U.S. patent application number 11/129586 was filed with the patent office on 2005-09-29 for screen for cdc7 inhibitors.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Amidon, Benjamin Stone, Bulawa, Christine Ellen.
Application Number | 20050214742 11/129586 |
Document ID | / |
Family ID | 23217598 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050214742 |
Kind Code |
A1 |
Amidon, Benjamin Stone ; et
al. |
September 29, 2005 |
Screen for CDC7 inhibitors
Abstract
Disclosed is a yeast cell whose genetic complement includes an
inactive allele of the yeast CDC7 gene, a first nucleic acid that
encodes a mammalian Cdc7 protein, and a second nucleic acid that
encodes a mammalian Dbf4 protein. The yeast cell is dependent on
the mammalian Cdc7 and Dbf4 proteins for viability. The yeast cell
can be used to identify potential anti-proliferative agents by
virtue of their inhibition of the mammalian Cdc7 and Dbf4 proteins.
In some embodiments, a control yeast cell which does not depend on
Cdc7 for viability is used in a secondary screen.
Inventors: |
Amidon, Benjamin Stone;
(Arlington, MA) ; Bulawa, Christine Ellen;
(Arlington, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23217598 |
Appl. No.: |
11/129586 |
Filed: |
May 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11129586 |
May 13, 2005 |
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10225323 |
Aug 21, 2002 |
|
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60313889 |
Aug 21, 2001 |
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Current U.S.
Class: |
435/4 ;
435/254.2 |
Current CPC
Class: |
C07K 14/4738
20130101 |
Class at
Publication: |
435/004 ;
435/254.2 |
International
Class: |
C12Q 001/00; C12N
001/18 |
Claims
What is claimed:
1. A yeast cell, the genetic complement of which comprises: a) an
inactive allele of the yeast CDC7 gene; b) a first nucleic acid
molecule that encodes a mammalian Cdc7 polypeptide; and c) a second
nucleic acid molecule that encodes a mammalian Dbf4
polypeptide.
2. The yeast cell of claim 1, wherein the mammalian Cdc7
polypeptide comprises SEQ ID NO: 19 with at least one conservative
amino acid substitution.
3. The yeast cell of claim 1, wherein the first nucleic acid
molecule comprises SEQ ID NO: 18.
4. The yeast cell of claim 1, wherein the mammalian Dbf4
polypeptide comprises SEQ ID NO:21 with at least one conservative
amino acid substitution.
5. The yeast cell of claim 1, wherein the second nucleic acid
molecule comprises SEQ ID NO:20.
6. A method for determining whether a test compound is an inhibitor
of mammalian Cdc7.4 activity, the method comprising: (i) obtaining
a yeast cell of claim 1; (ii) contacting the yeast cell with a test
compound; and (iii) assessing growth or cell cycle progression of
the yeast cell, wherein a decrease in growth or cell cycle
progression indicates that the test compound is an inhibitor of
mammalian Cdc7.4 activity.
7. The method of claim 6, wherein the Cdc7 polypeptide is human
Cdc7 polypeptide.
8. The method of claim 6, wherein the Ddf4 polypeptide is human
Ddf4 polypeptide.
9. The method of claim 6, wherein assessing comprises culturing the
yeast cell in a container.
10. The method of claim 9, wherein assessing comprises culturing
the yeast cell on a solid medium within the container and
monitoring the size of a colony formed by the yeast cell.
11. The method of claim 9, wherein assessing comprises culturing
the yeast cell in a liquid medium within the container and
determining the number of yeast cells in the liquid medium.
12. The method of claim 9, wherein assessing comprises culturing
the yeast cell in a liquid medium within the container and
measuring turbidity of the liquid medium.
13. The method of claim 6, wherein assessing comprises measuring
endogenous ATP levels of the yeast cell.
14. The method of claim 6, wherein assessing comprises
fluorescent-activated cell sorting based on DNA content.
15. The method of claim 6, further comprising assaying in vitro
mammalian Cdc7.4 kinase activity.
16. The method of claim 15, wherein assaying comprises determining
mammalian Cdc7.4 kinase activity for a minichromosome maintenance
protein substrate.
17. A method for determining whether a test compound is an
inhibitor of mammalian Cdc7.4 activity, the method comprising: (i)
contacting a test compound to a first yeast cell, the genetic
complement of which comprises: (a) an inactive allele of the yeast
CDC7 gene; (b) a first nucleic acid molecule that encodes a
mammalian Cdc7 polypeptide; and (c) a second nucleic acid molecule
that encodes a mammalian Dbf4 polypeptide; (ii) contacting the test
compound to a second yeast cell, the viability of which is
independent of a mammalian Cdc7 or Dbf4; and (iii) assessing growth
or cell cycle progression of the first and second yeast cells,
wherein a decrease in growth or cell cycle progression in the first
yeast cell relative to the second yeast strain indicates that the
test compound is an inhibitor of mammalian Cdc7.4 activity.
18. The method of claim 17, wherein the second yeast cell comprises
a mutant allele of CDC46.
19. The method of claim 17, wherein the Cdc7 polypeptide is human
Cdc7 polypeptide.
20. The method of claim 17, wherein the Dbf4 polypeptide is human
Dbf4 polypeptide.
21. The method of claim 6, further comprising: (iii) repeating (i)
and (ii) for multiple test compounds; (iv) identifying candidate
compounds that are inhibitors of mammalian Cdc7.4 activity; (v)
identifying and selecting a lead compound from the candidate
compounds, the lead compound being an inhibitor of Cdc7.4 activity;
and (vi) formulating the selected lead compound as an
anti-proliferative agent.
22. The method of claim 6, further comprising: (iii) repeating (i)
and (ii) for multiple test compounds; (iv) identifying candidate
compounds that are inhibitors of mammalian Cdc7.4 activity; (v)
isolating one or more lead compounds from the candidate compounds;
(vi) derivatizing the one or more lead compounds, thereby producing
derivatives of the lead compounds; (vii) identifying one or more
derivatives that are indicated as inhibitors of mammalian Cdc7.4
activity; and (viii) formulating one or more derivatives as an
anti-proliferative agent.
23. A composition comprising an anti-proliferative agent identified
by the method of claim 6 and a pharmaceutically acceptable
carrier.
24. A method of treating a subject having a proliferative disorder,
the method comprising administering to the subject the composition
of claim 23 in an amount effective to reduce cell proliferation
associated with the proliferative disorder.
25. A kit comprising: (1) a first yeast cell, the genetic
complement of which comprises a) an inactive allele of the yeast
CDC7 gene, b) a first nucleic acid that encodes a mammalian Cdc7
polypeptide, and c) a second nucleic acid that encodes a mammalian
Dbf4 polypeptide; and (2) a second yeast cell, the genetic
complement of which comprises a) an inactive allele of the yeast
CDC7 gene, and b) a mutant allele that bypasses the requirement for
Cdc7.4 activity.
26. The kit of claim 25, wherein the second yeast cell comprises a
mutant allele of CDC46.
27. The kit of claim 25, wherein the Cdc7 polypeptide is human Cdc7
polypeptide.
28. The kit of claim 25, wherein the Dbf4 polypeptide is human Dbf4
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application Ser. No. 60/313,889, filed on Aug. 21, 2001,
which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] This invention relates to methods for identifying
anti-proliferative agents for mammalian cells.
BACKGROUND
[0003] Neoplastic disorders such as cancer are characterized by
unregulated cell proliferation. Cancer cells progress through a
cycle of cellular events that include DNA replication and mitosis.
A common strategy for treating such a disorder is to administer
agents that inhibit either of these two events. Methotrexate, for
example, is an exemplary chemotherapeutic agent for treating
cancer. Methotrexate blocks DNA replication by inhibiting
dihydrofolate reductases, enzymes that are required for the
production of the nucleotide substrates for replication.
[0004] Eukaryotic DNA replication is a highly conserved process.
Eukaryotes from fungi to mammals utilize highly related proteins to
duplicate their genetic material. DNA replication can be separated
into two phases: initiation and DNA synthesis. During initiation,
the origin recognition complex (ORC) binds to specialized DNA
sequences termed "origins." ORC can recruit a hexameric complex of
MCM (minichromosome maintenance) proteins that may function as a
DNA helicase, which can unwind origins. The MCM complex in turn
recruits Cdc45 (cell division cycle-45) protein and other
downstream components. As a result, DNA polymerase machinery is
loaded onto the origin and DNA replication can be initiated.
[0005] In normal cells, these initiation events are precisely
regulated. One of the regulators is a complex formed of two
proteins, Cdc7 and Dbf4. Cdc7 is a serine-threonine kinase that
requires the additional subunit Dbf4 for activity. Both Cdc7 and
Dbf4 are essential for survival in Saccharomyces cerevisiae. The
requirement for either or both of these proteins is alleviated by
bob1-1, a mutant allele of the gene CDC46/MCM5.
[0006] Dbf4 interacts with origin DNA (Dowell et al., Science,
265:1243-1246, 1999). The levels of Dbf4 protein fluctuate during
the cell cycle to provide temporal regulation of Cdc7 kinase
activity.
[0007] Cdc7 interacts with Orc2 in addition to Dbf4 and its kinase
substrates (Hardy et al., Mol. Cell. Biol., 16:1832-1841, 1996).
Human Cdc7.4 complex phosphorylates Mcm2 (Jiang et al., EMBO J.,
18(20):5703-13, Oct. 15, 1999) to provide a modification which may
be required for MCM complex recruitment of Cdc45. Other substrates
of Cdc7.4 may include other MCM proteins, and the p180 primase
subunit.
SUMMARY
[0008] In both yeast and mammals, Cdc7 activity is required for
cell proliferation. The present invention is based, at least in
part, on the discovery that a yeast strain that is dependent upon
mammalian Cdc7 activity for viability (rather than an endogenous
Cdc7 activity) can be used to identify and/or determine whether a
test compound is useful as an anti-proliferative agent to treat
proliferative disorders in mammals. The yeast strain is modified by
impairing or deleting an endogenous component of Cdc7 activity, and
by including a heterologous component of mammalian Cdc7. If the
test compound inhibits mammalian Cdc7, then viability and/or growth
of the yeast strain is impaired. Such inhibition is an indication
that the test compound is useful for treating proliferative
disorders, e.g., cancer.
