U.S. patent application number 11/103068 was filed with the patent office on 2005-08-25 for hadhs as modifiers of the p21 pathway and methods of use.
This patent application is currently assigned to Exelixis, Inc.. Invention is credited to Belvin, Marcia, Friedman, Lori, Funke, Roel P., Li, Danxi, Plowman, Gregory D., Robertson, Stephanie A..
Application Number | 20050186200 11/103068 |
Document ID | / |
Family ID | 27405068 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186200 |
Kind Code |
A1 |
Friedman, Lori ; et
al. |
August 25, 2005 |
HADHs as modifiers of the p21 pathway and methods of use
Abstract
Human HADH genes are identified as modulators of the p21
pathway, and thus are therapeutic targets for disorders associated
with defective p21 function. Methods for identifying modulators of
p21, comprising screening for agents that modulate the activity of
HADH are provided.
Inventors: |
Friedman, Lori; (San
Francisco, CA) ; Plowman, Gregory D.; (San Carlos,
CA) ; Funke, Roel P.; (South San Francisco, CA)
; Belvin, Marcia; (Albany, CA) ; Li, Danxi;
(San Francisco, CA) ; Robertson, Stephanie A.;
(San Francisco, CA) |
Correspondence
Address: |
PATENT DEPT
EXELIXIS, INC.
170 HARBOR WAY
P.O. BOX 511
SOUTH SAN FRANCISCO
CA
94083-0511
US
|
Assignee: |
Exelixis, Inc.
|
Family ID: |
27405068 |
Appl. No.: |
11/103068 |
Filed: |
April 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11103068 |
Apr 11, 2005 |
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10191353 |
Jul 8, 2002 |
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6833635 |
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60305017 |
Jul 12, 2001 |
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60328491 |
Oct 10, 2001 |
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60357452 |
Feb 15, 2002 |
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Current U.S.
Class: |
424/130.1 ;
514/13.3; 514/18.9; 514/19.3 |
Current CPC
Class: |
G01N 33/502 20130101;
C12Q 2600/158 20130101; G01N 33/5008 20130101; A01K 2227/706
20130101; C12Q 1/6886 20130101; G01N 2333/914 20130101; G01N
33/57419 20130101; G01N 33/5011 20130101; G01N 33/5017 20130101;
G01N 2500/10 20130101; G01N 33/5748 20130101; G01N 33/5091
20130101; G01N 33/6872 20130101; G01N 2333/82 20130101; G01N 33/574
20130101; G01N 33/57496 20130101; G01N 33/57449 20130101 |
Class at
Publication: |
424/130.1 ;
514/012 |
International
Class: |
A61K 039/395; A61K
038/22 |
Claims
1-12. (canceled)
13. A method for modulating a p21 pathway of a cell comprising
contacting a cell defective in p21 function with a candidate
modulating agent that specifically binds to a purified or
recombinant HADH polypeptide comprising an amino acid sequence as
set forth in SEQ ID NO:4 3, whereby p21 function is restored,
wherein the defective p21 function is selected form the group
consisting of complete loss of p21 and overexpression of p21.
14. The method of claim 13 wherein the candidate modulating agent
is administered to a vertebrate animal predetermined to have a
disease or disorder resulting from a said defect in p21
function.
15. The method of claim 13 wherein the candidate modulating agent
is selected from the group consisting of an antibody and an
organic, non-peptide molecule, having a molecular weight less than
1000.
16-19. (canceled)
20. A method of modulating p21 pathway in a mammalian cell
comprising contacting the cell with an agent that specifically
binds a purified or recombinant HADH polypeptide comprising an
amino acid sequence as set forth in SEQ ID NO:3 or a purified or
recombinant HADH nucleic acid encoding said polypeptide.
21. The method of claim 20 wherein the agent is administered to a
mammalian animal predetermined to have a pathology associated with
the p21 pathway.
22. The method of claim 20 wherein the agent is a nucleic acid
modulator, an antibody, or an organic, non-peptide molecule, having
a molecular weight less than 1000.
23-25. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
application Nos. 60/305,017 filed Jul. 12, 2001, 60/328,491 filed
Oct. 10, 2001, and 60/357,452 filed Feb. 15, 2002. The contents of
the prior applications are hereby incorporated in their
entirety.
BACKGROUND OF THE INVENTION
[0002] The p21/CDKN1/WAF1/CIP1 protein(El-Deiry, W. S.; et al. Cell
75: 817-825, 1993; Harper, J. W.; et al. Cell 75: 805-816, 1993;
Huppi, Ket al. Oncogene 9: 3017-3020, 1994) is a cell cycle control
protein that inhibits cyclin-kinase activity, is tightly regulated
at the transcriptional level by p21, and mediates p21 suppression
of tumor cell growth. Along with p21, p21 appears to be essential
for maintaining the G2 checkpoint in human cells (Bunz, F.;
Dutriaux, A.; et al. Science 282:1497-1501, 1998). Sequences of P21
are well-conserved throughout evolution, and have been identified
in species as diverse as human (Genbank Identifier 13643057),
Drosophila melanogaster (GI# 1684911), Caenorhabditis elegans
(GI#4966283), and yeast (GI#2656016).
[0003] The hydrolytic dehalogenases catalyse a nucleophilic
displacement reaction, with water as the sole co-substrate. They
are divided into haloalkane dehalogenases and haloacid
dehalogenases (HAD). HADs belong to a large superfamily of
hydrolases with diverse substrate specificity, which also includes
epoxide hydrolases, phosphoglycolate phosphatases, histidinol
phosphate phosphatases, nitrophenyl phosphatases and numerous
putative proteins. The epoxide hydrolases (EH) add water to
epoxides, forming the corresponding diol. HADH (C20orf147) is a
member of the haloacid dehalogenase or epoxide hydrolase
family.
[0004] The ability to manipulate the genomes of model organisms
such as Drosophila provides a powerful means to analyze biochemical
processes that, due to significant evolutionary conservation, has
direct relevance to more complex vertebrate organisms. Due to a
high level of gene and pathway conservation, the strong similarity
of cellular processes, and the functional conservation of genes
between these model organisms and mammals, identification of the
involvement of novel genes in particular pathways and their
functions in such model organisms can directly contribute to the
understanding of the correlative pathways and methods of modulating
them in mammals (see, for example, Mechler BM et al., 1985 EMBO J.
4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K
L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M.
1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev
5: 44-50; and Booth DR. 1999 Cancer Metastasis Rev. 18: 261-284).
For example, a genetic screen can be carried out in an invertebrate
model organism having underexpression (e.g. knockout) or
overexpression of a gene (referred to as a "genetic entry point")
that yields a visible phenotype. Additional genes are mutated in a
random or targeted manner. When a gene mutation changes the
original phenotype caused by the mutation in the genetic entry
point, the gene is identified as a "modifier" involved in the same
or overlapping pathway as the genetic entry point. When the genetic
entry point is an ortholog of a human gene implicated in a disease
pathway, such as p21, modifier genes can be identified that may be
attractive candidate targets for novel therapeutics.
[0005] All references cited herein, including sequence information
in referenced Genbank identifier numbers and website references,
are incorporated herein in their entireties.
SUMMARY OF THE INVENTION
[0006] We have discovered genes that modify the p21 pathway in
Drosophila, and identified their human orthologs, hereinafter
referred to as HADH. The invention provides methods for utilizing
these p21 modifier genes and polypeptides to identify candidate
therapeutic agents that can be used in the treatment of disorders
associated with defective p21 function. Preferred HADH-modulating
agents specifically bind to HADH polypeptides and restore p21
function. Other preferred HADH-modulating agents are nucleic acid
modulators such as antisense oligomers and RNAi that repress HADH
gene expression or product activity by, for example, binding to and
inhibiting the respective nucleic acid (i.e. DNA or mRNA).
[0007] HADH-specific modulating agents may be evaluated by any
convenient in vitro or in vivo assay for molecular interaction with
an HADH polypeptide or nucleic acid. In one embodiment, candidate
p21 modulating agents are tested with an assay system comprising a
HADH polypeptide or nucleic acid. Candidate agents that produce a
change in the activity of the assay system relative to controls are
identified as candidate p21 modulating agents. The assay system may
be cell-based or cell-free. HADH-modulating agents include HADH
related proteins (e.g. dominant negative mutants, and
biotherapeutics); HADH-specific antibodies; HADH-specific antisense
oligomers and other nucleic acid modulators; and chemical agents
that specifically bind HADH or compete with HADH binding target. In
one specific embodiment, a small molecule modulator is identified
using a binding assay. In specific embodiments, the screening assay
system is selected from an apoptosis assay, a cell proliferation
assay, an angiogenesis assay, and a hypoxic induction assay.
[0008] In another embodiment, candidate p21 pathway modulating
agents are further tested using a second assay system that detects
changes in the p21 pathway, such as angiogenic, apoptotic, or cell
proliferation changes produced by the originally identified
candidate agent or an agent derived from the original agent. The
second assay system may use cultured cells or non-human animals. In
specific embodiments, the secondary assay system uses non-human
animals, including animals predetermined to have a disease or
disorder implicating the p21 pathway, such as an angiogenic,
apoptotic, or cell proliferation disorder (e.g. cancer).
[0009] The invention further provides methods for modulating the
p21 pathway in a mammalian cell by contacting the mammalian cell
with an agent that specifically binds a HADH polypeptide or nucleic
acid. The agent may be a small molecule modulator, a nucleic acid
modulator, or an antibody and may be administered to a mammalian
animal predetermined to have a pathology associated the p21
pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0010] An overexpression screen was carried out in Drosophila to
identify genes that interact with the cyclin dependent kinase
inhibitor, p21 (Bourne H R, et al., Nature (1990)
348(6297):125-132; Marshall CJ, Trends Genet (1991) 7(3):91-95).
Expression of the p21 gene in the eye causes deterioration of
normal eye morphology. The CG15771 gene was identified as a
modifier of the p21 pathway. Accordingly, vertebrate orthologs of
these modifiers, and preferably the human orthologs, HADH genes
(i.e., nucleic acids and polypeptides) are attractive drug targets
for the treatment of pathologies associated with a defective p21
signaling pathway, such as cancer.
