-Src and T339G Fyn. In agreement with the data for I338G
v-Src, (3g) was the most potent inhibitor analogue against T339G
Fyn, exhibiting an IC.sub.50 of 830 nM. This corresponds to greater
than 300 fold selectivity for T339G Fyn over WT Fyn. The
implication of this data is that multiple tyrosine kinases can be
systematically engineered to preferentially accept one inhibitor
analogue without the need to screen large libraries of putative
inhibitors.
[0260] Conclusion
[0261] In this report Applicant describes a novel approach to
selective protein kinase inhibition through the complementary
engineering of chemical sensitive kinases and rationally designed
inhibitors. Applicant demonstrates that high selectivity for the
target kinase can be achieved in whole cells, and that active site
inhibition of an oncogenic tyrosine kinase can be sufficient for
the disruption of a transformed cell morphology. Because the
approach is easily generalized, it should have far reaching
applications in deconvoluting signal transduction pathways as well
as validation of kinases as targets for drug design. The pace of
effective drug discovery is limited by the identification and
validation of important drug targets. This is not a trivial problem
in a milieu of 2000 homologous proteins. The use of chemical
sensitive mutants of protein kinases expands the capability to
probe the cellular and physiological effects of pharmacological
kinase inhibition. Since transfected cell lines and even "knock-in"
mice can now be generated rapidly, Applicant's approach should
greatly expedite the process of testing the effects of selective
inhibition of a given kinase in a whole cell or animal model. As
more inhibitor-bound protein kinase crystal structures become
available, this strategy will allow for the systematic
investigation of the effects of time and dose dependent inhibition
of any given kinase in the scope of an entire signal transduction
cascade.
[0262] Materials and Methods
[0263] Chemical Synthesis
[0264] All starting materials and synthetic reagents were purchased
from Aldrich unless otherwise noted. All compounds were
characterized by .sup.1H NMR and high resolution mass spectrometry.
4-Amino-l-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine (2) was
synthesized according to Hanefeld et al., (1996) J. Chem. Soc.
Perkin Trans. 1: 1545-1552.
[0265] General procedure for N-4 acylation of (2) (3a-3g). To a
solution of (2) (100 mg) dissolved in 2 ml pyridine was added 10
equivalents of the desired acyl chloride at 0C. The reaction
mixture was allowed to warm to room temperature and stirred for
twelve hours. The reaction was quenched by the addition of 25 ml
water. The resulting mixture was extracted with Et.sub.2O and the
combined Et.sub.2O extracts were washed with 1 N HCl and 5%
NaHCO.sub.3. The Et.sub.2O layer was dried over MgSO.sub.4 and
evaporated. The residue was purified by flash chromatography on 25
grams silica gel by elution with 1:1 Et.sub.2O/hexanes to yield
pure (3a-3g).
[0266]
4-cyclobutylamido-1-tert-butyl-3-phenylpyrazolo-[3,4-d]pyrimidine
(3a): yield 0.0116 grams (16%), white powder; HRMS (EI) molecular
ion calcd. for C.sub.20H.sub.23N.sub.5O 349.19049, found 349.18762;
.sup.1H NMR (300 MHz, CDCl.sub.3, ppm) d 1.86 (9H, s), 1.89-2.27
(6H, m), 3.58 (1H, m), 7.26-7.67 (5H, m), 8.69 (1H, s).
[0267]
4-cyclopentylamido-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine
(3b): yield 0.0456 grams (68%), white powder; HRMS (EI) molecular
ion calcd. for C.sub.21H.sub.25N.sub.5O 363.20615, found 363.20398;
.sup.1H NMR (270 MHz, CDCl.sub.3, ppm) d 1.41-1.91 (8H, m), 1.87
(9H, s), 2.97 (1H, m), 7.51-7.67 (5H, m), 8.70 (1H, s).