[0009] One exemplary screening method seeks to identify compounds
from a library of compounds that reduce the growth of yeast
dependent on a heterologous mammalian Cdc7, but does not reduce the
growth of yeast, whose viability is dependent on the endogenous
Cdc7 or is independent of Cdc7. The new methods are adaptable for
high-throughput screening.
[0010] Accordingly, the invention features a method for determining
whether a test compound is an inhibitor of mammalian Cdc7.4
activity. The method includes: (i) obtaining a yeast cell described
herein; (ii) contacting the yeast cell with a test compound; and
(iii) assessing growth or cell cycle progression of the yeast cell,
wherein a decrease in growth or cell cycle progression indicates
that the test compound is an inhibitor of mammalian Cdc7.Dbf4
activity. The genetic complement of the yeast cell includes: a) an
inactive allele of the yeast CDC7 gene or the yeast DBF4 gene; b) a
first nucleic acid molecule that encodes a mammalian Cdc7
polypeptide; and c) a second nucleic acid molecule that encodes a
mammalian Dbf4 polypeptide.
[0011] The mammalian Cdc7 polypeptide can be a human Cdc7, and can
have the amino acid sequence of SEQ ID NO: 19, optionally with at
least 1, e.g., 2, 3, 4, 6, 8, 10 or 20, or greater than 20,
conservative amino acid substitutions. Cdc7 polypeptides having
conservative amino acid substitutions retain one or more of the
activities of Cdc7polypeptides, e.g., serine threonine kinase
activity, the ability to associate with Dbf4, the ability to
interact with Orc2, and/or the ability (when complexed with Dbf4)
to phosphorylate Mcm2. The mammalian Dbf4 polypeptide can be a
human Dbf4, and can have the amino acid sequence of SEQ ID NO:21,
optionally with at least 1, e.g., 2, 3, 4, 6, 8, 10, 20, or greater
than 20, conservative amino acid substitutions. Dbf4 polypeptides
having conservative amino acid substitutions retain one or more of
the activities of Dbf4 polypeptides, e.g., to associate with Cdc7
and/or origin DNA, and the ability (when complexed with Cdc7) to
phosphorylate Mcm2.
[0012] The first nucleic acid molecule can comprise SEQ ID NO: 18
or degenerate variants thereof. The second nucleic acid molecule
can comprise SEQ ID NO:20, or degenerate variants thereof.
Degenerate variants of a nucleic acid sequence exist because of the
degeneracy of the amino acid code; thus, those sequences that vary
from the sequence represented by SEQ ID NO: 18 or SEQ ID NO:20, but
which nonetheless encode a Cdc7 or Dbf4 polypeptide, respectively,
are included within the invention.
[0013] The assessing can include culturing the yeast cell in a
container, e.g, a test tube or petri plate. The assessing can
include monitoring the size of a colony formed by the yeast cell;
determining, e.g., counting the exact number or estimating the
approximate number, of yeast cells in a container, measuring the
turbidity of the liquid in the container; measuring endogenous ATP
levels; or fluorescent-activated cell sorting based on DNA content.
The method can also further include assaying in vitro mammalian
Cdc7.4 kinase activity, e.g., determining whether an MCM protein
substrate is phosphorylated.
[0014] The invention also features a second method for determining
whether a test compound is an inhibitor of a mammalian Cdc7.4
activity. The method includes (i) contacting a test compound to a
first yeast cell, the genetic complement of which includes: (a) an
inactive allele of the yeast CDC7 gene; (b) a first nucleic acid
molecule that encodes a mammalian Cdc7 polypeptide; and (c) a
second nucleic acid molecule that encodes a mammalian Dbf4
polypeptide; (ii) contacting the test compound to a second yeast
cell, the viability of which is independent of a mammalian Cdc7 or
Dbf4; and (iii) assessing growth or cell cycle progression of the
first and second yeast cells, wherein a decrease in growth or cell
cycle progression in the first yeast cell relative to the second
yeast cell indicates that the test compound is an inhibitor of
mammalian Cdc7.4 activity. The second yeast cell can include a
mutant allele of CDC46, e.g., the bob1-1 allele. The first and
second yeast cells can each include distinct fluorescent markers,
e.g., markers that are spectrally distinct, e.g., green fluorescent
protein and yellow fluorescent protein. Further, the first and
second yeast cells can be related, e.g., isogenic. The Cdc7
polypeptide can be a human Cdc7 polypeptide. Likewise, the Dbf4
polypeptide can be a human Dbf4 polypeptide.
[0015] The method can further include: (iii) repeating (i) and (ii)
for multiple test compounds; (iv) identifying candidate compounds
that are indicated as inhibitors of mammalian Cdc7.4 activity; (v)
identifying, and selecting a lead compound from the candidate
compounds, the lead compound being an inhibitor of Cdc7.4 activity;
and (vi) formulating the selected lead compound as an
anti-proliferative agent.
[0016] In another implementation, the method can further include
(iii) repeating (i) and (ii) for multiple test compounds; (iv)
identifying candidate compounds that are inhibitors of mammalian
Cdc7.4 activity; (v) isolating one or more lead compounds from the
candidate compounds; (vi) derivatizing the one or more lead
compounds, thereby producing derivatives of the lead compounds;
(vii) identifying one or more derivatives that are indicated as
inhibitors of mammalian Cdc7.4 activity; and (viii) formulating one
or more derivatives as an anti-proliferative agent.
[0017] In another aspect, the invention features a composition that
includes an anti-proliferative agent identified by a method
described herein and a pharmaceutically acceptable carrier.
Further, a subject having a proliferative disorder can be treated
using a method that includes administering the composition to the
subject in an amount effective to reduce cell proliferation
associated with the proliferative disorder.
[0018] In another aspect, the invention features a yeast cell, the
genetic complement of which includes a) an inactive allele of the
yeast CDC7 gene or the yeast DBF4 gene; b) a first nucleic acid
molecule that encodes a mammalian Cdc7 polypeptide; and c) a second
nucleic acid molecule that encodes a mammalian Dbf4 polypeptide.
The yeast cell can be a haploid or diploid. The yeast can be, for
example, Saccharomyces cerevisiae or Schizosaccharomyces pombe.
[0019] The mammalian Cdc7 or Dbf4 can be; e.g., human, primate,
mouse,.rat, horse, dog, or cow Cdc7 or Dbf4. The mammalian Cdc7 or
Dbf4 can be at least 80, e.g., 85, 90, 95, or 100% identical to
human Cdc7 or Dbf4. In one embodiment, the mammalian Cdc7 is human
Cdc7, and the mammalian Dbf4 is human Dbf4. The first and second
nucleic acid molecules, which encode mammalian Cdc and Dbf4
polypeptides, respectively, can be at least 80, e.g., 85, 90, 95,
or 100% identical to nucleic acid sequences that encode human Cdc7
and Dbf4 polypeptides, respectively.
[0020] An example of human Cdc7 is SEQ ID NO: 19, as shown in FIG.
1 B. An example of a nucleic acid sequence that encodes human Cdc7
is SEQ ID NO: 18, as shown in FIG. 1A. Human Cdc7 sequences are
recited in GenBank Accession #AF015592. An example of human Dbf4 is
SEQ ID NO:21, as shown in FIG. 2B. An example of a nucleic acid
that encodes human Dbf4 is SEQ ID NO:20, as shown in FIG. 2A. Human
Dbf4 sequences are recited in GenBank Accession # AF160876.
[0021] In one embodiment, the first nucleic acid includes a nucleic
acid encoding the mammalian Cdc7 protein and a heterologous
promoter operably linked to the coding nucleic acid. The nucleic
acid can be integrated into a yeast chromosome, or on a plasmid.
The promoter can be constitutive or inducible. For example, the
promoter can be controllable by an exogenous agent, e.g., a steroid
hormone or an antibiotic, e.g., tetracycline.
[0022] The yeast cell can further include a nucleic acid that
encodes a marker protein, e.g., a fluorescent protein. The yeast
cell can further include a mutation in a drug transporter gene,
e.g., SNQ2 or PDR5. In some embodiments, the yeast cell further
includes a genetic alteration that allows viability of the yeast
cell independent of impaired endogenous Cdc7 activity and
heterologous Cdc7 activity. The genetic alteration can be a
genetically altered allele of CDC46. For example, it can be the
P83L allele.
[0023] In another aspect, the invention features a nucleic acid
that includes: a) a sequence encoding a mammalian Cdc7 or Dbf4
polypeptide; b) a promoter functional in a yeast cell and operably
linked to the sequence; and c) a marker sequence that is selectable
in a yeast cell. The promoter can include one or more TetR binding
sites. The marker sequence can be an auxotrophic marker, e.g.,
URA3, HIS3, TRP1, LEU2, ADE2, or ADE3. The marker sequence can also
confer resistance to an exogenous agent e.g., kanamycin.
[0024] The invention also features a kit that includes: (1) a first
yeast cell, the genetic complement of which includes a) an inactive
allele of the yeast CDC7 gene or the yeast DBF4 gene, b) a first
nucleic acid that encodes a mammalian Cdc7 polypeptide, and c) a
second nucleic acid that encodes a mammalian Dbf4 polypeptide; and
(2) a second yeast cell, the genetic complement of which includes
a) an inactive allele of the yeast CDC7 gene or the yeast DBF4
gene, and b) a mutant allele, which bypasses the requirement for
Cdc7.4 activity. The mammalian Cdc7 or Dbf4 polypeptides can be
human Cdc7 or Dbf4 polypeptides, respectively.
[0025] As used herein, "Cdc7.4" refers to the complex of Cdc7
kinase and its associated accessory factor Dbf4. An activity of
Cdc7.4 can include kinase activity directed towards its specific
substrates, or any other interaction in which the complex
participates to regulate DNA replication.