[0011] In vitro and in vivo methods of assessing HADH function are
provided herein. Modulation of the HADH or their respective binding
partners is useful for understanding the association of the p21
pathway and its members in normal and disease conditions and for
developing diagnostics and therapeutic modalities for p21 related
pathologies. HADH-modulating agents that act by inhibiting or
enhancing HADH expression, directly or indirectly, for example, by
affecting an HADH function such as enzymatic (e.g., catalytic) or
binding activity, can be identified using methods provided herein.
HADH modulating agents are useful in diagnosis, therapy and
pharmaceutical development.
[0012] Nucleic Acids and Polypeptides of the Invention
[0013] Sequences related to HADH nucleic acids and polypeptides
that can be used in the invention are disclosed in Genbank
(referenced by Genbank identifier (GI) number) as GI#s 11968366
(SEQ ID NO:1), 18490373 (SEQ ID NO:2), and 20405373 (SEQ ID NO:3)
for nucleic acid, and GI# 4902680 (SEQ ID NO:4) polypeptides.
Additionally, polypeptide sequence of SEQ ID NO:5 is a translation
of SEQ ID NO:2, and can be used in the invention.
[0014] HADHs are hydrolase proteins with hydrolase domains. The
term "HADH polypeptide" refers to a full-length HADH protein or a
functionally active fragment or derivative thereof. A "functionally
active" HADH fragment or derivative exhibits one or more functional
activities associated with a full-length, wild-type HADH protein,
such as antigenic or immunogenic activity, enzymatic activity,
ability to bind natural cellular substrates, etc. The functional
activity of HADH proteins, derivatives and fragments can be assayed
by various methods known to one skilled in the art (Current
Protocols in Protein Science (1998) Coligan et al., eds., John
Wiley & Sons, Inc., Somerset, N.J.) and as further discussed
below. For purposes herein, functionally active fragments also
include those fragments that comprise one or more structural
domains of an HADH, such as a hydrolase domain or a binding domain.
Protein domains can be identified using the PFAM program (Bateman
A., et al., Nucleic Acids Res, 1999, 27:260-2;
http://pfam.wustl.edu). For example, the hydrolase domain of HADH
from GI# 4902680 (SEQ ID NO:4) is located at approximately amino
acid residues 9-212 (PFAM 00702). Methods for obtaining HADH
polypeptides are also further described below. In some embodiments,
preferred fragments are functionally active, domain-containing
fragments comprising at least 25 contiguous amino acids, preferably
at least 50, more preferably 75, and most preferably at least 100
contiguous amino acids of SEQ ID NOs:4 or 5 (an HADH). In further
preferred embodiments, the fragment comprises the entire hydrolase
(functionally active) domain.
[0015] The term "HADH nucleic acid" refers to a DNA or RNA molecule
that encodes a HADH polypeptide. Preferably, the HADH polypeptide
or nucleic acid or fragment thereof is from a human, but can also
be an ortholog, or derivative thereof with at least 70% sequence
identity, preferably at least 80%, more preferably 85%, still more
preferably 90%, and most preferably at least 95% sequence identity
with HADH. Normally, orthologs in different species retain the same
function, due to presence of one or more protein motifs and/or
3-dimensional structures. Orthologs are generally identified by
sequence homology analysis, such as BLAST analysis, usually using
protein bait sequences. Sequences are assigned as a potential
ortholog if the best hit sequence from the forward BLAST result
retrieves the original query sequence in the reverse BLAST (Huynen
MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen Mass.
et al., Genome Research (2000) 10:1204-1210). Programs for multiple
sequence alignment, such as CLUSTAL (Thompson JD et al, 1994,
Nucleic Acids Res 22:4673-4680) may be used to highlight conserved
regions and/or residues of orthologous proteins and to generate
phylogenetic trees. In a phylogenetic tree representing multiple
homologous sequences from diverse species (e.g., retrieved through
BLAST analysis), orthologous sequences from two species generally
appear closest on the tree with respect to all other sequences from
these two species. Structural threading or other analysis of
protein folding (e.g., using software by ProCeryon, Biosciences,
Salzburg, Austria) may also identify potential orthologs. In
evolution, when a gene duplication event follows speciation, a
single gene in one species, such as Drosophila, may correspond to
multiple genes (paralogs) in another, such as human. As used
herein, the term "orthologs" encompasses paralogs. As used herein,
"percent (%) sequence identity" with respect to a subject sequence,
or a specified portion of a subject sequence, is defined as the
percentage of nucleotides or amino acids in the candidate
derivative sequence identical with the nucleotides or amino acids
in the subject sequence (or specified portion thereof), after
aligning the sequences and introducing gaps, if necessary to
achieve the maximum percent sequence identity, as generated by the
program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997)
215:403-410; http://blast.wustl.edulblast/README.html) with all the
search parameters set to default values. The HSP S and HSP S2
parameters are dynamic values and are established by the program
itself depending upon the composition of the particular sequence
and composition of the particular database against which the
sequence of interest is being searched. A % identity value is
determined by the number of matching identical nucleotides or amino
acids divided by the sequence length for which the percent identity
is being reported. "Percent (%) amino acid sequence similarity" is
determined by doing the same calculation as for determining % amino
acid sequence identity, but including conservative amino acid
substitutions in addition to identical amino acids in the
computation.
[0016] A conservative amino acid substitution is one in which an
amino acid is substituted for another amino acid having similar
properties such that the folding or activity of the protein is not
significantly affected. Aromatic amino acids that can be
substituted for each other are phenylalanine, tryptophan, and
tyrosine; interchangeable hydrophobic amino acids are leucine,
isoleucine, methionine, and valine; interchangeable polar amino
acids are glutamine and asparagine; interchangeable basic amino
acids are arginine, lysine and histidine; interchangeable acidic
amino acids are aspartic acid and glutamic acid; and
interchangeable small amino acids are alanine, serine, threonine,
cysteine and glycine.
[0017] Alternatively, an alignment for nucleic acid sequences is
provided by the local homology algorithm of Smith and Waterman
(Smith and Waterman, 1981, Advances in Applied Mathematics
2:482-489; database: European Bioinformatics Institute
http://www.ebi.ac.uk/MPsrch/; Smith and Waterman, 1981, J. of
Molec.Biol., 147:195-197; Nicholas et al., 1998, "A Tutorial on
Searching Sequence Databases and Sequence Scoring Methods"
(www.psc.edu) and references cited therein.; W. R. Pearson, 1991,
Genomics 11:635-650). This algorithm can be applied to amino acid
sequences by using the scoring matrix developed by Dayhoff
(Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986
Nucl. Acids Res. 14(6):6745-6763). The Smith-Waterman algorithm may
be employed where default parameters are used for scoring (for
example, gap open penalty of 12, gap extension penalty of two).
From the data generated, the "Match" value reflects "sequence
identity."
[0018] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of SEQ ID NOs:1, 2, or 3. The stringency of hybridization
can be controlled by temperature, ionic strength, pH, and the
presence of denaturing agents such as formamide during
hybridization and washing. Conditions routinely used are set out in
readily available procedure texts (e.g., Current Protocol in
Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons,
Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). In some embodiments, a nucleic acid molecule of the
invention is capable of hybridizing to a nucleic acid molecule
containing the nucleotide sequence of any one of SEQ ID NOs:1, 2,
or 3 under stringent hybridization conditions that comprise:
prehybridization of filters containing nucleic acid for 8 hours to
overnight at 65.degree. C. in a solution comprising 6.times.single
strength citrate (SSC) (1.times.SSC is 0.15 M NaCl, 0.015 M Na
citrate; pH 7.0), 5.times. Denhardt's solution, 0.05% sodium
pyrophosphate and 100 .mu.g/ml herring sperm DNA; hybridization for
18-20 hours at 65.degree. C. in a solution containing 6.times.SSC,
1.times. Denhardt's solution, 100 .mu.g/ml yeast tRNA and 0.05%
sodium pyrophosphate; and washing of filters at 65.degree. C. for 1
h in a solution containing 0.2.times.SSC and 0.1% SDS (sodium
dodecyl sulfate).
[0019] In other embodiments, moderately stringent hybridization
conditions are used that comprise: pretreatment of filters
containing nucleic acid for 6 h at 40.degree. C. in a solution
containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH7.5), 5 mM
EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA; hybridization for 18-20 h at 40.degree. C. in a
solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl
(pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml
salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by
washing twice for 1 hour at 55.degree. C. in a solution containing
2.times.SSC and 0.1% SDS.
[0020] Alternatively, low stringency conditions can be used that
comprise: incubation for 8 hours to overnight at 37.degree. C. in a
solution comprising 20% formamide, 5.times.SSC, 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured sheared salmon sperm DNA;
hybridization in the same buffer for 18 to 20 hours; and washing of
filters in 1.times.SSC at about 37.degree. C. for 1 hour.
[0021] Isolations Production, Expression, and Mis-Expression of
HADH Nucleic Acids and Polypeptides
[0022] HADH nucleic acids and polypeptides, useful for identifying
and testing agents that modulate HADH function and for other
applications related to the involvement of HADH in the p21 pathway.
HADH nucleic acids and derivatives and orthologs thereof may be
obtained using any available method. For instance, techniques for
isolating cDNA or genomic DNA sequences of interest by screening
DNA libraries or by using polymerase chain reaction (PCR) are well
known in the art. In general, the particular use for the protein
will dictate the particulars of expression, production, and
purification methods. For instance, production of proteins for use
in screening for modulating agents may require methods that
preserve specific biological activities of these proteins, whereas
production of proteins for antibody generation may require
structural integrity of particular epitopes. Expression of proteins
to be purified for screening or antibody production may require the
addition of specific tags (e.g., generation of fusion proteins).