[0268]
4-cyclohexylamido-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine
(3c): yield 0.0575 grams (84%), white powder; HRMS (EI) molecular
ion calcd. for C.sub.22H.sub.27N.sub.5O; .sup.1H NMR (270 MHz,
CDCl.sub.3, ppm) d 1.21-1.93 (1OH, m), 1.86 (9H, s), 2.43 (1H, m),
7.51-7.67 (5H, m), 8.70 (1H, s).
[0269]
4-2'-furylamido-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine
(3d): yield 0.0342 grams (60%), white powder; HRMS (EI) molecular
ion calcd. for C.sub.20H.sub.19N.sub.5O.sub.2 361.15407, found
361.15254; .sup.1H NMR (270 MHz, CDCl.sub.3, ppm) d 1.87 (911, s),
6.52 (1H, d), 7.23 (1H, d), 7.43-7.53 (5H, m), 7.95 (1H, s), 8.59
(1H, s).
[0270] 4-benzamido-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine
(3e): yield 0.1309 grams (56%), white powder; HRMS (EI) molecular
ion calcd. for C.sub.22H.sub.21N.sub.5O 371.17933, found 371.17324;
.sup.1H NMR (270 MHz, CDCl.sub.3, ppm) d 1.41-1.91 (8H, m),
7.22-8.11 (1OH, m), 8.48 (1H, s).
[0271] 4-(p-methyl)benzamido-1-tert-butyl-3-phenylpyrazolo
[3,4-d]pyrimidine (3f): yield 0.0751 grams (33%), white powder;
HRMS (EI) molecular ion calcd. for C.sub.23H.sub.23N.sub.5O
385.19499, found 385.18751; .sup.1H NMR (270 MHz, CDCl.sub.3, ppm)
d 1.88 (911, s), 2.42 (3H, s), 7.19 (2H, d), 7.41-8. 11(7H, m),
8.49 (1H, s).
[0272]
4-(p-tert-butyl)benzamido-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrim-
idine (3g): yield 0.1050 grams (42%), white powder; HRMS (EI)
molecular ion calcd. for C.sub.26H.sub.29N.sub.5O 427.23747, found
427.23474; .sup.1H NMR (270 MHz, CDCl.sub.3, ppm) d 1.35 (9H, s),
1.88 (9H, s), 7.38-7.99 (9H, m), 8.50 (1H, s).
[0273] General Procedure for the Reduction of N-4 Acyl Compounds to
N-4 Methylene Compounds (4b, 4d, 4e).
[0274] A round bottom flask was charged with 30 mg LiAlH.sub.4. The
flask was equipped with a pressure equalizing dropping funnel and
flushed with dry argon. The LiAlH.sub.4 was suspended in 3 ml THF
over an ice bath. Approximately 100 mg of the corresponding N-4
acyl (2) analogue was dissolved in 5 ml THF and added dropwise to
the suspension of LiAlH.sub.4. The reaction mixture was stirred for
thirty minutes on the ice bath and subsequently heated to reflux
for thirty minutes. The reaction was quenched by the sequential,
dropwise additions of 1 ml EtOAc, 1 ml water, and 1 ml 6 N NaOH.
After stirring for five minutes, the reaction mixture was filtered
through a celite pad, diluted with water and extracted with
Et.sub.2O. The Et.sub.2O extracts were combined, dried over
MgSO.sub.4, and evaporated. The residue was purified by flash
chromatography on 10 g silica gel by elution with 4:1
hexanes/EtOAc.
[0275] 4-cyclopentylmethylamino-1-tert-butyl-3-phenylpyrazolo
[3,4-d]pyrimidine (4b): yield 0.0649 grams (75%), clear oil; HRMS
(EI) molecular ion calcd. for C.sub.21H.sub.27N.sub.5 349.22691,
found 349.22420; .sup.1H NMR (270 MHz, CDCl.sub.3, ppm) d 1.16-2.14
(9H, m), 1.84 (9H, s), 3.54 (2H, d), 5.51(1H, s), 7.46-7.67 (5H,
m), 8.43 (1H, s).