[0026] A "yeast strain," as used herein, refers to a population of
fungal cells that each have substantially the same genome content.
Thus, the definition encompasses a population of cells that has a
subpopulation that includes a plasmid, which may be lost at some
frequency--provided that the plasmid is not specifically required
as detailed herein. The definition also encompasses a population of
yeast cells having minor variations such as loss or movement of a
transposon; incidental random mutation; and epigenetic variation.
The term "yeast cell" can encompass a population originating from
an original cell when appropriate, e.g., when a method requires
looking at more than one cell such as when measuring turbidity.
[0027] A "lead compound" is a test compound that impairs the growth
or viability of a yeast strain that depends on a mammalian Cdc7
activity. If desired, lead compounds can subsequently be
derivatized using conventional medicinal chemistry methods, as
described herein.
[0028] As used herein, the term "subject" is used throughout the
specification to describe an animal, human or non-human, to whom
treatment according to the methods of the present invention is
provided. Veterinary applications are included within the present
invention. The term includes but is not limited to mammals, e.g.,
humans, other primates, pigs, rodents such as mice and rats,
rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and
goats. The term "treatment" is used herein to describe delaying the
onset of, inhibiting, or alleviating the effect of a condition,
e.g., a proliferative disorder, e.g., cancer.
[0029] As used herein, the term "operably linked" means that a
nucleic acid sequence is connected to a promoter sequence in a
manner that allows for transcriptional expression of the nucleotide
sequence in vivo.
[0030] A "conservative amino acid substitution" is one in which an
amino acid residue is replaced with another amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0031] An "isolated nucleic acid" is a DNA or RNA that is not
immediately contiguous with both of the coding sequences with which
it is immediately contiguous (one on the 5' end and one on the 3'
end) in the naturally occurring genome of the organism from which
it is derived. Thus, in one embodiment, an isolated nucleic acid
includes some or all of the 5' non-coding (e.g., promoter)
sequences that are immediately contiguous to the coding sequence.
The term therefore includes, for example, a recombinant DNA that is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a-cDNA or
a genomic DNA fragment produced by PCR or restriction endonuclease
treatment) independent of other sequences. It also includes a
recombinant DNA that is part of a hybrid gene encoding an
additional polypeptide sequence. The term "isolated" can refer to a
nucleic acid or polypeptide that is substantially free of cellular
material, viral material, or culture medium (when produced by
recombinant DNA techniques), or chemical precursors or other
chemicals (when chemically synthesized). Moreover, an "isolated
nucleic acid fragment" is a nnucleic acid fragment that is not
naturally occurring as a fragment and would not be found in the
natural state.
[0032] As used herein, the terms "cancer,"0 "hyperproliferative,"
and "neoplastic" are used to describe cells having an abnormal
state or condition characterized by rapidly proliferating cell
growth. The terms include all types of cancerous growths and
oncogenic processes, metastatic tissues and malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. "Pathologic hyperproliferative" cells occur
in disease states characterized by malignant tumor growth. Examples
of non-pathologic hyperproliferative cells include proliferation of
cells associated with wound repair.
[0033] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). The length
of a reference sequence aligned for comparison purposes is at least
50% of the length of the reference sequence. The amino acid
residues or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid. residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein,
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology").
[0034] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0035] The comparison of sequences and determination of percent
identity between two sequences is accomplished using a mathematical
algorithm. The percent identity between two amino acid sequences is
determined using the Needleman and Wunsch (J. Mol. Biol.,
48:444-453 (1970)) algorithm, which has been incorporated into the
GAP program in the GCG software package (available on the internet
at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,
and a gap weight of 4 and a length weight of 6.
[0036] The present invention offers several advantages. For
example, various embodiments of the invention can readily be used
for high-throughput screening (HTS) of a wide variety of test
compounds. Thus, lead compounds can be selected from a large number
of test compounds. The assays described herein provide a high level
of sensitivity and are expected to detect a range of possible
inhibitors of mammalian Cdc7.4. Such inhibitors can be subsequently
modified using standard medicinal chemistry techniques and by
evaluating structure activity relationship (SAR) data. Because the
assays are cell-based, they can be used to identify
anti-proliferative agents that can efficiently enter eukaryotic
cells. Thus, the assays enable the identification of potent
anti-proliferative compounds and compounds of structural interest
that may have relatively modest potency, but have favorable cell
permeability properties.
[0037] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, technical manuals, and
other references mentioned herein are incorporated by reference in
their entirety. In case of conflict, the present application,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0038] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIGS. 1A-B are representations of the nucleic acid (SEQ ID
NO: 18) and polypeptide (SEQ ID NO: 19) sequences of human
Cdc7.
[0040] FIGS. 2A-B are representations of the nucleic acid (SEQ ID
NO:20) and,. polypeptide (SEQ ID NO:21) sequences of human
Dbf4.
[0041] FIG. 3 is a flow chart of an exemplary screening method.
[0042] FIG. 4 is a representation of a complementation assay
indicating that human HsCDC7 does not function in yeast.
[0043] FIG. 5 is a representation of a complementation assay
indicating that human HsCDC7 and human HsDBF4, in combination,
function in yeast.
[0044] FIGS. 6A-C are representations of an assay indicating the
requirement for the absence of doxycycline for tetO driven
co-expression of human HsCDC7 and human HsDBF4 in yeast.
DETAILED DESCRIPTION
[0045] The invention provides methods for determining whether a
test compound is an inhibitor of mammalian Cdc7.4, and methods for
identifying such inhibitors. A genetically-altered yeast strain
that depends on mammalian Cdc7.4 is used as a primary screen for
such compounds. Compounds that reduce the growth rate of this
specialized yeast strain are subjected to secondary screens to
eliminate compounds that reduce the growth rate by means other than
an inhibition of mammalian Cdc7.4. The remaining compounds are
identified as candidate compounds. The candidate compounds can be
subjected to in vitro and in vivo assays for efficacy as inhibitors
of mammalian Cdc7.4 and as anti-proliferative agents.
Primary Screen
[0046] A yeast strain whose viability depends on mammalian Cdc7.4
is constructed, e.g., as described in Example 1 or as referred to
as "Strain 1" (FIG. 3). Typically, human Cdc7 (HsCdc7) and human
Dbf4 (HsDbf4) are used. Nucleic acids encoding these polypeptides
can be amplified from mammalian, e.g., human, genomic DNA. A
wild-type diploid S. cerevisiae strain can be transformed with a
DNA cassette that disrupts one copy of the S. cerevisiae CDC7 gene.
The resulting heterozygous CDC7.sup.+/- strain is transformed with
a rescuing plasmid, e.g., a plasmid that expresses yeast CDC7. The
transformed heterozygote is sporulated. Haploid spores that have
the chromosomal cdc7.DELTA. and the rescuing plasmid are
identified. Plasmid shuffle techniques can then be used to replace
the rescuing plasmid with one or two constructs that include
nucleic acids encoding HsCdc7 and HsDbf4. The resulting yeast
strain is dependent on human Cdc7.4 activity (see below and FIG. 4
and FIG. 5).
[0047] The yeast strain dependent on HsCdc7 and. HsDbf4 can be used
in a variety of growth assays. For example, the strain can be grown
in liquid culture in a container such as a test tube, flask or well
of a microtitre plate. The container also includes the test
compound. The turbidity of the container can be compared with a
control container after a suitable incubation time. Alternatively,
the cells can be fixed and analyzed for DNA content, e.g., by
fluorescence-activated cell sorting. In a further alternative, the
abundance of a marker protein such as green fluorescent protein
(GFP) or an endogenous protein in the yeast can be assayed.
Generally, the determination of growth versus inhibition of growth
can be determined by a number of conventional methods including,
but not limited to, optical density, reporter gene assays, and
determination of endogenous ATP concentration.
[0048] In another example, the strain is used to create lawns on
petri plates. Filters containing the test compound are placed on
the plates. Halos forming in the lawn around the filters, which
indicate decreased and/or inhibited growth, can be identified and
measured.
[0049] The primary screen can identify at least two types of
compounds that inhibit the growth of the yeast strain that is
dependent on HsCdc7 and HsDbf4 activity. One type of compound
includes compounds that are candidate inhibitors of human Cdc7.Dbf4
activity, and hence cell proliferation. These compounds can be
formulated as pharmaceutical compositions to treat one or more
proliferative disorders, e.g., cancer. The second type of compound
includes compounds that are not candidates, but which impair growth
of the yeast strain for other reasons. Such compounds may be
anti-fungal compounds or compounds that interfere with the
heterologous expression system. These compounds can be
distinguished from the candidate inhibitors of human Cdc7.4
activity using the following secondary screens.
Secondary Screens
[0050] In one exemplary secondary screen, the growth and/or
proliferative activity of a yeast strain that does not depend on
any Cdc7 activity is assayed in the presence of the test compound
(FIG. 3). This control yeast strain can be similar or identical
(i.e., isogenic) to the strain used in the primary screen but
includes a bob1-1 mutant allele that bypasses the requirement for
Cdc7.4 activity (regardless of the species origin of Cdc7 or
Dbf4).
[0051] A secondary screen can be performed concurrently with the
primary screen. The strain that depends on HsCdc7 and HsDbf4 and
the control yeast strain can be assayed together in the presence of
a test compound. The two strains are differentially tagged with
different fluorescent proteins (e.g., GFP and derivatives of GFP,
e.g., with altered or enhanced fluorescent properties (Clontech
Laboratories, Inc. Calif.), such as yellow fluorescent protein
(YFP)). Fluorescent protein expression can be detected by
monitoring fluorescence emission upon excitation. The wavelengths
are selected depending on the fluorescent properties of the
utilized protein. The ratio in fluorescence of the two marker
proteins can be compared for the mixture grown in the presence of a
test compound relative to a control compound. This method can be
done in suspension (e.g., in a test tube or microtiter well), or on
a solid or semi-solid surface such as agarose or agar. The method
can include x-y translating the microtitre well or medium across a
scanning device. Fluorescent ratios for each relevant x-y position
can be stored. The system can also store references to compounds
applied to each position, and growth data for the screening strain
and the control strain.