Overexpression of an HADH protein for assays used to assess HADH
function, such as involvement in cell cycle regulation or hypoxic
response, may require expression in eukaryotic cell lines capable
of these cellular activities. Techniques for the expression,
production, and purification of proteins are well known in the art;
any suitable means therefore may be used (e.g., Higgins S J and
Hames B D (eds.) Protein Expression: A Practical Approach, Oxford
University Press Inc., New York 1999; Stanbury P F et al.,
Principles of Fermentation Technology, 2.sup.nd edition, Elsevier
Science, New York, 1995; Doonan S (ed.) Protein Purification
Protocols, Humana Press, New Jersey, 1996; Coligan J E et al,
Current Protocols in Protein Science (eds.), 1999, John Wiley &
Sons, New York). In particular embodiments, recombinant HADH is
expressed in a cell line known to have defective p21 function such
as HCT116 colon cancer cells available from American Type Culture
Collection (ATCC), Manassas, Va.). The recombinant cells are used
in cell-based screening assay systems of the invention, as
described further below.
[0023] The nucleotide sequence encoding an HADH polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native HADH gene
and/or its flanking regions or can be heterologous. A variety of
host-vector expression systems may be utilized, such as mammalian
cell systems infected with virus (e.g. vaccinia virus, adenovirus,
etc.); insect cell systems infected with virus (e.g. baculovirus);
microorganisms such as yeast containing yeast vectors, or bacteria
transformed with bacteriophage, plasmid, or cosmid DNA. A host cell
strain that modulates the expression of, modifies, and/or
specifically processes the gene product may be used.
[0024] To detect expression of the HADH gene product, the
expression vector can comprise a promoter operably linked to an
HADH gene nucleic acid, one or more origins of replication, and,
one or more selectable markers (e.g. thymidine kinase activity,
resistance to antibiotics, etc.). Alternatively, recombinant
expression vectors can be identified by assaying for the expression
of the HADH gene product based on the physical or functional
properties of the HADH protein in in vitro assay systems (e.g.
immunoassays).
[0025] The HADH protein, fragment, or derivative may be optionally
expressed as a fusion, or chimeric protein product (i.e. it is
joined via a peptide bond to a heterologous protein sequence of a
different protein), for example to facilitate purification or
detection. A chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other using standard methods and expressing the
chimeric product. A chimeric product may also be made by protein
synthetic techniques, e.g. by use of a peptide synthesizer
(Hunkapiller et al., Nature (1984) 310:105-111).
[0026] Once a recombinant cell that expresses the HADH gene
sequence is identified, the gene product can be isolated and
purified using standard methods (e.g. ion exchange, affinity, and
gel exclusion chromatography; centrifugation; differential
solubility; electrophoresis, cite purification reference).
Alternatively, native HADH proteins can be purified from natural
sources, by standard methods (e.g. immunoaffinity purification).
Once a protein is obtained, it may be quantified and its activity
measured by appropriate methods, such as immunoassay, bioassay, or
other measurements of physical properties, such as
crystallography.
[0027] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of HADH or
other genes associated with the p21 pathway. As used herein,
mis-expression encompasses ectopic expression, over-expression,
under-expression, and non-expression (e.g. by gene knock-out or
blocking expression that would otherwise normally occur).
[0028] Genetically Modified Animals
[0029] Animal models that have been genetically modified to alter
HADH expression may be used in in vivo assays to test for activity
of a candidate p21 modulating agent, or to further assess the role
of HADH in a p21 pathway process such as apoptosis or cell
proliferation. Preferably, the altered HADH expression results in a
detectable phenotype, such as decreased or increased levels of cell
proliferation, angiogenesis, or apoptosis compared to control
animals having normal HADH expression. The genetically modified
animal may additionally have altered p21 expression (e.g. p21
knockout). Preferred genetically modified animals are mammals such
as primates, rodents (preferably mice), cows, horses, goats, sheep,
pigs, dogs and cats. Preferred non-mammalian species include
zebrafish, C. elegans, and Drosophila. Preferred genetically
modified animals are transgenic animals having a heterologous
nucleic acid sequence present as an extrachromosomal element in a
portion of its cells, i.e. mosaic animals (see, for example,
techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763.)
or stably integrated into its germ line DNA (i.e., in the genomic
sequence of most or all of its cells). Heterologous nucleic acid is
introduced into the germ line of such transgenic animals by genetic
manipulation of, for example, embryos or embryonic stem cells of
the host animal.
[0030] Methods of making transgenic animals are well-known in the
art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci.
USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and
Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle
bombardment see U.S. Pat. No., 4,945,050, by Sandford et al.; for
transgenic Drosophila see Rubin and Spradling, Science (1982)
218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see
Berghammer A. J. et al., A Universal Marker for Transgenic Insects
(1999) Nature 402:370-371; for transgenic Zebrafish see Lin S.,
Transgenic Zebrafish, Methods Mol. Biol. (2000);136:375-3830); for
microinjection procedures for fish, amphibian eggs and birds see
Houdebine and Chourrout, Experientia (1991) 47:897-905; for
transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and
for culturing of embryonic stem (ES) cells and the subsequent
production of transgenic animals by the introduction of DNA into ES
cells using methods such as electroporation, calcium phosphate/DNA
precipitation and direct injection see, e.g., Teratocarcinomas and
Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,
IRL Press (1987)). Clones of the nonhuman transgenic animals can be
produced according to available methods (see Wilmut, I. et al.
(1997) Nature, 385:810-813; and PCT International Publication Nos.
WO 97/07668 and WO 97/07669).
[0031] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous HADH gene that results in a decrease of
HADH function, preferably such that HADH expression is undetectable
or insignificant. Knock-out animals are typically generated by
homologous recombination with a vector comprising a transgene
having at least a portion of the gene to be knocked out. Typically
a deletion, addition or substitution has been introduced the
transgene to functionally disrupt it. The transgene can be a human
gene (e.g., from a human genomic clone) but more preferably is an
ortholog of the human gene derived from the transgenic host
species. For example, a mouse HADH gene is used to construct a
homologous recombination vector suitable for altering an endogenous
HADH gene in the mouse genome. Detailed methodologies for
homologous recombination in mice are available (see Capecchi,
Science (1989) 244:1288-1292; Joyner et al., Nature (1989)
338:153-156). Procedures for the production of non-rodent
transgenic mammals and other animals are also available (Houdebine
and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288;
Simms et al., Bio/Technology (1988) 6:179-183). In a preferred
embodiment, knock-out animals, such as mice harboring a knockout of
a specific gene, may be used to produce antibodies against the
human counterpart of the gene that has been knocked out (Claesson M
H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al.,
(1995) J Biol. Chem. 270:8397-400).
[0032] In another embodiment, the transgenic animal is a "knock-in"
animal having an alteration in its genome that results in altered
expression (e.g., increased (including ectopic) or decreased
expression) of the HADH gene, e.g., by introduction of additional
copies of HADH, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
HADH gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0033] Transgenic nonhuman animals can also be produced that
contain selected systems allowing for regulated expression of the
transgene. One example of such a system that may be produced is the
cre/loxP recombinase system of bacteriophage P 1 (Lakso et al.,
PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxp
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase. Another example of a recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a
preferred embodiment, both Cre-LoxP and Flp-Frt are used in the
same system to regulate expression of the transgene, and for
sequential deletion of vector sequences in the same cell (Sun X et
al (2000) Nat Genet 25:83-6).
[0034] The genetically modified animals can be used in genetic
studies to further elucidate the p21 pathway, as animal models of
disease and disorders implicating defective p21 function, and for
in vivo testing of candidate therapeutic agents, such as those
identified in screens described below. The candidate therapeutic
agents are administered to a genetically modified animal having
altered HADH function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered HADH
expression that receive candidate therapeutic agent.
[0035] In addition to the above-described genetically modified
animals having altered HADH function, animal models having
defective p21 function (and otherwise normal HADH function), can be
used in the methods of the present invention. For example, a p21
knockout mouse can be used to assess, in vivo, the activity of a
candidate p21 modulating agent identified in one of the in vitro
assays described below. p21 knockout mouse are described in the
literature (Umanoff H, et al., Proc Natl Acad Sci USA 1995 Feb.
28;92(5):1709-13). Preferably, the candidate p21 modulating agent
when administered to a model system with cells defective in p21
function, produces a detectable phenotypic change in the model
system indicating that the p21 function is restored, i.e., the
cells exhibit normal cell cycle progression.
[0036] Modulating Agents
[0037] The invention provides methods to identify agents that
interact with and/or modulate the function of HADH and/or the p21
pathway. Such agents are useful in a variety of diagnostic and
therapeutic applications associated with the p21 pathway, as well
as in further analysis of the HADH protein and its contribution to
the p21 pathway. Accordingly, the invention also provides methods
for modulating the p21 pathway comprising the step of specifically
modulating HADH activity by administering a HADH-interacting or
-modulating agent.
[0038] In a preferred embodiment, HADH-modulating agents inhibit or
enhance HADH activity or otherwise affect normal HADH function,
including transcription, protein expression, protein localization,
and cellular or extra-cellular activity. In a further preferred
embodiment, the candidate p21 pathway--modulating agent
specifically modulates the function of the HADH. The phrases
"specific modulating agent", "specifically modulates", etc., are
used herein to refer to modulating agents that directly bind to the
HADH polypeptide or nucleic acid, and preferably inhibit, enhance,
or otherwise alter, the function of the HADH. The term also
encompasses modulating agents that alter the interaction of the
HADH with a binding partner or substrate (e.g. by binding to a
binding partner of an HADH, or to a protein/binding partner
complex, and inhibiting function).
[0039] Preferred HADH-modulating agents include small molecule
compounds; HADH-interacting proteins, including antibodies and
other biotherapeutics; and nucleic acid modulators such as
antisense and RNA inhibitors. The modulating agents may be
formulated in pharmaceutical compositions, for example, as
compositions that may comprise other active ingredients, as in
combination therapy, and/or suitable carriers or excipients.
Techniques for formulation and administration of the compounds may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., 19.sup.th edition.