[0276]
4-2'-furylmethylamino-1-tert-butyl-3-phenylpyrazolol[3,4-d]pyrimidi-
ne (4d): yield 0.0620 grams (66%), beige powder; HRMS (EI)
molecular ion calcd. for C.sub.20H.sub.21N.sub.5O 347.17483, found
347.17330; .sup.-H NMR (270 MHz, CDCl.sub.3, ppm) d 1.83 (9H, s),
4.75 (2H, d), 5.64 (1H, s), 6.25 (2H, d), 7.34-7.63 (6H, m), 8.45
(1H, s).
[0277] 4-benzylamino-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine
(4e): yield 0.0520 grams (54%), white powder; HRMS (EI) molecular
ion calcd. for C.sub.22H.sub.23N.sub.5 357.19559, found 357.19303;
.sup.1H NMR (270 MHz, CDCl.sub.3, ppm) d 1.82 (9H, s), 4.76 (2H,
d), 5.63 (1H, s), 7.28-7.63 (10H, m), 8.44 (1H, s).
[0278] Protein Expression and Purification
[0279] Site directed mutagenesis and cloning of the genes for the
glutathione-S-transferase fusion proteins of WT v-Src SHI domain,
I338G v-Src SHI, WT Fyn, T339G Fyn, and WT Abl into the pGEX-KT
plasmid was carried out as described previously. These kinases were
expressed in DH5.alpha. E. coli and purified on immobilized
glutathione beads (Sigma). PKA was purchased (Pierce) and used
without further purification. PKCd was expressed as the 6-His
construct using the Bac-to-Bac (expression system (pFastBac B
vector). PKCd was purified using a QIAexpress Ni-NTA agarose
column.
[0280] In vitro Kinase Inhibition Assay
[0281] IC.sub.50 for putative kinase inhibitors were determined by
measuring the counts per minute (cpm) of [.sup.32P] transferred to
an optimized peptide substrate for src family kinases (IYGEFKKK).
Various concentrations of inhibitor were incubated with 50 mM Tris
(pH 8.0), 10 mM MgCl.sub.2, 1.6 mM glutathione, 1 mg/ml BSA, 133 mM
IYGEFKKK, 3.3% DMSO, 0.05 mM kinase and 11 nM (2 mCi)
[.gamma.-.sup.32P]ATP (6000 Ci/mmol, NEN) in a total volume of 30
ml for thirty minutes. Reaction mixtures (25 ml) were spotted onto
a phosphocellulose disk, immersed in 10% HOAc, and washed with 0.5%
H.sub.3PO.sub.4. The transfer of [.sup.32P] was measured by
standard scintillation counting. IC.sub.50 was defined to be the
concentration of inhibitor at which the cpm was 50% of the control
disk. When the IC.sub.50 fell between two measured concentrations
it was calculated based on the assumption of an inversely
proportional relationship between inhibitor concentration and cpm
between the two data points. Because the solubility limit of the
inhibitor analogues in aqueous solutions is (300 .mu.M, IC.sub.50
values of 250 .mu.M are approximate as full titrations to the upper
limit of inhibition could not be tested). 1 C.sub.50 for non-src
family kinases were measured equivalently with the following
exceptions. Kemtide (Pierce, 133 mg/ml) was used as the substrate
for PKA. An optimized Abl substrate (EAIYAAPFAKKK, 133 mg/ml) was
used for Abl assays. PKCd assays were performed in the presence of
17 ng/ml diacyl glycerol Sigma) and 17 ng/ml phosphatidyl serine
(Sigma) with 170 ng/ml histone (Sigma) as the kinase substrate.