[0052] Test compounds that are not invalidated by one of the
secondary screens are candidate inhibitors of human Cdc7.Dbf4
activity. An inhibitor of human Cdc7.4 activity may function by one
of the following exemplary mechanisms: (1) direct inhibition of
kinase complex activity; (2) inhibition of substrate recognition;
(3) inhibition of the interaction between HsCdc7 and HsDbf4; (4)
inhibition of kinase activation; and (5) inhibition of recruitment
of HsDbf4 (and complex) to origin of DNA replication. An inhibitor
of one or more of these activities can be used to prevent mammalian
cell proliferation in vivo or in vitro.
In Vitro Assays
[0053] Candidate test compounds can be screened in vitro by
assaying the kinase activity of purified HsCdc7 (i.e., human Cdc7
or SEQ ID NO: 19) and HsDbf4 (i.e., human Dbf4 or SEQ ID NO:21),
e.g., as described in Masai et al. J. Biol. Chem. 275:29042-52
(2000). The nucleic acid sequence encoding the two proteins can be
cloned into baculovirus expression vectors. One of the two
constructs can include a translational fusion of the coding
sequence of HsCdc7 or HsDbf4 to a purification tag, e.g.,
glutathione-S-transferase or hexa-histidine. The two constructs can
be co-infected into Sf9 cells to express the proteins. The tagged
HsCdc7.HsDbf4 protein complex is then purified using an affinity
column. Optionally, additional chromatographic steps can be used to
purify the complex.
[0054] Similarly, tagging and baculovirus expression systems can be
used to purify, substrates, such as human Mcm2 or a complex of any
two or more of human Mcm2, Mcm4, Mcm6, and Mcm7. In addition,
substrates such as S. cerevisiae Mcm2 or a complex of any two or
more of S. cerevisiae Mcm2, Mcm4, Mcm6, and Mcm7 can be
overexpressed and purified from S. cerevisiae.
[0055] To assay a test compound, reaction mixtures are set up with
various concentrations of the candidate compound and appropriate
controls. The standard reaction mixtures can include 40 mM
Hepes.KOH (pH 7.6), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM
.beta.-glycerophosphate, 1 mM NaF, 2 mM dithiothreitol, 10 mM
magnesium acetate, 80 .mu.g/ml bovine serum albumin, 0.1 mM ATP, 1
.mu.Ci of [.gamma.-.sup.32P]ATP, 0.1-0.5 .mu.g of MCM2 (or
MCM2-4-6-7 complex) and 50 ng of human HsCdc7.HsDbf4 kinase complex
(.mu.g of protein are per 25 .mu.l of volume). The reaction is
initiated by the addition of either the cold and labeled. ATP or
the HsCdc7-HsDbf4 complex. The reaction can be incubated, e.g., at
30.degree. C. for 30 minutes. The reaction can be stopped by heat
inactivation or the addition of an SDS-PAGE sample buffer.
Phosphorylation of the substrate can be measured by
immunoprecipitating the substrate onto beads or by separation of
the substrate, e.g., on an SDS-PAGE gel.
Test Compounds
[0056] The invention provides a method for screening a test
compound useful in the prevention or treatment of tumor metastasis.
A "test compound" can be any chemical compound, for example, a
macromolecule (e.g., a polypeptide, a protein complex, or a nucleic
acid) or a small molecule (e.g., an amino acid, a nucleotide, an
organic or inorganic compound). The test compound can have a
formula weight of less than about 10,000 grams per mole, less than
5,000 grams per mole, less than 1,000 grams per mole, or less than
about 500 grams per mole. The test compound can be naturally
occurring (e.g., an herb or a natural product), synthetic, or can
include both natural and synthetic components. Examples of small
molecules include peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, and small molecules, such as
organic or inorganic compounds, e.g., heteroorganic or
organometallic compounds.
[0057] The test compound or compounds can be screened individually
or in parallel. An example of the parallel screening is a high
throughput drug screen of large libraries of chemicals. Such
libraries of test compounds can be generated or purchased, e.g.,
from Chembridge Corp., San Diego, Calif. Libraries can be designed
to cover a diverse range of compounds. For example, a library can
include 500, 1000, 10,000, 50,000, or 100,000 or more unique
compounds. Alternatively, prior experimentation and anecdotal
evidence can suggest a class or category of compounds of enhanced
potential. A library can be designed and synthesized to cover such
a class of chemicals.
[0058] Examples of methods for the synthesis of molecular libraries
can be found in the literature, for example in: DeWitt et al.
(1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994)
Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J.
Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et
al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.
(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al.
(1994) J. Med. Chem. 37:1233.
[0059] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad
Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol.
Biol. 222:301-310; Ladner supra.).
[0060] Regardless of the method used for screening, compounds that
alter the growth or proliferative capacity of a yeast strain
dependent on human Cdc7.Dbf4 are considered candidate
anti-proliferative compounds. Candidate anti-proliferative
compounds can be retested on cancer cells, e.g., in vitro, or
tested on animals, e.g., animals that are models for cancer.
Candidate compounds that are positive in a retest are considered to
be anti-proliforative agents, and can also be used as "lead"
compounds to be further optimized and derivatized. A lead compound
can be a compound that impairs the growth or viability of a yeast
strain that depends on a mammalian Cdc7 activity such that the
ratio of A to B is at least 1.2, e.g., 1.5, 2.0, 3.0, or 5.0, where
A is the percentage increase in the doubling time of the yeast
strain in the presence of the lead compound relative to the same
strain in the absence of the compound, and B is the percentage
increase in doubling time of a control strain that does not depend
on a mammalian Cdc7 activity relative to the same control strain in
the absence of the compound.
[0061] Once a lead compound has been identified, standard
principles of medicinal chemistry can be used to produce
derivatives of the compound. Derivatives can be screened for
improved pharmacological properties, for example, efficacy,
pharmaco-kinetics, stability, solubility, and clearance. The
moieties responsible for a compound's activity in the assays
described above can be delineated by examination of
structure-activity relationships (SAR) as is commonly practiced in
the art. A person of ordinary skill in pharmaceutical chemistry
could modify moieties on a lead compound and measure the effects of
the modification on the efficacy of the compound to thereby produce
derivatives with increased potency. For an example, see Nagarajan
et al. (1988) J. Antibiot. 41: 1430-8. Furthermore, if the
biochemical target of the lead compound is known or determined, the
structure of the target and the lead compound can inform the design
and optimization of derivatives. Molecular modeling software is
commercially available (e.g., Molecular Simulations, Inc.) for this
purpose.
Use of Human Cdc7.Dbf4 Inhibitors
[0062] A compound identified as a human Cdc7.4 inhibitor can be
used as an anti-proliferative agent to inhibit cell proliferation
and/or division of a mammalian cell, e.g., a human cell. Because
human Cdc7.4 activity is required to initiate DNA replication in
human cells, inhibition of its activity would prevent cells from
advancing through the cell cycle, thereby inhibiting cell
proliferation and division.
[0063] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, or metastatic
disorders. Specific examples of such disorders include: a
fibrosarcoma, myosarcoma, endotheliosarcoma, gastric cancer,
esophageal cancer, rectal cancer, pancreatic cancer, breast cancer,
ovarian cancer, prostate cancer, uterine cancer, cancer of the head
and neck, skin cancer, brain cancer, squamous cell carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinoma, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular cancer, small cell lung
carcinoma, non-small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, medulloblastoma, craniopharyngioma,
melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or
Kaposi sarcoma.
Formulation
[0064] A composition containing an effective amount of a human
Cdc7.Dbf4 inhibitor can be administered (e.g., topically, orally,
nasally, buccally, subcutaneously, or intraperitoneally) to an
organism in a method of treatment. Treatment typically includes
administering an effective amount of the composition to a subject
in need of such treatment, thereby inhibiting cell proliferation in
the subject. Such a composition typically contains from about 0.1
to 90% by weight (e.g., 1 to 20% or 1 to 10%) of the
anti-proliferative agent of the invention in a pharmaceutically
acceptable carrier.
[0065] Solid formulations of the compositions for oral
administration may contain suitable carriers or excipients such
gelatin, lactose, acacia, sucrose, kaolin, mannitol, dicalcium
phosphate, calcium carbonate, sodium chloride, or alginic acid.
Disintegrators that can be used include, without limitation,
micro-crystalline cellulose, corn starch, sodium starch glycolate
and alginic acid. Tablet binders that may be used include acacia,
methylcellulose, sodium carboxymethylcellulose,
polyvinylpyrrolidone (Povidone.TM.), hydroxypropyl methylcellulose,
sucrose, starch, and ethylcellulose. Lubricants that may be used
include magnesium stearates, stearic acid, silicone fluid, talc,
waxes, oils, and colloidal silica.
[0066] Liquid formulations of the compositions for oral
administration prepared in water or other aqueous vehicles may
contain various suspending agents such as methylcellulose,
alginates, tragacanth, pectin, kelgin, carrageenan, acacia,
polyvinylpyrrolidone, and polyvinyl alcohol. The liquid
formulations may also include solutions, emulsions, syrups arid
elixirs containing, together with the active compound(s), wetting
agents, sweeteners, and coloring and flavoring agents. Various
liquid and powder formulations can be prepared by conventional
methods for inhalation into the lungs of the mammal to be
treated.
[0067] Injectable formulations of the compositions may contain
various carriers such as vegetable oils, dimethylacetanide,
dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl
myristate, ethanol and polyols (glycerol, propylene glycol, liquid
polyethylene glycol, and the like). For intravenous injections,
water soluble versions of the compounds may be administered by the
drip method, wherein a composition containing the compound and a
physiologically acceptable excipient is infused into a subject.
