Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF SCREENING FOR THERAPEUTIC AGENTS USING
NOVEL APOPTOSIS-MODULATING PROTEINS
TECHNICAL FIELD
This invention relates to methods of screening for therapeutic agents using
novel proteins with apoptosis-modulating activity.
BACKGROUND ART
Apoptosis is a normal physiologic process that leads to individual cell
death. This process of programmed cell death is involved in a variety of
normal
and pathogenic biological events and can be induced by a number of unrelated
stimuli. Changes in the biological regulation of apoptosis also occur during
aging
and are responsible for many of the conditions and diseases related to aging.
Recent studies of apoptosis have implied that a common metabolic pathway
leading to cell death may be initiated by a wide variety of signals, including
hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing
radiation and infection by human immunodeficiency virus (HIV). Wyllie (1980)
Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun.
118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al.
(1988) Biochem. Biophys. Res. Commun. 155:324-33 1; Kruman et al. (1991) J.
Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunology Today
12:102; and Sheppard and Ascher (1992) J. AIDS 5:143. Agents that modulate
the biological control of apoptosis thus have therapeutic utility in a wide
variety of
conditions.
Apoptotic cell death is characterized by cellular shrinkage, chromatin
condensation, cytoplasmic blebbing, increased membrane permeability and
interchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; and
Cohen and Duke (1992) Ann. Rev. Immunol. 10:267. The blebs, small,
membrane-encapsulated spheres that pinch off of the surface of apoptotic
cells,
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may continue to produce superoxide radicals which damage surrounding cell
tissue and may be involved in inflammatory processes.
The Bcl-2 gene was discovered at the common chromosomal translocation
site t(14:18) in follicular lymphomas and results in aberrant over-expression
of
bcl-2. Tsujimoto et al. (1984) Science 226:1097-1099; and Cleary et al. (1986)
Cell 47:19-28. The normal function of bcl-2 is the prevention of apoptosis;
unregulated expression of bcl-2 in B cells is thought to lead to increased
numbers
of proliferating B cells which may be a critical factor in the development of
lymphoma. McDonnell and Korsmeyer (1991) Nature 349:254-256; and, for
review see, Edgington (1993) Bio/Tech. 11:787-792. Bcl-2 is also capable of
blocking of y irradiation-induced cell death. Sentman et al. (1991) Cell
67:879-
888; and Strassen (1991) Cell 67:889-899. It is now known that bcl-2 inhibits
most types of apoptotic cell death and is thought to function by regulating an
antioxidant pathway at sites of free radical generation. Hockenbery et al.
(1993)
Cell 75:241-25 1.
Apoptosis, a normal cellular event, can also be induced by pathological
conditions and a variety of injuries. Apoptosis is involved in a wide variety
of
conditions including, but not limited to: cardiovascular disease; cancer
regression;
immunoregulation; viral diseases; anemia; neurological disorders;
gastrointestinal
disorders such as diarrhea and dysentery; diabetes; hair loss; rejection of
organ
transplants; prostate hypertrophy; obesity; ocular disorders; stress; and
aging.
Bcl-2 belongs to a family of proteins of which some have been cloned and
sequenced. Williams and Smith (1993) Cell 74:777-779. Various Bcl-2 members
have the ability to associate with one another as heterodimers. Oltvai et al.
(1993)
Cell 74:609-619; and Sato et al. (1994) Proc. Natl. Acad. Sci. USA 91:9238-
9242.
Additionally, BHRF1 displays a 25% sequence identity to Bcl-2 (Cleary et al.
(1986) Cell 47:19-28) and has been shown by gene transfer studies to protect B
=
cells from apoptosis. Henderson et al. (1993) Proc. Natl. Acad. Sci. USA =
90:8479-8483.
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The herpesvirus family of viruses typically produce latent and recurrent
infections. Herpesvirus genomes are composed of sequences with a short and a
long region. Herpesvirus particles have a diameter from 180 nm to 200 nm.
Many particles do not contain envelopes. Typically, the DNA is wrapped around
.5 an associated protein. The herpesvirus has a tendency to persist in a
quiescent
state for irregular periods of time.