[0282] Murine B Cell Assay
[0283] Splenic lymphocytes were isolated from 6-20 week old Balb/c
or C57/B6 mice. The cells were washed out of the spleen into RPMI
media containing 1 mg/ml DNase I and the red blood cells were lysed
in 17 mM tris-ammonium chloride (pH 7.2). Approximately
4.times.10.sup.6 cells were incubated at 37.degree. C. for thirty
minutes with 100 mM of (3g) or (2) in 1.1% DMSO. B cell stimulation
was initiated by the addition of 2 mg of goat anti-mouse IgM
(Jackson ImmunoResearch) and subsequent incubation for five minutes
at 37.degree. C. The cells were isolated by centrifugation (13,000
rpm, two minutes) and lysed (lysis buffer: 1% Triton X-100, 50 mM
Tris (pH 7.4), 2 mM EDTA, 150 mM NaCl, 100 mM PMSF, 2 mM sodium
orthovanadate, 10 mg/ml leupeptin, 10 mg/ml apoprotin). The
cellular debris was then pelleted at 13,000 rpm for fifteen
minutes. Cellular proteins were separated by 10% polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane by
Western blotting. Phosphotyrosine containing proteins were
visualized by immunoblotting with anti-phosphotyrosine antibody
(Upstate Biotechnology).
[0284] Retroviral Infection of NIH3T3 Fibroblasts
[0285] Genes encoding WT and I338G v-Src were transfected into a
packaging cell line and NIH3T3 fibroblasts were retrovirally
infected using the pBabe retroviral vector and a puromycin (2.5
mg/ml) selectable marker as described (Shah et al., (1992) Proc.
Natl. Acad. Sci. USA 94:3565-3570. WT and I338G v-Src transformed
cells were cultured in DMEM/10% BCS containing 2.5 mg/ml
puromycin.
[0286] Inhibition of v-Src in NIH3T3 Fibroblasts
[0287] Non-transformed NIH3T3 cells, WT v-Src transformed NIH3T3
cells, and I338G v-Src transformed NIH3T3 cells were incubated at
37.degree. C. with 1.1% DMSO or 100 mM (3g) in 1.1% DMSO. After
twelve hours, the cells were washed with PBS and lysed (lysis
buffer: 1% Triton X-100, 50 mM tris (pH 7.4), 2 mM EDTA, 150 mM
NaCl, 100 mM phenylmethylsulphonyl fluoride, 2 mM sodium
orthovanadate, 10 mg/ml leupeptin, 10 mg/ml apoprotin). The lysate
was clarified by centrifugation at 13,000 rpm for fifteen minutes.
Lysate protein concentrations were normalized and equal volumes of
the lysate were resolved electrophoretically and analyzed for
phosphotyrosine content as described above.
[0288] Microscopy
[0289] Non-transformed, WT v-Src transformed, and I338G v-Src
transformed NIH-3T3 fibroblasts were grown in DMEM/10% BCS on
tissue culture treated slides. v-Src expressing cells were treated
with either 1.1% DMSO or 100 mM (3g) in 1.1% DMSO. After
forty-eight hours cells were photographed at 400.times.
magnification on an Nikon TMS light microscope. Immediately
following light microscopy, the cells were fixed for twenty minutes
in 3.7% formaldehyde/PBS and permeabilized for sixty seconds in
0.2% Triton X-100 in PBS. Permeabilized cells were incubated with
200 ng/ml phalloidin-FITC/PBS for twenty minutes. Slides were
rinsed with PBS and polymerized actin was visualized by
fluorescence microscopy at 600.times. magnification on a Zeiss
fluorescence microscope.
Example 17
[0290] Confirming Retention of Protein Substrate Specificity and
Biological Activity
[0291] This could be carried out as described in (Shah et al.,
(1997) Proc. Natl. Acad. Sci. USA 94:3565-3570). Further, the
stereo typed role of v-Src in the oncogenic transformation of
NIH-3T3 cells can be determined by observing the morphological
change in cells expressing v-Src. The NIH-3T3 cells expressing
mutant I338G v-Src display the identical morphological features of
cells expressing wild-type v-Src which are dramatically distinct
from NIH-3T3 cells which do not express either v-Src kinase,
confirming that the I338G mutation does not lead to any loss or
gain of biological function of normal v-Src. Further, an assay for
the ability of NIH-3T3 cells to grow without "contact inhibition"
can be measured in a cell culture based assay containing agarose, a
viscous growth medium. The wild-type v-Src and mutant v-Src
expressing NIH3T3 cells display the exact same ability to form
large growth colonies in this stereotyped assay as well, further
confirming their identical functions (including substrate
specificity, kinetics, cell distribution, etc.) in fibroblasts.