Physiologically acceptable excipients may include, for example, 5%
dextrose, 0.9% saline, Ringer's solution or other suitable
excipients. For intramuscular preparations, a sterile formulation
of a suitable soluble salt form of the compounds can be dissolved
and administered in a pharmaceutical excipient such as
Water-for-Injection, 0.9% saline, or 5% glucose solution. A
suitable insoluble form of the compound may be prepared and
administered as a suspension in an aqueous base or a
pharmaceutically acceptable oil base, such as an ester of a long
chain fatty acid (e.g., ethyl oleate).
[0068] A topical semi-solid ointment formulation typically contains
a concentration of the active ingredient from about 0.1 to 20%
wt/vol (e.g., 0.1 to 2% wt/vol of essentially pure material) in a
carrier such as a pharmaceutical cream base. Various formulations
for topical use include drops, tinctures, lotions, creams,
solutions, and ointments containing the active ingredient and
various supports and vehicles.
Dosage Determination
[0069] An appropriate dosage for treatment is determined using
standard techniques. For the purposes of inhibiting cell
proliferation in a subject, an effective amount of an inhibitor is
the amount or dose which is required to ameliorate a neoplasia
symptom in a subject. Determination of the amount or dose required
to treat an individual subject is routine to one skilled in the
art, e.g., a physician, pharmacist, or researcher. First, the
toxicity and therapeutic efficacy of the compound is determined.
Routine protocols are available for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population) in non-human
animals. The therapeutic index is measured as the ratio of the
LD.sub.50/ED.sub.50. Suitable ratios are greater than about 2, 5,
10, 50, or 100. Compounds, formulations, and methods of
administration with high therapeutic indices can be determined, as
such treatments have little toxicity at dosages which provide high
efficacy. Compounds with toxic or undesirable side effects can be
used, e.g., using means to deliver the compound to the affected
tissue, i.e., tumor site or proliferative site (e.g., bone
marrow).
[0070] In formulating a dosage range for use in humans, the
effective dose of an inhibitor can be estimated from studies with
an animal model for a proliferative disorder. For example,
therapeutically effective dosages in cell culture assays are about
5 ng/ml, 50 ng/ml, 500 ng/ml, 5 .mu.g/ml, and 50 .mu.g/ml of
inhibitor. A dose can be formulated in an animal to achieve a
circulating plasma concentration of inhibitor that falls in this
range. An exemplary dose produces a plasma concentration which
exceeds the IC.sub.50 (i.e., the concentration of the test compound
which achieves a half-maximal inhibition of a symptom) as
determined in cell culture assays. The circulating plasma
concentration can be determined, for example, by obtaining a blood
sample, and by analyzing the sample with high performance liquid
chromatography or mass spectroscopy.
[0071] Alternatively, the dose can be estimated from tests in an
animal model, e.g., a mouse or primate model for a proliferative
disorder. If, for example, the model is a nude mouse have
xenografted human tumor cells and an alleviation of symptoms is
observed when mice receive a compound in their drinking water at
doses of about 4 .mu.g/day, 10 .mu.g/day, 20 .mu.g/day, 40
.mu.g/day, 60 .mu.g/day, and 80 .mu.g/day, then an appropriate dose
for treating human patients can be approximately 0.4 mg kg.sup.-1
day.sup.-1, 1 mg kg.sup.-1 day.sup.-1, 2 mg kg.sup.-1 day.sup.-1, 4
mg kg.sup.-1 day.sup.-1, 6 mg kg.sup.-1 day.sup.-1, or
approximately 8 mg kg.sup.-1 day.sup.-1. Depending on the method of
administration, the appropriate dose can vary, e.g., from about 100
.mu.g kg.sup.-1 day.sup.-1 to about 500 mg kg.sup.-1 day.sup.-1, 1
mg kg.sup.-1 day.sup.-1 to about 100 mg kg.sup.-1 day.sup.-1, or 5
mg kg.sup.-1 day.sup.-1 to about 50 mg kg.sup.-1 day.sup.-1. The
dose for a patient can be optimized while the patient is under care
of a physician, pharmacist, or researcher. For example, a
relatively low dose of the anti-proliferative agent can be
administered initially. The patient can be monitored for symptoms
of neoplastic activity as described below. The dose can be
increased until an appropriate response is obtained. In addition,
the specific dose level for any particular subject can vary
depending on the age, body weight, general health, gender, and diet
of the subject, the time of administration, the route of
administration, the rate of excretion, and other drugs provided in
combination.
[0072] The efficacy of a dose of an anti-proliferative agent or any
other treatment can be determined in a subject. For example, the
subject can be monitored for clinical symptoms. Subjects can also
be directly monitored for changes in neoplastic activity, e.g.,
tumor growth and/or metastasis can be monitored, for example, by
labeling and imaging techniques, surgery, or physical examination.
For example, blood or tissue samples can be obtained from the
subject during treatment, and the level of antigens or cells
associated with neoplasia can be monitored. Alternatively,
histopathologic analysis of samples can be used to determine the
efficacy of the agent.
[0073] Without further elaboration, one skilled in the art can,
based on the above disclosure and the examples discussed below,
utilize the present invention to its fullest extent. The following
examples are to be construed as merely illustrative of how one
skilled in the art can produce and use a yeast strain to identify
an anti-proliferative agent, and does not limit of the remainder of
the disclosure.
EXAMPLES
Example 1
Construction of a Yeast Strain Dependent on Human CDC7 and DBF4
[0074] MMB2334, an example of Strain 1, has the following genotype:
cdc7.DELTA.::KAN snq2.DELTA.::HIS3
pdr5.DELTA.::HIS3+p411-tetO:HsCDC7+p41- 5-tetO:HsDSBF4. A yeast
cdc7.DELTA. strain that expresses the HsCDC7 and HsDBF4 was
constructed as follows.
Creating cdc7.DELTA.::KAN Deletion Strain
[0075] The entire open reading frame of CDC7 was deleted using
PCR-mediated gene deletion by the method of Wach, et al. ((1994)
Yeast 10: 1793-1808). Primers oBA234 and oBA235 were used to
generate a 1.6 kb PCR product that was transformed into MMB1489
using conventional methods (Sherman, et al. (1979) Methods in Yeast
Genetics). The following conditions were used for all PCR reactions
unless otherwise specified: 94.degree. C. 2 minutes; 25 cycles of
94.degree. C. 30 seconds, 50.degree. C. seconds, 72.degree. C. 4
minutes; then a final incubation at 72.degree. C. for 10 minutes.
Amplification of correctly sized products was confirmed by
visualizing resolved agarose gels stained with ethidium
bromide.
[0076] Transformants were selected on rich media supplemented with
200 .mu.g/ml G418 antibiotic. The complete deletion of the CDC7
open reading frame was confirmed by PCR using oBA235 and oBA236
(primer 500 bp upstream of START). This strain was transformed with
pRS416-CDC7 (described below), then sporulated and tetrads
dissected to obtain a haploid cdc7.DELTA.::KAN carrying
pRS416-CDC7.
Cloning of S. cerevisiae CDC7
[0077] A 2.2kb fragment containing the entire CDC7 gene was
amplified from S. cerevisiae genomic DNA by PCR using primers
oBA263 and oBA264 and subcloned into the KpnI-EcoRI sites of
pRS416. This plasmid is used to generate pRS416-CDC7, and cover a
genomic deletion of the yeast CDC7 gene.
Construction of Plasmids p411-tetO and p415-tetO
[0078] The entire tetracycline repressible promoter cassette was
subcloned from pCM188 into the PvuII sites of pRS411 and pRS416 to
generate p411-tetO and p415-tetO, respectively. These plasmids were
constructed to permit tetracycline repressible heterologous
expression of coding nucleic acids in S. cerevisiae with a
prototrophic marker other than that in pCM188.
Cloning of Human CDC7 (HsCdc7)
[0079] The HsCDC7 mRNA sequence (recited in GenBank Accession
#AF015592) encodes the HsCdc7 protein. The complete HsCDC7 open
reading frame (ORF) (GenBank Accession #AF015592) was amplified by
PCR and subcloned into the NotI site of p411-tetO. The orientation
of the subcloned DNA fragment was confirmed by restriction digests
and the nucleotide sequence verified by sequence analysis. The
cloned ORF is used in a plasmid to introduce HsCdc7 into yeast
cells.
Cloning of Human DBF4 (HsDbf4)
[0080] The HsDBF4 mRNA sequence is available at GenBank (Accession
# AF160876). The entire HsDBF4 ORF was amplified by PCR and
subdloned into the NotI site of p415-tetO. The orientation of the
subcloned DNA fragment was confirmed by restriction digests and the
nucleotide sequence verified by sequence analysis. The cloned ORF
is used in a plasmid to introduce HsDbf4 into yeast cells.
[0081] Referring to FIG. 4, a plate growth assay demonstrated that
tetO-HsCDC7 does not complement cdc7.DELTA.. MMB2166 was
transformed with p415-tetO-CDC7, p415-tetO, or p415-tetO-HsCDC7.
Each open reading frame is under the control of a tetracycline
responsive promoter. Two independent transformants were patched to
non-selective media and then replica plated to plates containing
5-FOA. Only strains where the cdc7.DELTA. mutation is complemented
by the transformed plasmid grow, e.g., a plasmid expressing yeast
CDC7 (first column, FIG. 4). Expression of HsCDC7 alone (under
control of the tetO promoter) does not complement the cdc7.DELTA.
(third column, FIG. 4).
[0082] Referring to FIG. 5, a plate growth assay demonstrated that
co-expression of HsCDC7 and HsDBF4 complements cdc7.DELTA.. MMB2166
was transformed with p415-tetO-CDC7 independently, p411-HsCDC7
independently, and co-transformed with both p411-tetO-HsCDC7 and
p415-tetO-HsDBF4. Two independent transformants of each of the
three variations were tested for complementation of the cdc7.DELTA.
mutant phenotype. Only the strains transformed with nucleic acids
encoding both HsCDC7 and HsDBF4 (or the yeast CDC7) are viable.
This result indicates that both HsCDC7 and HsDBF4 are required to
complement cdc7.DELTA..