All references cited herein, both supra and infra, are hereby incorporated
by reference herein.
SUMMARY OF THE INVENTION
Methods of screening for pharmaceutical agents that stimulate, as well as
pharmaceutical agents that inhibit Bak and Bak-2 protein activity levels are
provided. The methods include combining a Bak protein and a viral protein
under
conditions in which they interact to form a test sample, exposing the test
sample to
a potential therapeutic agent and monitoring the interaction of the proteins.
Potential therapeutic agents which disrupt the interaction compared to control
test
samples to which no agent has been added are selected for further study.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the Bak cDNA nucleotide sequence and amino acid
sequence encoded thereby.
Figure 2 shows the sequence of the Bak-2 cDNA and flanking sequences
and the corresponding predicted amino acid sequence of the Bak-2 protein.
Figure 3 shows the interactions of Bak and Flag-Bak (F-Bak) fusion
proteins with Epstein-Barr virus BHRF-1 protein. In column A, lane 1 depicts
the
results obtained from in vitro co-translated proteins F-Bak/BHRF-1 and lane 2
= 25 depicts F-Bak/BHRF-1 proteins bound to anti-FLAG agarose. In column B,
the
lanes are the same with the exception that the Bak protein is Bak-2.
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DISCLOSURE OF THE INVENTION
The present invention provides methods of screening for potential anti-
viral therapeutic agents. The proteins encoded by nucleotide sequences
encoding
the novel bcl-2 homologs, Bak and Bak-2 proteins have been found to interact
with the Epstein Barr Virus (EBV) protein BHRF1 indicating that Bak proteins
contribute to the pathogenicity of the disease. BHRF1 is an EBV early lytic
cycle
protein. Pearson et al. (1987) Virol. 160:151-161. The invention encompasses
methods containing the steps of exposing the Bak proteins and viral proteins,
or
functional portions thereof, to potential therapeutic agents and monitoring
the
interaction of the proteins. The invention further utilizes recombinant cells
and
transgenic animals expressing the cloned Bak or Bak-2 genes.
The cloning and analysis of Bak genes and proteins are described in detail
in commonly owned WO application PCT/US94/13930. Bak genes and proteins
are also described in Kiefer et al. (1995) Nature 374:736. The nucleotide and
predicted amino acid residue sequences of Bak protein are shown in Figure 1;
and
those of Bak-2 are shown in Figure 2. Bak mRNA has been detected in a variety
of human organs and tissues by Northern blot analysis. These organs include
liver; heart; skeletal muscle; lung; kidney; and pancreas.
These references also disclose that the Bak proteins are capable of
modulating apoptosis. In a lymphoblastoid cell line, expression of Bak protein
was shown to decrease Fas-mediated apoptosis. In a mouse progenitor B cell
line,
FL5.12, Bak-2 protein and a derivative of Bak protein decrease IL-3-induced
apoptosis whereas Bak protein increased apoptosis. Thus, depending on the cell
type, the derivative of Bak protein, and the method of induction of apoptosis,
apoptosis can be modulated in a highly specific manner by controlling the
concentration of Bak proteins.
As used herein, the term "Bak gene(s)" refers to the nucleic acid molecules
described herein and in PCT/US94/13930, "the Bak protein(s)" refers to the =
proteins encoded thereby. The nucleotides include, but are not limited to, the
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cDNA and complementary DNA, genome-derived DNA and synthetic or semi-
synthetic DNA or RNA. The nucleotide sequence of the Bak cDNA with the
location of restriction endonuclease sites is shown in Figure 1.
The nucleotide sequence of Bak-2 cDNA, along with the predicted amino
5 acid sequence of Bak-2 protein and the locations of restriction endonuclease
recognition sites, is given in Figure 2. The Bak gene is on human chromosome 6
and the Bak-2 gene is on human chromosome 20. There is also a member of the
family, Bak-3, which is on human chromosome 11. Bak-3 appears to be a
pseudogene. Fluorescence in situ hybridization (FISH) indicated an approximate
location of the Bak gene to be at 6p2l-23.