[0040] Small Molecule Modulators
[0041] Small molecules, are often preferred to modulate function of
proteins with enzymatic function, and/or containing protein
interaction domains. Chemical agents, referred to in the art as
"small molecule" compounds are typically organic, non-peptide
molecules, having a molecular weight less than 10,000, preferably
less than 5,000, more preferably less than 1,000, and most
preferably less than 500. This class of modulators includes
chemically synthesized molecules, for instance, compounds from
combinatorial chemical libraries. Synthetic compounds may be
rationally designed or identified based on known or inferred
properties of the HADH protein or may be identified by screening
compound libraries. Alternative appropriate modulators of this
class are natural products, particularly secondary metabolites from
organisms such as plants or fungi, which can also be identified by
screening compound libraries for HADH-modulating activity. Methods
for generating and obtaining compounds are well known in the art
(Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther
J, Science (2000) 151:1947-1948).
[0042] Small molecule modulators identified from screening assays,
as described below, can be used as lead compounds from which
candidate clinical compounds may be designed, optimized, and
synthesized. Such clinical compounds may have utility in treating
pathologies associated with the p21 pathway. The activity of
candidate small molecule modulating agents may be improved
several-fold through iterative secondary functional validation, as
further described below, structure determination, and candidate
modulator modification and testing. Additionally, candidate
clinical compounds are generated with specific regard to clinical
and pharmacological properties. For example, the reagents may be
derivatized and re-screened using in vitro and in vivo assays to
optimize activity and minimize toxicity for pharmaceutical
development.
[0043] Protein Modulators
[0044] Specific HADH-interacting proteins are useful in a variety
of diagnostic and therapeutic applications related to the p21
pathway and related disorders, as well as in validation assays for
other HADH-modulating agents. In a preferred embodiment,
HADH-interacting proteins affect normal HADH function, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In another embodiment,
HADH-interacting proteins are useful in detecting and providing
information about the function of HADH proteins, as is relevant to
p21 related disorders, such as cancer (e.g., for diagnostic
means).
[0045] An HADH-interacting protein may be endogenous, i.e. one that
naturally interacts genetically or biochemically with an HADH, such
as a member of the HADH pathway that modulates HADH expression,
localization, and/or activity. HADH-modulators include dominant
negative forms of HADH-interacting proteins and of HADH proteins
themselves. Yeast two-hybrid and variant screens offer preferred
methods for identifying endogenous HADH-interacting proteins
(Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A
Practical Approach, eds. Glover D. & Hames B. D (Oxford
University Press, Oxford, England), pp. 169-203; Fashema S F et
al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999)
3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29;
and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative
preferred method for the elucidation of protein complexes (reviewed
in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates JR
3.sup.rd, Trends Genet (2000) 16:5-8).
[0046] An HADH-interacting protein may be an exogenous protein,
such as an HADH-specific antibody or a T-cell antigen receptor
(see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using
antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold
Spring Harbor Laboratory Press). HADH antibodies are further
discussed below.
[0047] In preferred embodiments, an HADH-interacting protein
specifically binds an HADH protein. In alternative preferred
embodiments, an HADH-modulating agent binds an HADH substrate,
binding partner, or cofactor.
[0048] Antibodies
[0049] In another embodiment, the protein modulator is an HADH
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify HADH modulators. The antibodies can also be used
in dissecting the portions of the HADH pathway responsible for
various cellular responses and in the general processing and
maturation of the HADH.
[0050] Antibodies that specifically bind HADH polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of HADH polypeptide, and more preferably,
to human HADH. Antibodies may be polyclonal, monoclonal (mAbs),
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. Epitopes of HADH
which are particularly antigenic can be selected, for example, by
routine screening of HADH polypeptides for antigenicity or by
applying a theoretical method for selecting antigenic regions of a
protein (Hopp and Wood (1981), Proc. Natl. Acad. Sci. U.S.A.
78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;
Sutcliffe et al., (1983) Science 219:660-66) to the amino acid
sequence shown in SEQ ID NOs:4 or 5. Monoclonal antibodies with
affinities of 10.sup.8 M.sup.-1 preferably 10.sup.9 M.sup.-1 to
10.sup.10 M.sup.-1, or stronger can be made by standard procedures
as described (Harlow and Lane, supra; Goding (1986) Monoclonal
Antibodies: Principles and Practice (2d ed) Academic Press, New
York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577).
Antibodies may be generated against crude cell extracts of HADH or
substantially purified fragments thereof. If HADH fragments are
used, they preferably comprise at least 10, and more preferably, at
least 20 contiguous amino acids of an HADH protein. In a particular
embodiment, HADH-specific antigens and/or immunogens are coupled to
carrier proteins that stimulate the immune response. For example,
the subject polypeptides are covalently coupled to the keyhole
limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in
Freund's complete adjuvant, which enhances the immune response. An
appropriate immune system such as a laboratory rabbit or mouse is
immunized according to conventional protocols.
[0051] The presence of HADH-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding HADH polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0052] Chimeric antibodies specific to HADH polypeptides can be
made that contain different portions from different animal species.
For instance, a human immunoglobulin constant region may be linked
to a variable region of a murine mAb, such that the antibody
derives its biological activity from the human antibody, and its
binding specificity from the murine fragment. Chimeric antibodies
are produced by splicing together genes that encode the appropriate
regions from each species (Morrison et al., Proc. Natl. Acad. Sci.
(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608;
Takeda et al., Nature (1985) 31:452-454). Humanized antibodies,
which are a form of chimeric antibodies, can be generated by
grafting complementary-determining regions (CDRs) (Carlos, T. M.,
J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a
background of human framework regions and constant regions by
recombinant DNA technology (Riechmann LM, et al., 1988 Nature 323:
323-327). Humanized antibodies contain .about.10% murine sequences
and .about.90% human sequences, and thus further reduce or
eliminate immunogenicity, while retaining the antibody
specificities (Co MS, and Queen C. 1991 Nature 351: 501-501;
Morrison SL. 1992 Ann. Rev. Immun. 10:239-265). Humanized
antibodies and methods of their production are well-known in the
art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and
6,180,370).
[0053] HADH-specific single chain antibodies which are recombinant,
single chain polypeptides formed by linking the heavy and light
chain fragments of the Fv regions via an amino acid bridge, can be
produced by methods known in the art (U.S. Pat. No. 4,946,778;
Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad.
Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989)
334:544-546).
[0054] Other suitable techniques for antibody production involve in
vitro exposure of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in phage or
similar vectors (Huse et al., Science (1989) 246:1275-1281). As
used herein, T-cell antigen receptors are included within the scope
of antibody modulators (Harlow and Lane, 1988, supra).
[0055] The polypeptides and antibodies of the present invention may
be used with or without modification. Frequently, antibodies will
be labeled by joining, either covalently or non-covalently, a
substance that provides for a detectable signal, or that is toxic
to cells that express the targeted protein (Menard S, et al., Int
J. Biol Markers (1989) 4:131-134). A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, fluorescent emitting lanthanide metals,
chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,
recombinant immunoglobulins may be produced (U.S. Pat. No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered
and reach their targets by conjugation with membrane-penetrating
toxin proteins (U.S. Pat. No. 6,086,900).
[0056] When used therapeutically in a patient, the antibodies of
the subject invention are typically administered parenterally, when
possible at the target site, or intravenously. The therapeutically
effective dose and dosage regimen is determined by clinical
studies. Typically, the amount of antibody administered is in the
range of about 0.1 mg/kg--to about 10 mg/kg of patient weight. For
parenteral administration, the antibodies are formulated in a unit
dosage injectable form (e.g., solution, suspension, emulsion) in
association with a pharmaceutically acceptable vehicle. Such
vehicles are inherently nontoxic and non-therapeutic. Examples are
water, saline, Ringer's solution, dextrose solution, and 5% human
serum albumin. Nonaqueous vehicles such as fixed oils, ethyl
oleate, or liposome carriers may also be used. The vehicle may
contain minor amounts of additives, such as buffers and
preservatives, which enhance isotonicity and chemical stability or
otherwise enhance therapeutic potential. The antibodies'
concentrations in such vehicles are typically in the range of about
1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further
described in the literature (U.S. Pat. No. 5,859,206;
WO0073469).
[0057] Nucleic Acid Modulators
[0058] Other preferred HADH-modulating agents comprise nucleic acid
molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit HADH activity. Preferred nucleic
acid modulators interfere with the function of the HADH nucleic
acid such as DNA replication, transcription, translocation of the
HADH RNA to the site of protein translation, translation of protein
from the HADH RNA, splicing of the HADH RNA to yield one or more
mRNA species, or catalytic activity which may be engaged in or
facilitated by the HADH RNA.
[0059] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to an HADH mRNA
to bind to and prevent translation, preferably by binding to the 5'
untranslated region. HADH-specific antisense oligonucleotides,
preferably range from at least 6 to about 200 nucleotides. In some
embodiments the oligonucleotide is preferably at least 10, 15, or
20 nucleotides in length. In other embodiments, the oligonucleotide
is preferably less than 50, 40, or 30 nucleotides in length. The
oligonucleotide can be DNA or RNA or a chimeric mixture or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appending groups such as peptides, agents that
facilitate transport across the cell membrane,
hybridization-triggered cleavage agents, and intercalating
agents.
[0060] In another embodiment, the antisense oligomer is a
phosphothioate morpholino oligomer (PMO). PMOs are assembled from
four different morpholino subunits, each of which contain one of
four genetic bases (A, C, G, or T) linked to a six-membered
morpholine ring. Polymers of these subunits are joined by non-ionic
phosphodiamidate intersubunit linkages. Details of how to make and
use PMOs and other antisense oligomers are well known in the art
(e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide
and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J,
and Weller D. 1997 Antisense Nucleic Acid Drug Dev.:7:187-95; U.S.
Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).
[0061] Alternative preferred HADH nucleic acid modulators are
double-stranded RNA species mediating RNA interference (RNAi). RNAi
is the process of sequence-specific, post-transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene.
Methods relating to the use of RNAi to silence genes in C. elegans,
Drosophila, plants, and humans are known in the art (Fire A, et
al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363
(1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490
(2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119
(2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A.
et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature
404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);
Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M.,
et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;
Elbashir SM, et al., 2001 Nature 411:494-498).