[0083] Referring to FIGS. 6A, 6B, and 6C, complementation of
cdc7.DELTA. by tetO driven co-expression of HsCdc7 and HsDbf4 is
doxycycline sensitive. MMB2327 (#1), MMB2333 (#13) and multiple
isolates of MMB2334 (#'s 14, 16, 17 and 18) were streaked onto
Synthetic medium in the absence (FIG. 6A) or presence (FIG. 6B) of
(5 .mu.g/ml) doxycycline. Only strains with a copy of yeast CDC7
(under control of the CDC7 promoter) are able to grow in the
presence of doxycycline.
Example 2
Construction of a Control bob1-1 Strain
[0084] Although CDC7 is an essential gene, a documented point
mutation, bob1-1, is capable of completely bypassing CDC7 function.
The allele bob1-1 is a mutant gene that has a single point
mutation: P83L. The mutation bypasses the cell cycle arrest caused
by loss of CDC7 function. The bob1-1 mutation was introduced into a
yeast strain dependent on human CDC7 and DBF4.
[0085] Primers oBA255 and oBA258 were used to amplify a 0.63 kb PCR
product that contained the first 270 bp of BOB1 along with 360 bp
of the upstream promoter region. oBA258 contained a single G to A
point mutation that produces a P83L single amino acid mutation.
Primers oBA257 and oBA259 were used to generate a PCR product that
amplified 570 bp downstream of the point mutation. These PCR
products were subcloned into pRS303 at the XhoI-NotI sites. This
plasmid was cut with SpeI and transformed into MMB2166.
Transformants were selected on synthetic medium lacking histidine
and scored for resistance to 5-fluoroorotic acid (5-FOA).
[0086] MMB2170 was crossed with MMB2326 to generate a heterozygous
diploid at both the CDC7 and BOB1 loci. This diploid strain was
sporulated, tetrads dissected and germinated spores were scored for
the appropriate prototrophic markers and phenotypes. Crossing the
appropriate haploid strains resulting from the previous cross
generated diploids homozygous at both loci. Such diploid strains
were co-transformed with p411-tetO-HsCDC7 and p415-tetO-HsDBF4 and
strains that grew in the presence of 5-FOA were used. MMB2334 and
MMB2491 were further confirmed by doxycycline (80 .mu.g/ml)
sensitive and resistant growth, respectively.
[0087] The resulting strain, MMB2491, an example of a control yeast
strain, has the following genotype: cdc7.DELTA.::KAN
snq2.DELTA.::HIS3pdr5.DELTA.::HIS3
bob1-1::HIS3+p411-tetO:HsCDC7+p415-tet- O:HsDSBF4.
[0088] The plasmids listed in Table I are exemplary nucleic acids
and vector hucleic acids that can be used as described herein.
Likewise, the yeast strains listed in Table II are exemplary yeast
strains that can be used for the methods described herein. Table
III lists oligonucleotides that can be used to construct the
strains and nucleic acids described herein. Their sequences are
also provided.
1TABLE I Plasmids Plasmid Source Markers, notes pRS416 ATCC, #87521
CEN, URA3, amp pRS416-CDC7 Millennium Pharmaceuticals, Inc CEN,
URA3, amp pRS411 ATCC, #87521 CEN, MET15, amp p411-tetO Millennium
Pharmaceuticals, Inc CEN, MET15, amp p411-tetO-HsCDC7 Millennium
Pharmaceuticals, Inc CEN, MET15, amp pRS415 ATCC, #87520 CEN, LEU2,
amp p415-tetO Millennium Pharmaceuticals, Inc CEN, LEU2, amp
p415-tetO-CDC7 Millennium Pharmaceuticals, Inc CEN, LEU2, amp
p415-tetO-HsCDC7 Millennium Pharmaceuticals, Inc CEN, LEU2, amp
p415-tetO-HsDBF4 Millennium Pharmaceuticals, Inc CEN, LEU2, amp
pRS303 ATCC, #77138 Int, HIS3, amp pRS303-bob1-1 Millennium
Pharmaceuticals, Inc Int, HIS3, amp pFA6-kanMX4 Wach, et al. (1994)
Yeast 10: PCR template for 1793-1808 G418-selectable deletions
pCM188 Gari, et al. (1997) Yeast 13: CEN, URA3, 837-848 tetO2
promoter, amp
[0089]
2TABLE II Yeast Strains Strain Relevant genotype BY4743* MATa/a
ura3 leu2 his3 met15/MET15 LYS2/lys2 MMB1489 MATa/.alpha. ura3 leu2
his3 MET15/met15 lys2/LYS2 snq2 pdr5 MMB2166 MATa cdc7 +
pRS416-CDC7 MMB2326 MAT.alpha. cdc7 + pRS416-CDC7 MMB2327 MATa cdc7
+ pRS416-CDC7 + p-415-tetO-CDC7 MMB2328 MATa cdc7 + pRS416-CDC7 +
p-411-tetO-HsCDC7 MMB2333 MATa/.alpha. cdc7 + pRS416-CDC7 +
p-411-tetO-HsCDC7 + p415-tetO-HsDBF4 MMB2334 MATa/a cdc7 +
p411-tetO-HsCDC7 + p415-tetO-HsDBF4 MMB2170 MATa cdc7 bob1-1
MMB2491 MATa/a cdc7 bob1-1 + p411-tetO-HsCDC7 + p415-tetO-HsDBF4
*ATCC, #201390, all other strains are from Millennium
Pharmaceuticals, Inc.
[0090]
3TABLE III Oligonucleotide primers Oligo # Sequence (5' - 3')
oBA234 GGAAAGAGGCAGTTTCGAAGTAGAACAATCA- TAATGACAAGCAA (SEQ ID NO:1)
AACGCGTACGCTGCAGGTCGAC oBA235
AGAACATCCTTATCGAGCAAATCTGCCTCGCTTGAGCTGACAACG (SEQ ID NO:2)
ATCGATGAATTCGAGCTCG oBA236 TGACCATGACAGTGTAGG (SEQ ID NO:3) oBA255
GTCACTCGAGCCCTTTATTCTACCC (SEQ ID NO:4) oBA257
GAAACTATCAGACGAACTTTCAGATATCATTCCATTATTCG (SEQ ID NO:5) oBA258
CGAATAATGGAATGATATCTGAAAGTTCGTCTGATAGTTTC (SEQ ID NO:6) oBA259
GTCAGCGGCCGCTACTGGAACCAGTTCTGGG (SEQ ID NO:7) oBA263
GCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCTGAC- C (SEQ ID NO:8)
ATGACAGTGTAGG oBA264 CGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCTAAGTA
(SEQ ID NO:9) TCGTCTGCACCTGTGC oBA271
TCATCCCAAGCTAGCGTAGTCAGGAACGTCATA- TGGATAGGCGCC (SEQ ID NO:10)
GCTCATATCTTTAAAAAATGG oBA273
CTAACCGTTGAGGTCTTCCTCACTGATCAATTTCTGTTCAGTAAAT (SEQ ID NO:11)
GTAGAAGTTGAAGG oBA296 CGTGAATGTAAGCGTGACATAACTAATTACAT-
GATGCGGCCCTCCt (SEQ ID NO:12) caAGCGTAATCTGGAACGTC oBA297
ACGCAAACACAAATACACACACTAAATTACCGGATCAATTCGGG (SEQ ID NO:13)
GATGGAGGCGTCTTTGGGGATTC oBA298 GCAGAAGAAGCTTTGTTGCATCCAT-
TTTTTAAAGATATGAGCTTG (SEQ ID NO:14) ATCTTTTACCCATACGAT oBA299
ACGCAAACACAAATACACACACTAAATTACCGGATCAATTCGGG (SEQ ID NO:15)
GATGAACTCCGGAGCCATGAGG oBA300
ACAGCGTTTTTCTCGTCCCCTTCAACTTCTACATTTACTGGCTTTCT (SEQ ID NO:16)
CTGCGGCCGCTCTGAG oBA301 CGTGAATGTAAGCGTGACATAACTAATTACATG-
ATGCGGCCCTCC (SEQ ID NO:17) TCATCCACTAGTGCGGCCGCT
Example 3
Screening for Mammalian Cdc7 Inhibitors
[0091] The strain MMB2334, which has the following genotype:
cdc7.DELTA.::KAN snq2.DELTA.::HIS3
pdr5.DELTA.::HIS3+p411-tetO:HsCDC7+p41- 5-tetO:HsDSBF4, is used as
the yeast strain for the primary screen. A culture containing the
strain is used to seed wells of a microtitre plate. Dilutions of
test compounds from a chemical library are applied to each well in
duplicate. Control wells are used to monitor the effects of buffer
alone and other control solvents. After 24 hours of incubation at
30.degree. C., the endogenous ATP levels of the cells in each well
is measured. Wells that grew substantially slower as indicated by a
lower level of endogenous ATP than the control wells are
identified.
[0092] MMB2491, which has the following genotype: cdc7.DELTA.::KAN
snq2.DELTA.::HIS3 pdr5.DELTA.::HIS3
bob1-1::HIS3+p411-tetO:HsCDC7+p415-te- tO:HsDSBF4, is used for the
secondary screen. Compounds that were identified in the primary
screen are rescreened in the same format, except using a starter
culture of yeast from MMB2491 strain. Compounds that do not inhibit
the growth of this strain are identified. Such compounds are
candidate inhibitors of human Cdc7.4.
OTHER EMBODIMENTS
[0093] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. For example, any mammalian Cdc7 or Dbf4 can
be used in place of human Cdc7 and Dbf4. Moreover, mutant,
truncated and other modified forms of a mammalian Cdc7 and Dbf4 can
be used so long as they are able to complement yeast Cdc7 activity
and the yeast cdc7 mutant phenotype. Accordingly, other embodiments
are within the scope of the following claims.