Example 18
[0292] Confirmation that the Orthogonal Inhibitor does not Inhibit
Wild-type Kinases in Cells which express Multiple Tyrosine
Kinases
[0293] To confirm Applicant's initial assays regarding the
orthogonal nature of compound (3) in purified kinases described in
Example 2 Applicant conducted inhibition experiments using whole
cells (see FIG. 4, two left lanes). Anti-phosphotyrosine blots of
pyrazolo pyrimidine (2-6) (25 .mu.LM) treated NIH3T3 cells
expressing v-Src kinase were performed by lysing cells in modified
RIPA buffer according to the method of Coussens et al., (1985)
Biol. 2753-2763. Cells were also treated for various times before
lysis and antiphosphotyrosine detection. Proteins were separated by
12.5% SDS-PAGE and transferred to Protran BA85
(Schleicher-Schuell). The blot was probed with the
antiphosphotyrosine monoclonal antibody 4G10 (obtained from Dr.
Brian Druker, Oregon Health Sciences Center) and the bound antibody
was detected via enhanced chemiluminescence (Pierce) following
treatment with HRP-coupled goatanti-mouse antibody (VWR) according
to the manufacturer's instructions.
Example 19
[0294] Identifying the Substrates
[0295] A schematic representation of an experimental approach to
identifying v-Src substrates is outlined in FIG. 1 and the data
showing experimental validation is in FIG. 4. The assays were
performed by making anti-phosphotyrosine blots of
pyrazolopyrimidine (2-6) (25 .mu.M) treated NIH3T3 cells expressing
either v-Src or v-Src (I338G) kinases were performed by lysing
cells in modified RIPA buffer according to the method of Coussens
et al., (1985) Biol. 2753-2763. Cells were also treated for various
times (in a cell culture CO.sub.2 incubator) before lysis and
anti-phosphotyrosine detection. Proteins were separated by 12.5%
SDS-PAGE and transferred to Protran BA85 (Schleicher-Schuell).
[0296] The blot was probed with the anti-phosphotyrosine monoclonal
antibody 4G10 and the bound antibody was detected via enhanced
chemiluminescence (Pierce) following treatment with HRP-coupled
goat-anti-mouse antibody (VWR) according to the manufacturer's
instructions. As discussed in Example 7, the two left lanes in FIG.
4 show the same phosphoprotein band pattern indicating that the
orthogonal inhibitor (3) does not inhibit wild type v-Src kinase.
The series of lanes in the right gel show a prominent band in the
bottom of the gel (corresponding to protein molecular weight 3 kDa)
which is lost after treatment with 100 .mu.M of compound (3). This
specific inhibition of one phosphoprotein is a hallmark of a
specific kinase inhibitor. The specificity of the inhibition is
confirmed in the last lanes of the gel where the inhibitor is
diluted and the phosphorylation of the 36 kDa band reappears when
the inhibitor concentration is lower that 5 pM (the measured
IC.sub.50 in vitro is 5 pM, see text). This protein has been
tentatively identified based on its unique molecular weight, as a
protein called annexin II, an actin binding protein, of unknown
function.
[0297] This invention may be embodied in other forms or carried out
in other ways without departing from the spirit or essential
characteristics thereof. The present disclosure is therefore to be
considered as in all respects illustrative and not restrictive, the
scope of the invention being indicated by the appended claims, and
all changes which come within the meaning and range of equivalency
are intended to be embraced therein.
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