[0062] Nucleic acid modulators are commonly used as research
reagents, diagnostics, and therapeutics. For example, antisense
oligonucleotides, which are able to inhibit gene expression with
exquisite specificity, are often used to elucidate the function of
particular genes (see, for example, U.S. Pat. No. 6,165,790).
Nucleic acid modulators are also used, for example, to distinguish
between functions of various members of a biological pathway. For
example, antisense oligomers have been employed as therapeutic
moieties in the treatment of disease states in animals and man and
have been demonstrated in numerous clinical trials to be safe and
effective (Milligan J F, et al, Current Concepts in Antisense Drug
Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al.,
Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,
Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the
invention, an HADH-specific nucleic acid modulator is used in an
assay to further elucidate the role of the HADH in the p21 pathway,
and/or its relationship to other members of the pathway. In another
aspect of the invention, an HADH-specific antisense oligomer is
used as a therapeutic agent for treatment of p21-related disease
states.
[0063] Assay Systems
[0064] The invention provides assay systems and screening methods
for identifying specific modulators of HADH activity. As used
herein, an "assay system" encompasses all the components required
for performing and analyzing results of an assay that detects
and/or measures a particular event. In general, primary assays are
used to identify or confirm a modulator's specific biochemical or
molecular effect with respect to the HADH nucleic acid or protein.
In general, secondary assays further assess the activity of a HADH
modulating agent identified by a primary assay and may confirm that
the modulating agent affects HADH in a manner relevant to the p21
pathway. In some cases, HADH modulators will be directly tested in
a secondary assay.
[0065] In a preferred embodiment, the screening method comprises
contacting a suitable assay system comprising an HADH polypeptide
with a candidate agent under conditions whereby, but for the
presence of the agent, the system provides a reference activity
(e.g. binding activity), which is based on the particular molecular
event the screening method detects. A statistically significant
difference between the agent-biased activity and the reference
activity indicates that the candidate agent modulates HADH
activity, and hence the p21 pathway.
[0066] Primary Assays
[0067] The type of modulator tested generally determines the type
of primary assay.
[0068] Primary Assays for Small Molecule Modulators
[0069] For small molecule modulators, screening assays are used to
identify candidate modulators. Screening assays may be cell-based
or may use a cell-free system that recreates or retains the
relevant biochemical reaction of the target protein (reviewed in
Sittampalam GS et al., Curr Opin Chem Biol (1997) 1:384-91 and
accompanying references). As used herein the term "cell-based"
refers to assays using live cells, dead cells, or a particular
cellular fraction, such as a membrane, endoplasmic reticulum, or
mitochondrial fraction. The term "cell free" encompasses assays
using substantially purified protein (either endogenous or
recombinantly produced), partially purified or crude cellular
extracts. Screening assays may detect a variety of molecular
events, including protein-DNA interactions, protein-protein
interactions (e.g., receptor-ligand binding), transcriptional
activity (e.g., using a reporter gene), enzymatic activity (e.g.,
via a property of the substrate), activity of second messengers,
immunogenicty and changes in cellular morphology or other cellular
characteristics. Appropriate screening assays may use a wide range
of detection methods including fluorescent, radioactive,
colorimetric, spectrophotometric, and amperometric methods, to
provide a read-out for the particular molecular event detected.
[0070] Cell-based screening assays usually require systems for
recombinant expression of HADH and any auxiliary proteins demanded
by the particular assay. Appropriate methods for generating
recombinant proteins produce sufficient quantities of proteins that
retain their relevant biological activities and are of sufficient
purity to optimize activity and assure assay reproducibility. Yeast
two-hybrid and variant screens, and mass spectrometry provide
preferred methods for determining protein-protein interactions and
elucidation of protein complexes. In certain applications, when
HADH-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the HADH protein may be assayed by various known methods
such as substrate processing (e.g. ability of the candidate
HADH-specific binding agents to function as negative effectors in
HADH-expressing cells), binding equilibrium constants (usually at
least about 10.sup.7 M.sup.-1, preferably at least about 10.sup.8
M.sup.-1, more preferably at least about 10.sup.9 M.sup.-1), and
immunogenicity (e.g. ability to elicit HADH specific antibody in a
heterologous host such as a mouse, rat, goat or rabbit). For
enzymes and receptors, binding may be assayed by, respectively,
substrate and ligand processing.
[0071] The screening assay may measure a candidate agent's ability
to specifically bind to or modulate activity of a HADH polypeptide,
a fusion protein thereof, or to cells or membranes bearing the
polypeptide or fusion protein. The HADH polypeptide can be full
length or a fragment thereof that retains functional HADH activity.
The HADH polypeptide may be fused to another polypeptide, such as a
peptide tag for detection or anchoring, or to another tag. The HADH
polypeptide is preferably human HADH, or is an ortholog or
derivative thereof as described above. In a preferred embodiment,
the screening assay detects candidate agent-based modulation of
HADH interaction with a binding target, such as an endogenous or
exogenous protein or other substrate that has HADH-specific binding
activity, and can be used to assess normal HADH gene function.
[0072] Suitable assay formats that may be adapted to screen for
HADH modulators are known in the art. Preferred screening assays
are high throughput or ultra high throughput and thus provide
automated, cost-effective means of screening compound libraries for
lead compounds (Fernandes P B, Curr Opin Chem Biol (1998)
2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In
one preferred embodiment, screening assays uses fluorescence
technologies, including fluorescence polarization, time-resolved
fluorescence, and fluorescence resonance energy transfer. These
systems offer means to monitor protein-protein or DNA-protein
interactions in which the intensity of the signal emitted from
dye-labeled molecules depends upon their interactions with partner
molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4;
Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem
Biol (2000) 4:445-451).
[0073] A variety of suitable assay systems may be used to identify
candidate HADH and p21 pathway modulators (e.g. U.S. Pat. Nos.
5,550,019 and 6,133,437 (apoptosis assays), among others). Specific
preferred assays are described in more detail below.
[0074] Hydrolase assays. Hydrolases catalyze the hydrolysis of a
substrate such as esterases, lipases, peptidases, nucleotidases,
and phosphatases, among others. Enzyme activity assays may be used
to measure hydrolase activity. The activity of the enzyme is
determined in presence of excess substrate, by
spectrophotometrically measuring the rate of appearance of reaction
products. High throughput arrays and assays for hydrolases are
known to those skilled in the art (Park CB and Clark DS (2002)
Biotech Bioeng 78:229-235).
[0075] Apoptosis assays. Assays for apoptosis may be performed by
terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP
nick end labeling (TUNEL) assay. The TUNEL assay is used to measure
nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et
al., 1994, Nature 371, 346), by following the incorporation of
fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747).
Apoptosis may further be assayed by acridine orange staining of
tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41).
An apoptosis assay system may comprise a cell that expresses an
HADH, and that optionally has defective p21 function (e.g. p21 is
over-expressed or under-expressed relative to wild-type cells). A
test agent can be added to the apoptosis assay system and changes
in induction of apoptosis relative to controls where no test agent
is added, identify candidate p21 modulating agents. In some
embodiments of the invention, an apoptosis assay may be used as a
secondary assay to test a candidate p21 modulating agents that is
initially identified using a cell-free assay system. An apoptosis
assay may also be used to test whether HADH function plays a direct
role in apoptosis. For example, an apoptosis assay may be performed
on cells that over- or under-express HADH relative to wild type
cells. Differences in apoptotic response compared to wild type
cells suggests that the HADH plays a direct role in the apoptotic
response. Apoptosis assays are described further in U.S. Pat. No.
6,133,437.
[0076] Cell proliferation and cell cycle assays. Cell proliferation
may be assayed via bromodeoxyuridine (BRDU) incorporation. This
assay identifies a cell population undergoing DNA synthesis by
incorporation of BRDU into newly-synthesized DNA. Newly-synthesized
DNA may then be detected using an anti-BRDU antibody (Hoshino et
al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J.
Immunol. Meth. 107, 79), or by other means.
[0077] Cell Proliferation may also be examined using
[.sup.3H]-thymidine incorporation (Chen, J., 1996, Oncogene
13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This
assay allows for quantitative characterization of S-phase DNA
syntheses. In this assay, cells synthesizing DNA will incorporate
[.sup.3H]-thymidine into newly synthesized DNA. Incorporation can
then be measured by standard techniques such as by counting of
radioisotope in a scintillation counter (e.g., Beckman LS 3800
Liquid Scintillation Counter).
[0078] Cell proliferation may also be assayed by colony formation
in soft agar (Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). For example, cells transformed with HADH are seeded
in soft agar plates, and colonies are measured and counted after
two weeks incubation.
[0079] Involvement of a gene in the cell cycle may be assayed by
flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud
Phys Chem Med 49:237-55). Cells transfected with an HADH may be
stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson).
[0080] Accordingly, a cell proliferation or cell cycle assay system
may comprise a cell that expresses an HADH, and that optionally has
defective p21 function (e.g. p21 is over-expressed or
under-expressed relative to wild-type cells). A test agent can be
added to the assay system and changes in cell proliferation or cell
cycle relative to controls where no test agent is added, identify
candidate p21 modulating agents. In some embodiments of the
invention, the cell proliferation or cell cycle assay may be used
as a secondary assay to test a candidate p21 modulating agents that
is initially identified using another assay system such as a
cell-free kinase assay system. A cell proliferation assay may also
be used to test whether HADH function plays a direct role in cell
proliferation or cell cycle. For example, a cell proliferation or
cell cycle assay may be performed on cells that over- or
under-express HADH relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the HADH plays a direct role in cell proliferation or cell
cycle.