Sequence CWU 1
1
21 1 66 DNA Artificial Sequence Primer 1 ggaaagaggc agtttcgaag
tagaacaatc ataatgacaa gcaaaacgcg tacgctgcag 60 gtcgac 66 2 64 DNA
Artificial Sequence Primer 2 agaacatcct tatcgagcaa atctgcctcg
cttgagctga caacgatcga tgaattcgag 60 ctcg 64 3 18 DNA Artificial
Sequence Primer 3 tgaccatgac agtgtagg 18 4 25 DNA Artificial
Sequence Primer 4 gtcactcgag ccctttattc taccc 25 5 41 DNA
Artificial Sequence Primer 5 gaaactatca gacgaacttt cagatatcat
tccattattc g 41 6 41 DNA Artificial Sequence Primer 6 cgaataatgg
aatgatatct gaaagttcgt ctgatagttt c 41 7 31 DNA Artificial Sequence
Primer 7 gtcagcggcc gctactggaa ccagttctgg g 31 8 58 DNA Artificial
Sequence Primer 8 gcgcgcgtaa tacgactcac tatagggcga attgggtacc
tgaccatgac agtgtagg 58 9 61 DNA Artificial Sequence Primer 9
cgctctagaa ctagtggatc ccccgggctg caggaattct aagtatcgtc tgcacctgtg
60 c 61 10 66 DNA Artificial Sequence Primer 10 tcatcccaag
ctagcgtagt caggaacgtc atatggatag gcgccgctca tatctttaaa 60 aaatgg 66
11 60 DNA Artificial Sequence Primer 11 ctaaccgttg aggtcttcct
cactgatcaa tttctgttca gtaaatgtag aagttgaagg 60 12 66 DNA Artificial
Sequence Primer 12 cgtgaatgta agcgtgacat aactaattac atgatgcggc
cctcctcaag cgtaatctgg 60 aacgtc 66 13 67 DNA Artificial Sequence
Primer 13 acgcaaacac aaatacacac actaaattac cggatcaatt cggggatgga
ggcgtctttg 60 gggattc 67 14 63 DNA Artificial Sequence Primer 14
gcagaagaag ctttgttgca tccatttttt aaagatatga gcttgatctt ttacccatac
60 gat 63 15 66 DNA Artificial Sequence Primer 15 acgcaaacac
aaatacacac actaaattac cggatcaatt cggggatgaa ctccggagcc 60 atgagg 66
16 63 DNA Artificial Sequence Primer 16 acagcgtttt tctcgtcccc
ttcaacttct acatttactg gctttctctg cggccgctct 60 gag 63 17 66 DNA
Artificial Sequence Primer 17 cgtgaatgta agcgtgacat aactaattac
atgatgcggc cctcctcatc cactagtgcg 60 gccgct 66 18 1725 DNA Homo
sapiens 18 atggaggcgt ctttggggat tcagatggat gagccaatgg ctttttctcc
ccagcgtgac 60 cggtttcagg ctgaaggctc tttaaaaaaa aacgagcaga
attttaaact tgcaggtgtt 120 aaaaaagata ttgagaagct ttatgaagct
gtaccacagc ttagtaatgt gtttaagatt 180 gaggacaaaa ttggagaagg
cactttcagc tctgtttatt tggccacagc acagttacaa 240 gtaggacctg
aagagaaaat tgctctaaaa cacttgattc caacaagtca tcctataaga 300
attgcagctg aacttcagtg cctaacagtg gctggggggc aagataatgt catgggagtt
360 aaatactgct ttaggaagaa tgatcatgta gttattgcta tgccatatct
ggagcatgag 420 tcgtttttgg acattctgaa ttctctttcc tttcaagaag
tacgggaata tatgcttaat 480 ctgttcaaag ctttgaaacg cattcatcag
tttggtattg ttcaccgtga tgttaagccc 540 agcaattttt tatataatag
gcgcctgaaa aagtatgcct tggtagactt tggtttggcc 600 caaggaaccc
atgatacgaa aatagagctt cttaaatttg tccagtctga agctcagcag 660
gaaaggtgtt cacaaaacaa atcccacata atcacaggaa acaagattcc actgagtggc
720 ccagtaccta aggagctgga tcagcagtcc accacaaaag cttctgttaa
aagaccctac 780 acaaatgcac aaattcagat taaacaagga aaagacggaa
aggagggatc tgtaggcctt 840 tctgtccagc gctctgtttt tggagaaaga
aatttcaata tacacagctc catttcacat 900 gagagccctg cagtgaaact
catgaagcag tcaaagactg tggatgtact gtctagaaag 960 ttagcaacaa
aaaagaaggc tatttctacg aaagttatga atagtgctgt gatgaggaaa 1020
actgccagtt cttgcccagc tagcctgacc tgtgactgct atgcaacaga taaagtttgt
1080 agtatttgcc tttcaaggcg tcagcaggtt gcccctaggg caggtacacc
aggattcaga 1140 gcaccagagg tcttgacaaa gtgccccaat caaactacag
caattgacat gtggtctgca 1200 ggtgtcatat ttctttcttt gcttagtgga
cgatatccat tttataaagc aagtgatgat 1260 ttaactgctt tggcccaaat
tatgacaatt aggggatcca gagaaactat ccaagctgct 1320 aaaacttttg
ggaaatcaat attatgtagc aaagaagttc cagcacaaga cttgagaaaa 1380
ctctgtgaga gactcagggg tatggattct agcactccca agttaacaag tgatatacaa
1440 gggcatgctt ctcatcaacc agctatttca gagaagactg accataaagc
ttcttgcctc 1500 gttcaaacac ctccaggaca atactcaggg aattcattta
aaaaggggga tagtaatagc 1560 tgtgagcatt gttttgatga gtataatacc
aatttagaag gctggaatga ggtacctgat 1620 gaagcttatg acctgcttga
taaacttcta gatctaaatc cagcttcaag aataacagca 1680 gaagaagctt
tgttgcatcc attttttaaa gatatgagct tgtga 1725 19 574 PRT Homo sapiens
19 Met Glu Ala Ser Leu Gly Ile Gln Met Asp Glu Pro Met Ala Phe Ser
1 5 10 15 Pro Gln Arg Asp Arg Phe Gln Ala Glu Gly Ser Leu Lys Lys
Asn Glu 20 25 30 Gln Asn Phe Lys Leu Ala Gly Val Lys Lys Asp Ile
Glu Lys Leu Tyr 35 40 45 Glu Ala Val Pro Gln Leu Ser Asn Val Phe
Lys Ile Glu Asp Lys Ile 50 55 60 Gly Glu Gly Thr Phe Ser Ser Val
Tyr Leu Ala Thr Ala Gln Leu Gln 65 70 75 80 Val Gly Pro Glu Glu Lys
Ile Ala Leu Lys His Leu Ile Pro Thr Ser 85 90 95 His Pro Ile Arg
Ile Ala Ala Glu Leu Gln Cys Leu Thr Val Ala Gly 100 105 110 Gly Gln
Asp Asn Val Met Gly Val Lys Tyr Cys Phe Arg Lys Asn Asp 115 120 125
His Val Val Ile Ala Met Pro Tyr Leu Glu His Glu Ser Phe Leu Asp 130
135 140 Ile Leu Asn Ser Leu Ser Phe Gln Glu Val Arg Glu Tyr Met Leu
Asn 145 150 155 160 Leu Phe Lys Ala Leu Lys Arg Ile His Gln Phe Gly
Ile Val His Arg 165 170 175 Asp Val Lys Pro Ser Asn Phe Leu Tyr Asn
Arg Arg Leu Lys Lys Tyr 180 185 190 Ala Leu Val Asp Phe Gly Leu Ala
Gln Gly Thr His Asp Thr Lys Ile 195 200 205 Glu Leu Leu Lys Phe Val
Gln Ser Glu Ala Gln Gln Glu Arg Cys Ser 210 215 220 Gln Asn Lys Ser
His Ile Ile Thr Gly Asn Lys Ile Pro Leu Ser Gly 225 230 235 240 Pro
Val Pro Lys Glu Leu Asp Gln Gln Ser Thr Thr Lys Ala Ser Val 245 250
255 Lys Arg Pro Tyr Thr Asn Ala Gln Ile Gln Ile Lys Gln Gly Lys Asp
260 265 270 Gly Lys Glu Gly Ser Val Gly Leu Ser Val Gln Arg Ser Val
Phe Gly 275 280 285 Glu Arg Asn Phe Asn Ile His Ser Ser Ile Ser His
Glu Ser Pro Ala 290 295 300 Val Lys Leu Met Lys Gln Ser Lys Thr Val
Asp Val Leu Ser Arg Lys 305 310 315 320 Leu Ala Thr Lys Lys Lys Ala
Ile Ser Thr Lys Val Met Asn Ser Ala 325 330 335 Val Met Arg Lys Thr
Ala Ser Ser Cys Pro Ala Ser Leu Thr Cys Asp 340 345 350 Cys Tyr Ala
Thr Asp Lys Val Cys Ser Ile Cys Leu Ser Arg Arg Gln 355 360 365 Gln
Val Ala Pro Arg Ala Gly Thr Pro Gly Phe Arg Ala Pro Glu Val 370 375
380 Leu Thr Lys Cys Pro Asn Gln Thr Thr Ala Ile Asp Met Trp Ser Ala
385 390 395 400 Gly Val Ile Phe Leu Ser Leu Leu Ser Gly Arg Tyr Pro
Phe Tyr Lys 405 410 415 Ala Ser Asp Asp Leu Thr Ala Leu Ala Gln Ile
Met Thr Ile Arg Gly 420 425 430 Ser Arg Glu Thr Ile Gln Ala Ala Lys
Thr Phe Gly Lys Ser Ile Leu 435 440 445 Cys Ser Lys Glu Val Pro Ala
Gln Asp Leu Arg Lys Leu Cys Glu Arg 450 455 460 Leu Arg Gly Met Asp
Ser Ser Thr Pro Lys Leu Thr Ser Asp Ile Gln 465 470 475 480 Gly His