[0081] Angiogenesis. Angiogenesis may be assayed using various
human endothelial cell systems, such as umbilical vein, coronary
artery, or dermal cells. Suitable assays include Alamar Blue based
assays (available from Biosource International) to measure
proliferation; migration assays using fluorescent molecules, such
as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture
inserts to measure migration of cells through membranes in presence
or absence of angiogenesis enhancer or suppressors; and tubule
formation assays based on the formation of tubular structures by
endothelial cells on Matrigel.RTM. (Becton Dickinson). Accordingly,
an angiogenesis assay system may comprise a cell that expresses an
HADH, and that optionally has defective p21 function (e.g. p21 is
over-expressed or under-expressed relative to wild-type cells). A
test agent can be added to the angiogenesis assay system and
changes in angiogenesis relative to controls where no test agent is
added, identify candidate p21 modulating agents. In some
embodiments of the invention, the angiogenesis assay may be used as
a secondary assay to test a candidate p21 modulating agents that is
initially identified using another assay system. An angiogenesis
assay may also be used to test whether HADH function plays a direct
role in cell proliferation. For example, an angiogenesis assay may
be performed on cells that over- or under-express HADH relative to
wild type cells. Differences in angiogenesis compared to wild type
cells suggests that the HADH plays a direct role in
angiogenesis.
[0082] Hypoxic induction. The alpha subunit of the transcription
factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor
cells following exposure to hypoxia in vitro. Under hypoxic
conditions, HIF-1 stimulates the expression of genes known to be
important in tumour cell survival, such as those encoding glyolytic
enzymes and VEGF. Induction of such genes by hypoxic conditions may
be assayed by growing cells transfected with HADH in hypoxic
conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated
in a Napco 7001 incubator (Precision Scientific)) and normoxic
conditions, followed by assessment of gene activity or expression
by Taqman.RTM.. For example, a hypoxic induction assay system may
comprise a cell that expresses an HADH, and that optionally has a
mutated p21 (e.g. p21 is over-expressed or under-expressed relative
to wild-type cells). A test agent can be added to the hypoxic
induction assay system and changes in hypoxic response relative to
controls where no test agent is added, identify candidate p21
modulating agents. In some embodiments of the invention, the
hypoxic induction assay may be used as a secondary assay to test a
candidate p21 modulating agents that is initially identified using
another assay system. A hypoxic induction assay may also be used to
test whether HADH function plays a direct role in the hypoxic
response. For example, a hypoxic induction assay may be performed
on cells that over- or under-express HADH relative to wild type
cells. Differences in hypoxic response compared to wild type cells
suggests that the HADH plays a direct role in hypoxic
induction.
[0083] Cell adhesion. Cell adhesion assays measure adhesion of
cells to purified adhesion proteins, or adhesion of cells to each
other, in presence or absence of candidate modulating agents.
Cell-protein adhesion assays measure the ability of agents to
modulate the adhesion of cells to purified proteins. For example,
recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and
used to coat the wells of a microtiter plate. The wells used for
negative control are not coated. Coated wells are then washed,
blocked with 1% BSA, and washed again. Compounds are diluted to
2.times.final test concentration and added to the blocked, coated
wells. Cells are then added to the wells, and the unbound cells are
washed off. Retained cells are labeled directly on the plate by
adding a membrane-permeable fluorescent dye, such as calcein-AM,
and the signal is quantified in a fluorescent microplate
reader.
[0084] Cell-cell adhesion assays measure the ability of agents to
modulate binding of cell adhesion proteins with their native
ligands. These assays use cells that naturally or recombinantly
express the adhesion protein of choice. In an exemplary assay,
cells expressing the cell adhesion protein are plated in wells of a
multiwell plate. Cells expressing the ligand are labeled with a
membrane-permeable fluorescent dye, such as BCECF, and allowed to
adhere to the monolayers in the presence of candidate agents.
Unbound cells are washed off, and bound cells are detected using a
fluorescence plate reader.
[0085] High-throughput cell adhesion assays have also been
described. In one such assay, small molecule ligands and peptides
are bound to the surface of microscope slides using a microarray
spotter, intact cells are then contacted with the slides, and
unbound cells are washed off. In this assay, not only the binding
specificity of the peptides and modulators against cell lines are
determined, but also the functional cell signaling of attached
cells using immunofluorescence techniques in situ on the microchip
is measured (Falsey J R et al., Bioconjug Chem. 2001
May-Jun;12(3):346-53).
[0086] Primary Assays for Antibody Modulators
[0087] For antibody modulators, appropriate primary assays test is
a binding assay that tests the antibody's affinity to and
specificity for the HADH protein. Methods for testing antibody
affinity and specificity are well known in the art (Harlow and
Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay
(ELISA) is a preferred method for detecting HADH-specific
antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
[0088] Primary Assays for Nucleic Acid Modulators
[0089] For nucleic acid modulators, primary assays may test the
ability of the nucleic acid modulator to inhibit or enhance HADH
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing HADH expression in like populations of
cells (e.g., two pools of cells that endogenously or recombinantly
express HADH) in the presence and absence of the nucleic acid
modulator. Methods for analyzing mRNA and protein expression are
well known in the art. For instance, Northern blotting, slot
blotting, ribonuclease protection, quantitative RT-PCR (e.g., using
the TaqMan.RTM., PE Applied Biosystems), or microarray analysis may
be used to confirm that HADH mRNA expression is reduced in cells
treated with the nucleic acid modulator (e.g., Current Protocols in
Molecular Biology (1994) Ausubel FM et al., eds., John Wiley &
Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999)
26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H
and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Protein
expression may also be monitored. Proteins are most commonly
detected with specific antibodies or antisera directed against
either the HADH protein or specific peptides. A variety of means
including Western blotting, ELISA, or in situ detection, are
available (Harlow E and Lane D, 1988 and 1999, supra).
[0090] Secondary Assays
[0091] Secondary assays may be used to further assess the activity
of HADH-modulating agent identified by any of the above methods to
confirm that the modulating agent affects HADH in a manner relevant
to the p21 pathway. As used herein, HADH-modulating agents
encompass candidate clinical compounds or other agents derived from
previously identified modulating agent. Secondary assays can also
be used to test the activity of a modulating agent on a particular
genetic or biochemical pathway or to test the specificity of the
modulating agent's interaction with HADH.
[0092] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express HADH) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate HADH-modulating agent results
in changes in the p21 pathway in comparison to untreated (or mock-
or placebo-treated) cells or animals. Certain assays use
"sensitized genetic backgrounds", which, as used herein, describe
cells or animals engineered for altered expression of genes in the
p21 or interacting pathways.
[0093] Cell-Based Assays
[0094] Cell based assays may use a variety of a cell line known to
have defective p21 function such as HCT116 colon cancer cells
available from American Type Culture Collection (ATCC), Manassas,
Va.). Cell based assays may detect endogenous p21 pathway activity
or may rely on recombinant expression of p21 pathway components.
Any of the aforementioned assays may be used in this cell-based
format. Candidate modulators are typically added to the cell media
but may also be injected into cells or delivered by any other
efficacious means.
[0095] Animal Assays
[0096] A variety of non-human animal models of normal or defective
p21 pathway may be used to test candidate HADH modulators. Models
for defective p21 pathway typically use genetically modified
animals that have been engineered to mis-express (e.g.,
over-express or lack expression in) genes involved in the p21
pathway. Assays generally require systemic delivery of the
candidate modulators, such as by oral administration, injection,
etc.
[0097] In a preferred embodiment, p21 pathway activity is assessed
by monitoring neovascularization and angiogenesis. Animal models
with defective and normal p21 are used to test the candidate
modulator's affect on HADH in Matrigel.RTM. assays. Matrigel.RTM.
is an extract of basement membrane proteins, and is composed
primarily of laminin, collagen IV, and heparin sulfate
proteoglycan. It is provided as a sterile liquid at 4.degree. C.,
but rapidly forms a solid gel at 37.degree. C. Liquid Matrigel.RTM.
is mixed with various angiogenic agents, such as bFGF and VEGF, or
with human tumor cells which over-express the HADH. The mixture is
then injected subcutaneously(SC) into female athymic nude mice
(Taconic, Germantown, N.Y.) to support an intense vascular
response. Mice with Matrigel.RTM. pellets may be dosed via oral
(PO), intraperitoneal (IP), or intravenous (IV) routes with the
candidate modulator. Mice are euthanized 5-12 days post-injection,
and the Matrigel.RTM. pellet is harvested for hemoglobin analysis
(Sigma plasma hemoglobin kit). Hemoglobin content of the gel is
found to correlate the degree of neovascularization in the gel.
[0098] In another preferred embodiment, the effect of the candidate
modulator on HADH is assessed via tumorigenicity assays. In one
example, xenograft human tumors are implanted SC into female
athymic mice, 6-7 week old, as single cell suspensions either from
a pre-existing tumor or from in vitro culture. The tumors which
express the HADH endogenously are injected in the flank,
1.times.10.sup.5 to 1.times.10.sup.7 cells per mouse in a volume of
100 .mu.L using a 27 gauge needle. Mice are then ear tagged and
tumors are measured twice weekly. Candidate modulator treatment is
initiated on the day the mean tumor weight reaches 100 mg.
Candidate modulator is delivered IV, SC, IP, or PO by bolus
administration. Depending upon the pharmacokinetics of each unique
candidate modulator, dosing can be performed multiple times per
day. The tumor weight is assessed by measuring perpendicular
diameters with a caliper and calculated by multiplying the
measurements of diameters in two dimensions. At the end of the
experiment, the excised tumors maybe utilized for biomarker
identification or further analyses. For immunohistochemistry
staining, xenograft tumors are fixed in 4% paraformaldehyde, 0.1M
phosphate, pH 7.2, for 6 hours at 4.degree. C., immersed in 30%
sucrose in PBS, and rapidly frozen in isopentane cooled with liquid
nitrogen.
[0099] Diagnostic and Therapeutic Uses
[0100] Specific HADH-modulating agents are useful in a variety of
diagnostic and therapeutic applications where disease or disease
prognosis is related to defects in the p21 pathway, such as
angiogenic, apoptotic, or cell proliferation disorders.
Accordingly, the invention also provides methods for modulating the
p21 pathway in a cell, preferably a cell pre-determined to have
defective p21 function, comprising the step of administering an
agent to the cell that specifically modulates HADH activity.
Preferably, the modulating agent produces a detectable phenotypic
change in the cell indicating that the p21 function is restored,
i.e., for example, the cell undergoes normal proliferation or
progression through the cell cycle.