Ala Ser His Gln Pro Ala Ile Ser Glu Lys Thr Asp His Lys 485 490 495
Ala Ser Cys Leu Val Gln Thr Pro Pro Gly Gln Tyr Ser Gly Asn Ser 500
505 510 Phe Lys Lys Gly Asp Ser Asn Ser Cys Glu His Cys Phe Asp Glu
Tyr 515 520 525 Asn Thr Asn Leu Glu Gly Trp Asn Glu Val Pro Asp Glu
Ala Tyr Asp 530 535 540 Leu Leu Asp Lys Leu Leu Asp Leu Asn Pro Ala
Ser Arg Ile Thr Ala 545 550 555 560 Glu Glu Ala Leu Leu His Pro Phe
Phe Lys Asp Met Ser Leu 565 570 20 2025 DNA Homo sapiens 20
atgaactccg gagccatgag gatccacagt aaaggacatt tccagggtgg aatccaagtc
60 aaaaatgaaa aaaacagacc atctctgaaa tctctgaaaa ctgataacag
gccagaaaaa 120 tccaaatgta agccactttg gggaaaagta ttttaccttg
acttaccttc tgtcaccata 180 tctgaaaaac ttcaaaagga cattaaggat
ctgggagggc gagttgaaga atttctcagc 240 aaagatatca gttatcttat
ttcaaataag aaggaagcta aatttgcaca aaccttgggt 300 cgaatttctc
ctgtaccaag tccagaatct gcatatactg cagaaaccac ttcacctcat 360
cccagccatg atggaagttc atttaagtca ccagacacag tgtgtttaag cagaggaaaa
420 ttattagttg aaaaagctat caaggaccat gattttattc cttcaaatag
tatattatca 480 aatgccttgt catggggagt aaaaattctt catattgatg
acattagata ctacattgaa 540 caaaagaaaa aagagttgta tttactcaag
aaatcaagta cttcagtaag agatgggggc 600 aaaagagttg gtagtggtgc
acaaaaaaca agaacaggaa gactcaaaaa gccttttgta 660 aaggtggaag
atatgagcca actttatagg ccattttatc ttcagctgac caatatgcct 720
tttataaatt attctattca gaagccctgc agtccatttg atgtagacaa gccatctagt
780 atgcaaaagc aaactcaggt taaactaaga atccaaacag atggcgataa
gtatggtgga 840 acctcaattc aactccagtt gaaagagaag aagaaaaaag
gatattgtga atgttgcttg 900 cagaaatatg aagatctaga aactcacctt
ctaagtgagc aacacagaaa ctttgcacag 960 agtaaccagt atcaagttgt
tgatgatatt gtatctaagt tagtttttga ctttgtggaa 1020 tatgaaaagg
acacacctaa aaagaaaaga ataaaataca gtgttggatc cctttctcct 1080
gtttctgcaa gtgtcctgaa aaagactgaa caaaaggaaa aagtggaatt gcaacatatt
1140 tctcagaaag attgccagga agatgataca acagtgaagg agcagaattt
cctgtataaa 1200 gagacccagg aaactgaaaa aaagctcctg tttatttcag
agcccatccc ccacccttca 1260 aatgaattga gagggcttaa tgagaaaatg
agtaataaat gttccatgtt aagtacagct 1320 gaagatgaca taagacagaa
ttttacacag ctacctctac ataaaaacaa acaggaatgc 1380 attcttgaca
tttccgaaca cacattaagt gaaaatgact tagaagaact aagggtagat 1440
cactataaat gtaacataca ggcatctgta catgtttctg atttcagtac agataatagt
1500 ggatctcaac caaaacagaa gtcagatact gtgctttttc cagcaaagga
tctcaaggaa 1560 aaggaccttc attcaatatt tactcatgat tctggtctga
taacaataaa cagttcacaa 1620 gagcacctaa ctgttcaggc aaaggctcca
ttccatactc ctcctgagga acccaatgaa 1680 tgtgacttca agaatatgga
tagtttacct tctggtaaaa tacatcgaaa agtgaaaata 1740 atattaggac
gaaatagaaa agaaaatctg gaaccaaatg ctgaatttga taaaagaact 1800
gaatttatta cacaagaaga aaacagaatt tgtagttcac cggtacagtc tttactagac
1860 ttgtttcaga ctagtgaaga gaaatcagaa tttttgggtt tcacaagcta
cacagaaaag 1920 agtggtatat gcaatgtttt agatatttgg gaagaggaaa
attcagataa tctgttaaca 1980 gcgtttttct cgtccccttc aacttctaca
tttactggct tttag 2025 21 674 PRT Homo sapiens 21 Met Asn Ser Gly
Ala Met Arg Ile His Ser Lys Gly His Phe Gln Gly 1 5 10 15 Gly Ile
Gln Val Lys Asn Glu Lys Asn Arg Pro Ser Leu Lys Ser Leu 20 25 30
Lys Thr Asp Asn Arg Pro Glu Lys Ser Lys Cys Lys Pro Leu Trp Gly 35
40 45 Lys Val Phe Tyr Leu Asp Leu Pro Ser Val Thr Ile Ser Glu Lys
Leu 50 55 60 Gln Lys Asp Ile Lys Asp Leu Gly Gly Arg Val Glu Glu
Phe Leu Ser 65 70 75 80 Lys Asp Ile Ser Tyr Leu Ile Ser Asn Lys Lys
Glu Ala Lys Phe Ala 85 90 95 Gln Thr Leu Gly Arg Ile Ser Pro Val
Pro Ser Pro Glu Ser Ala Tyr 100 105 110 Thr Ala Glu Thr Thr Ser Pro
His Pro Ser His Asp Gly Ser Ser Phe 115 120 125 Lys Ser Pro Asp Thr
Val Cys Leu Ser Arg Gly Lys Leu Leu Val Glu 130 135 140 Lys Ala Ile
Lys Asp His Asp Phe Ile Pro Ser Asn Ser Ile Leu Ser 145 150 155 160
Asn Ala Leu Ser Trp Gly Val Lys Ile Leu His Ile Asp Asp Ile Arg 165
170 175 Tyr Tyr Ile Glu Gln Lys Lys Lys Glu Leu Tyr Leu Leu Lys Lys
Ser 180 185 190 Ser Thr Ser Val Arg Asp Gly Gly Lys Arg Val Gly Ser
Gly Ala Gln 195 200 205 Lys Thr Arg Thr Gly Arg Leu Lys Lys Pro Phe
Val Lys Val Glu Asp 210 215 220 Met Ser Gln Leu Tyr Arg Pro Phe Tyr
Leu Gln Leu Thr Asn Met Pro 225 230 235 240 Phe Ile Asn Tyr Ser Ile
Gln Lys Pro Cys Ser Pro Phe Asp Val Asp 245 250 255 Lys Pro Ser Ser
Met Gln Lys Gln Thr Gln Val Lys Leu Arg Ile Gln 260 265 270 Thr Asp
Gly Asp Lys Tyr Gly Gly Thr Ser Ile Gln Leu Gln Leu Lys 275 280 285
Glu Lys Lys Lys Lys Gly Tyr Cys Glu Cys Cys Leu Gln Lys Tyr Glu 290
295 300 Asp Leu Glu Thr His Leu Leu Ser Glu Gln His Arg Asn Phe Ala
Gln 305 310 315 320 Ser Asn Gln Tyr Gln Val Val Asp Asp Ile Val Ser
Lys Leu Val Phe 325 330 335 Asp Phe Val Glu Tyr Glu Lys Asp Thr Pro
Lys Lys Lys Arg Ile Lys 340 345 350 Tyr Ser Val Gly Ser Leu Ser Pro
Val Ser Ala Ser Val Leu Lys Lys 355 360 365 Thr Glu Gln Lys Glu Lys
Val Glu Leu Gln His Ile Ser Gln Lys Asp 370 375 380 Cys Gln Glu Asp
Asp Thr Thr Val Lys Glu Gln Asn Phe Leu Tyr Lys 385 390 395 400 Glu
Thr Gln Glu Thr Glu Lys Lys Leu Leu Phe Ile Ser Glu Pro Ile 405 410
415 Pro His Pro Ser Asn Glu Leu Arg Gly Leu Asn Glu Lys Met Ser Asn
420 425 430 Lys Cys Ser Met Leu Ser Thr Ala Glu Asp Asp Ile Arg Gln
Asn Phe 435 440 445 Thr Gln Leu Pro Leu His Lys Asn Lys Gln Glu Cys
Ile Leu Asp Ile 450 455 460 Ser Glu His Thr Leu Ser Glu Asn Asp Leu
Glu Glu Leu Arg Val Asp 465 470 475 480 His Tyr Lys Cys Asn Ile Gln
Ala Ser Val His Val Ser Asp Phe Ser 485 490 495 Thr Asp Asn Ser Gly
Ser Gln Pro Lys Gln Lys Ser Asp Thr Val Leu 500 505 510 Phe Pro Ala
Lys Asp Leu Lys Glu Lys Asp Leu His Ser Ile Phe Thr 515 520 525 His
Asp Ser Gly Leu Ile Thr Ile Asn Ser Ser Gln Glu His Leu Thr 530 535
540 Val Gln Ala Lys Ala Pro Phe His Thr Pro Pro Glu Glu Pro Asn Glu
545 550 555 560 Cys Asp Phe Lys Asn Met Asp Ser Leu Pro Ser Gly Lys
Ile His Arg 565 570 575 Lys Val Lys Ile Ile Leu Gly Arg Asn Arg Lys
Glu Asn Leu Glu Pro 580 585 590 Asn Ala Glu Phe Asp Lys Arg Thr Glu
Phe Ile Thr Gln Glu Glu Asn 595 600 605 Arg Ile Cys Ser Ser Pro Val
Gln Ser Leu Leu Asp Leu Phe Gln Thr 610 615 620 Ser Glu Glu Lys Ser
Glu Phe Leu Gly Phe Thr Ser Tyr Thr Glu Lys 625 630 635 640 Ser Gly
Ile Cys Asn Val Leu Asp Ile Trp Glu Glu Glu Asn Ser Asp 645 650 655
Asn Leu Leu Thr Ala Phe Phe Ser Ser Pro Ser Thr Ser Thr Phe Thr 660
665 670 Gly Phe
* * * * *