[0101] The discovery that HADH is implicated in p21 pathway
provides for a variety of methods that can be employed for the
diagnostic and prognostic evaluation of diseases and disorders
involving defects in the p21 pathway and for the identification of
subjects having a predisposition to such diseases and
disorders.
[0102] Various expression analysis methods can be used to diagnose
whether HADH expression occurs in a particular sample, including
Northern blotting, slot blotting, ribonuclease protection,
quantitative RT-PCR, and microarray analysis. (e.g., Current
Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John
Wiley & Sons, Inc., chapter 4; Freeman W M et al.,
Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001,
33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,
12:41-47). Tissues having a disease or disorder implicating
defective p21 signaling that express an HADH, are identified as
amenable to treatment with an HADH modulating agent. In a preferred
application, the p21 defective tissue overexpresses an HADH
relative to normal tissue. For example, a Northern blot analysis of
mRNA from tumor and normal cell lines, or from tumor and matching
normal tissue samples from the same patient, using full or partial
HADH cDNA sequences as probes, can determine whether particular
tumors express or overexpress HADH. Alternatively, the TaqMan.RTM.
is used for quantitative RT-PCR analysis of HADH expression in cell
lines, normal tissues and tumor samples (PE Applied
Biosystems).
[0103] Various other diagnostic methods may be performed, for
example, utilizing reagents such as the HADH oligonucleotides, and
antibodies directed against an HADH, as described above for: (1)
the detection of the presence of HADH gene mutations, or the
detection of either over- or under-expression of HADH mRNA relative
to the non-disorder state; (2) the detection of either an over- or
an under-abundance of HADH gene product relative to the
non-disorder state; and (3) the detection of perturbations or
abnormalities in the signal transduction pathway mediated by
HADH.
[0104] Thus, in a specific embodiment, the invention is drawn to a
method for diagnosing a disease in a patient, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for HADH expression; c)
comparing results from step (b) with a control; and d) determining
whether step (c) indicates a likelihood of disease. Preferably, the
disease is cancer, most preferably colon or ovarian cancer. The
probe may be either DNA or protein, including an antibody.
EXAMPLES
[0105] The following experimental section and examples are offered
by way of illustration and not by way of limitation.
[0106] I: Drosophila p21 Screen
[0107] An overexpression screen was carried out in Drosophila to
identify genes that interact with the cyclin dependent kinase
inhibitor, p21 (Bourne H R, et al., Nature (1990)
348(6297):125-132; Marshall CJ, Trends Genet (1991) 7(3):91-95).
Expression of the p21 gene in the eye causes deterioration of
normal eye morphology. Modifiers of the eye phenotype were
identified as members of the p21 pathway. CG 15771 was a suppressor
of the small eye defect.
[0108] BLAST analysis (Altschul et al., supra) was employed to
identify Targets from Drosophila modifiers. For example,
arepresentative sequence from HADH (GI# 4902680, SEQ ID NO:4)
shares 34% amino acid identity with the Drosophila CG 15771.
[0109] Various domains, signals, and functional subunits in
proteins were analyzed using the PSORT (Nakai K., and Horton P.,
Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting
signals and prediction of subcellular localization, Adv. Protein
Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids
Res, 1999, 27:260-2; http://pfam.wustl.edu), SMART (Ponting C P, et
al., SMART: identification and annotation of domains from signaling
and extracellular protein sequences. Nucleic Acids Res. 1999 Jan.
1;27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne,
and Anders Krogh: A hidden Markov model for predicting
transmembrane helices in protein sequences. In Proc. of Sixth Int.
Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J.
Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen Menlo Park, Calif.: AAAI Press, 1998), and clust (Remm M,
and Sonnhammer E. Classification of transmembrane protein families
in the Caenorhabditis elegans genome and identification of human
orthologs. Genome Res. 2000 November; 10(11): 1679-89) programs.
For example, the hydrolase domain of HADH from GI# 4902680 (SEQ ID
NO:4) is located at approximately amino acid residues 9-212 (PFAM
00702).
[0110] Detailed functional analysis of SEQ ID NO:5 indicated that
HADH appears to be a member of the family of the HADH
aspartyl-phosphate utilizing phosphohydrolases/phosphotransferases.
We used Threading algorithm (Proceryon, NY) to identify structure
family relationship of HADH. Threading alignment identified several
key residues for members of this family: D12, T16, T131, N132,
K164, D189, T193, D194. In SEQ ID NO:5, D12 is highly conserved,
and is the site of phosphorylation; T16 is somewhat variable, and
appears to impact rate of autohydrolysis of the D-phosphate; T131
likely involved in coordination of phosphate; K164 likely involved
in activating the water molecule that hydrolyzes the acyl
intermediate, and/or involved in coordination of oxygen in
acyl-phosphate/stabilization of phosphorylated state; D189,D194
likely coordinate Mg or other metal cation. (Mg or other metal
cations are not conserved in epoxide hydrolases and dehalogenases,
but are conserved and required for the activity of
phosphohydrolases/phosphotransferases).
[0111] II. High-Throughput In Vitro Fluorescence Polarization
Assay
[0112] Fluorescently-labeled HADH peptide/substrate are added to
each well of a 96-well microtiter plate, along with a test agent in
a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH
7.6). Changes in fluorescence polarization, determined by using a
Fluorolite FPM-2 Fluorescence Polarization Microtiter System
(Dynatech Laboratories, Inc), relative to control values indicates
the test compound is a candidate modifier of HADH activity.
[0113] III. High-Throughput In Vitro Binding Assay.
[0114] .sup.33P-labeled HADH peptide is added in an assay buffer
(100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl.sub.2, 1% glycerol, 0.5%
NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of
protease inhibitors) along with a test agent to the wells of a
Neutralite-avidin coated assay plate and incubated at 25.degree. C.
for 1 hour. Biotinylated substrate is then added to each well and
incubated for 1 hour. Reactions are stopped by washing with PBS,
and counted in a scintillation counter. Test agents that cause a
difference in activity relative to control without test agent are
identified as candidate p21 modulating agents.
[0115] IV. Immunoprecipitations and Immunoblotting
[0116] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the HADH
proteins are plated on 10-cm dishes and transfected on the
following day with expression constructs. The total amount of DNA
is kept constant in each transfection by adding empty vector. After
24 h, cells are collected, washed once with phosphate-buffered
saline and lysed for 20 min on ice in 1 ml of lysis buffer
containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20
mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, protease inhibitors (complete,
Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris
is removed by centrifugation twice at 15,000.times.g for 15 min.
The cell lysate is incubated with 25 .mu.l of M2 beads (Sigma) for
2 h at 4.degree. C. with gentle rocking.
[0117] After extensive washing with lysis buffer, proteins bound to
the beads are solubilized by boiling in SDS sample buffer,
fractionated by SDS-polyacrylamide gel electrophoresis, transferred
to polyvinylidene difluoride membrane and blotted with the
indicated antibodies. The reactive bands are visualized with
horseradish peroxidase coupled to the appropriate secondary
antibodies and the enhanced chemiluminescence (ECL) Western
blotting detection system (Amersham Pharmacia Biotech).
[0118] V. Expression Analysis
[0119] All cell lines used in the following experiments are NCI
(National Cancer Institute) lines, and are available from ATCC
(American Type Culture Collection, Manassas, Va. 20110-2209).
Normal and tumor tissues were obtained from Impath, UC Davis,
Clontech, Stratagene, and Ambion.
[0120] TaqMan analysis was used to assess expression levels of the
disclosed genes in various samples.
[0121] RNA was extracted from each tissue sample using Qiagen
(Valencia, Calif.) RNeasy kits, following manufacturer's protocols,
to a final concentration of 50 ng/.mu.l. Single stranded cDNA was
then synthesized by reverse transcribing the RNA samples using
random hexamers and 500 ng of total RNA per reaction, following
protocol 4304965 of Applied Biosystems (Foster City, Calif.,
http://www.appliedbiosystems.com/).
[0122] Primers for expression analysis using TaqMan assay (Applied
Biosystems, Foster City, Calif.) were prepared according to the
TaqMan protocols, and the following criteria:
[0123] a) primer pairs were designed to span introns to eliminate
genomic contamination, and
[0124] b) each primer pair produced only one product.
[0125] Taqman reactions were carried out following manufacturer's
protocols, in 25 .mu.l total volume for 96-well plates and 10 .mu.l
total volume for 384-well plates, using 300 nM primer and 250 nM
probe, and approximately 25 ng of cDNA. The standard curve for
result analysis was prepared using a universal pool of human cDNA
samples, which is a mixture of cDNAs from a wide variety of tissues
so that the chance that a target will be present in appreciable
amounts is good. The raw data were normalized using 18S rRNA
(universally expressed in all tissues and cells).
[0126] For each expression analysis, tumor tissue samples were
compared with matched normal tissues from the same patient. A gene
was considered overexpressed in a tumor when the level of
expression of the gene was 2 fold or higher in the tumor compared
with its matched normal sample. In cases where normal tissue was
not available, a universal pool of cDNA samples was used instead.
In these cases, a gene was considered overexpressed in a tumor
sample when the difference of expression levels between a tumor
sample and the average of all normal samples from the same tissue
type was greater than 2 times the standard deviation of all normal
samples (i.e., Tumor--average(all normal samples)>2.times.STDEV-
(all normal samples)).
[0127] HADH GI#11968366 (SEQ ID NO:1) was overexpressed in 10 of 30
matched colon tumors, and 2 of 7 matched ovarian tumors. A
modulator identified by an assay described herein can be further
validated for therapeutic effect by administration to a tumor in
which the gene is overexpressed. A decrease in tumor growth
confirms therapeutic utility of the modulator. Prior to treating a
patient with the modulator, the likelihood that the patient will
respond to treatment can be diagnosed by obtaining a tumor sample
from the patient, and assaying for expression of the gene targeted
by the modulator. The expression data for the gene(s) can also be
used as a diagnostic marker for disease progression. The assay can
be performed by expression analysis as described above, by antibody
directed to the gene target, or by any other available detection
method.
Sequence CWU 1
1
4 1 2352 DNA Homo sapiens 1 aggctacggt tcgcgccagc ggccggcgct
atggggctga gccgcgtgcg ggcggttttc 60 tttgacttgg acaacactct
catcgacacg gccggggcga gcaggagagg catgttggag 120 gtgataaaac
tcttacaatc aaaataccat tataaagaag aggctgaaat catctgtgat 180
aaagttcaag ttaaactcag caaggaatgt tttcatcctt acaatacatg cattactgat
240 ttaaggactt cacattggga agaagcaatc caggaaacaa aaggtggtgc
agccaataga 300 aaattggctg aagaatgtta tttcctttgg aaatctacac
gtttacagca tatgacacta 360 gcagaagacg tcaaagccat gcttactgaa
cttcgaaagg aggtccgcct acttctatta 420 acgaatgggg acagacagac
ccagagggag aagattgagg cttgtgcctg tcagtcctat 480 tttgacgctg
ttgttgtagg tggagagcag agagaggaga aaccagcacc gtccatattt 540
tattactgct gcaatcttct cggagtacaa cctggggact gtgtgatggt cggtgacaca
600 ttagaaaccg acatccaagg aggcctcaat gcaggattga aagcaacagt
ctggatcaat 660 aaaaatggaa tagtgccact gaagtcctcc ccagttccgc
attacatggt ttcttctgtg 720 ctagagttac ctgctctctt acaaagtata
gactgcaaag tcagtatgtc cacttaaagc 780 acataaaagg gcatgattat
gaatgttaga atcaatttgc tgagtatgaa ataagaaaag 840 ttagggcact
ccacttatga taatccagct ctaagaataa ttttacttat gatttaatgg 900
ccaatatttt gaaggtcttc ccaaccctat tgcttctaag ttgtaacaac caaccattga
960 gtggtactta tatctgaaaa ttcagattgc atgaattcag gtcagtagta
tagcccagaa 1020 aatttaagga aatatattat ttgttagtct gtatctggag
ctttttaaaa ttatgttatt 1080 aatcttttag tatcttggct gcataatgcc
aagcaggatt gctttacaca tggatgcaca 1140 aatgtaaggt ttatcttctg
gcttaaaaat agatattttt aaaaaataga ttttctaaaa 1200 cacagattta
tgaaagcaag tgaatctggt taatatgaaa taagtactaa gtcacatgca 1260
aatcaaggta ttatatagtg aaattatttt gcatattttg aaaacataaa ccatagtttt
1320 tgcctacttt ggatgtatac tttcttttat gaacctgatt tttctgtatg
acattttttt 1380 ttttttcaga gggcagggag caatttttct atggcatgtg
acagattcct ccagttagaa 1440 aaagctgtta aaatcaacac atggtgctct
tttaccgtga cattttctca cctgtgcaca 1500 gtgagccgat agcttccttt
tagtcttcac ctctcaagga aatgttttta ctgtcttttc 1560 ccagacacac
agtggggttg agggagctag gctgttttgc tagagataat tgcaaggcac 1620
gtggcactaa aagtcatttt tcttctgtgg atccataaga ggaacatttc ctcagtgtag
1680 cctaacaatg cagcccccaa tctgttcctt tttttttttg aaatgggatc
tctgtcgccc 1740 aggttggagt gccatggcac catctcggct cactgcaacc
tctgcctcct gagctcaagt 1800 gatcctccca cctcagcctc ccaagggtgt
gtgtgactac aagtacacac taccacgccc 1860 agctaatgtg tttttttgta
gagatgggat tttgccatgt tgcccaggct ggtctcgaat 1920 tcctggattc
aagtgatcct cccacctcag cctcctaaag tcctaggatt ataggcatga 1980
gccactgtgc ctggccctct catctgatag aaaattagat tttgctatga gccatttcct
2040 gagggccaat ttaatactcg tgtgactctt cttagagtta ccatctgcct
taaatttcct 2100 ctgtttttca cattcttgga aatatatcat tgttttgcaa
atttctatat ctaattcagg 2160 gtttaccagg agcttaataa ttaatggcta
catagcaagg catcgtcttg gaaccggaga 2220 attttctcta gactattagg
ctagacagtc tcatgattat actaaccaaa cctggagtaa 2280 agtggttgaa
aaaaaagaaa gtataaaggg gcttattaaa gtggttaata aatatgaaaa 2340
aaaaaaaaaa aa 2352 2 892 DNA Homo sapiens 2 ccacgcgtcc ggccggcgct
atggggctga gccgcgtgcg ggcggttttc tttgacttgg 60 acaacactct
catcgacacg gccggggcga gcaggagagg catgttggag gtgataaaac 120
tcttacaatc aaaataccat tataaagaag aggctgaaat catctgtgat aaagttcaag
180 ttaaactcag caaggaatgt tttcatcctt acaatacatg cattactgat
ttaaggactt 240 cacattggga agaagcaatc caggaaacaa aaggtggtgc
agccaataga aaattggctg 300 aagaatgtta tttcctttgg aaatctacac
gtttacagca tatgacacta gcagaagacg 360 tcaaagccat gcttactgaa
cttcgaaagg aggtccgcct acttctatta acgaatgggg 420 acagacagac
ccagagggag aagattgagg cttgtgcctg tcagtcctat tttgacgctg 480
ttgttgtagg tggagagcag agagaggaga aaccagcacc gtccatattt tattactgct
540 gcaatcttct cggagtacaa cctggggact gtgtgatggt cggtgacaca
ttagaaaccg 600 acatccaagg aggcctcaat gcaggattga aagcaacagt
ctggatcaat aaaaatggaa 660 tagtgccact gaagttctcc ccagttccgc
attacatggc ttcttctgtg ctagagttac 720 ctgctctctt acaaagcata
gactgcaaag tcagtatgtc cacttaaagc acataacaag 780 ggcatgatta
tgaatgttaa aatcaatttt gcctgagtat gcaatacaaa aagttagggc 840
actccccttt atgataatcc agcttctaag aatcattttc acttaatgat tt 892 3 212
PRT Homo sapiens 3 Ala Gly Ala Met Gly Leu Ser Arg Val Arg Ala Val
Phe Phe Asp Leu 1 5 10 15 Asp Asn Thr Leu Ile Asp Thr Ala Gly Ala
Ser Arg Arg Gly Met Leu 20 25 30 Glu Val Ile Lys Leu Leu Gln Ser
Lys Tyr His Tyr Lys Glu Glu Ala 35 40 45 Glu Ile Ile Cys Asp Lys
Val Gln Val Lys Leu Ser Lys Glu Cys Phe 50 55 60 His Pro Tyr Asn
Thr Cys Ile Thr Asp Leu Arg Thr Ser His Trp Glu 65 70 75 80 Glu Ala
Ile Gln Glu Thr Lys Gly Gly Ala Ala Asn Arg Lys Leu Ala 85 90 95
Glu Glu Cys Tyr Phe Leu Trp Lys Ser Thr Arg Leu Gln His Met Thr 100
105 110 Leu Ala Glu Asp Val Lys Ala Met Leu Thr Glu Leu Arg Lys Glu
Val 115 120 125 Arg Leu Leu Leu Leu Thr Asn Gly Asp Arg Gln Thr Gln
Arg Glu Lys 130 135 140 Ile Glu Ala Cys Ala Cys Gln Ser Tyr Phe Asp
Ala Val Val Val Gly 145 150 155 160 Gly Glu Gln Arg Glu Glu Lys Pro
Ala Pro Ser Ile Phe Tyr Tyr Cys 165 170 175 Cys Asn Leu Leu Gly Val
Gln Pro Gly Asp Cys Val Met Val Gly Asp 180 185 190 Thr Leu Glu Thr
Asp Ile Gln Gly Gly Leu Asn Ala Gly Leu Lys Ala 195 200 205 Thr Val
Trp Ile 210 4 248 PRT Homo sapiens 4 Met Gly Leu Ser Arg Val Arg
Ala Val Phe Phe Asp Leu Asp Asn Thr 1 5 10 15 Leu Ile Asp Thr Ala
Gly Ala Ser Arg Arg Gly Met Leu Glu Val Ile 20 25 30 Lys Leu Leu
Gln Ser Lys Tyr His Tyr Lys Glu Glu Ala Glu Ile Ile 35 40 45 Cys
Asp Lys Val Gln Val Lys Leu Ser Lys Glu Cys Phe His Pro Tyr 50 55
60 Asn Thr Cys Ile Thr Asp Leu Arg Thr Ser His Trp Glu Glu Ala Ile
65 70 75 80 Gln Glu Thr Lys Gly Gly Ala Ala Asn Arg Lys Leu Ala Glu
Glu Cys 85 90 95 Tyr Phe Leu Trp Lys Ser Thr Arg Leu Gln His Met
Thr Leu Ala Glu 100 105 110 Asp Val Lys Ala Met Leu Thr Glu Leu Arg
Lys Glu Val Arg Leu Leu 115 120 125 Leu Leu Thr Asn Gly Asp Arg Gln
Thr Gln Arg Glu Lys Ile Glu Ala 130 135 140 Cys Ala Cys Gln Ser Tyr
Phe Asp Ala Val Val Val Gly Gly Glu Gln 145 150 155 160 Arg Glu Glu
Lys Pro Ala Pro Ser Ile Phe Tyr Tyr Cys Cys Asn Leu 165 170 175 Leu
Gly Val Gln Pro Gly Asp Cys Val Met Val Gly Asp Thr Leu Glu 180 185
190 Thr Asp Ile Gln Gly Gly Leu Asn Ala Gly Leu Lys Ala Thr Val Trp
195 200 205 Ile Asn Lys Asn Gly Ile Val Pro Leu Lys Ser Ser Pro Val
Pro His 210 215 220 Tyr Met Val Ser Ser Val Leu Glu Leu Pro Ala Leu
Leu Gln Ser Ile 225 230 235 240 Asp Cys Lys Val Ser Met Ser Thr
245
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References