The invention includes the use of modified Bak DNA sequences such as
deletions, substitutions and additions particularly in the non-coding regions
of
genomic DNA. Such changes are useful to facilitate cloning and modify gene
expression. Any DNA which encodes a portion of a Bak protein sufficient to
bind
to BHRFl or any other suitable viral protein is suitable for use herein. As
described below, various fusion proteins are suitable for use herein.
Various substitutions can be made within the coding region that either do
not alter the amino acid residues encoded or result in conservatively
substituted
amino acid residues. Nucleotide substitutions that do not alter the amino acid
residues encoded are useful for optimizing gene expression in different
systems.
Suitable substitutions are known to those of skill in the art and are made,
for
instance, to reflect preferred codon usage in the particular expression
systems.
The invention encompasses the use of functionally equivalent variants and
derivatives of Bak genes which may enhance, decrease or not significantly
affect
the properties of Bak proteins. For instance, changes in the DNA sequence that
do
not change the encoded amino acid sequence, as well as those that result in
conservative substitutions of amino acid residues, one or a few amino acid
deletions or additions, and substitution of amino acid residues by amino acid
analogs are those which will not significantly affect its properties.
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Amino acid residues which can be conservatively substituted for one
another include but are not limited to: glycine/alanine;
valine/isoleucine/leucine;
asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine;
lysine/arginine; and phenylalanine/tyrosine. Any conservative amino acid
substitution which does not significantly affect the properties of Bak
proteins is
encompassed by the present invention.
Techniques for nucleic acid manipulation useful for the practice of the
present invention are described in a variety of references, including, but not
limited to, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, eds.
Sambrook et al., Cold Spring Harbor Laboratory Press (1989); and Current
Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and
Wiley-Interscience: New York (1987) and periodic updates.
The coding regions of Bak genes can also be ligated into expression
vectors capable of stably integrating into other cell types including but not
limited
to cardiomyocytes, neural cell lines such as GTI-7 and TNF sensitive cells
such as
the human colon adenocarcinoma cell line HT29 so as to provide a variety of
assay systems to monitor the regulation of apoptosis by Bak proteins.
As used herein, "BHRF1" or "viral proteins" encompasses the full length
EBV protein and portions or derivations thereof sufficient to bind to Bak
proteins
or portions or derivatives thereof. Such proteins include, but are not limited
to,
homologous proteins expressed by any virus, particularly various forms of
herpes
and herpes-like viruses, such as cytomegalovirus and varicella zoster.
The interaction between a Bak protein and viral protein such as BHRF-1
can be produced by adding purified proteins together. Preferably, however, the
proteins are cotranscribed and translated under conditions that allow protein-
protein interactions. Co-translation can be performed in vitro or in vivo in
whole
cells expressing native or recombinant Bak proteins and viral proteins. Any
suitable recombinant expression vectors may be used. The Bak proteins can also
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be separately translated and then combined under conditions that allow for
protein-protein interactions.
Methods of monitoring protein interactions are known in the art, any
method is suitable for use herein. Preferably, co-precipitation is used. The
ability
of an antibody to precipitate one of the proteins or an immunological tag
fused
thereto is used to immunoprecipitate the protein and the immunoprecipitate is
monitored for the presence of both proteins. Methods of co-precipitation are
known in the art and are described in the examples below. Any other method in
the art is suitable for use herein, including, but not limited to, protein
interactive
trapping, such as GST fusion protein immobilization on glutathione columns
and,
ELISA. Immunological tags are often incorporated into fusion proteins and
including, for instance, FLAG, hemagglutinin and glutathione-S transferase.
Purification or isolation of Bak proteins expressed either by the
recombinant DNA or from biological sources such as tissues can be accomplished
by any method known in the art. Protein purification methods are known in the
art. Generally, substantially purified proteins are those which are free of
other,
contaminating cellular substances, particularly proteins. Preferably, the
purified
Bak proteins are more than eighty percent pure and, most preferably, more than
ninety-five percent pure. For clinical use as described below, the Bak
proteins are
preferably highly purified, at least about ninety-nine percent pure, and free
of
pyrogens and other contaminants.
Suitable methods of protein purification are known in the art and include,
but are not limited to, affinity chromatography, immunoaffinity
chromatography,
size exclusion chromatography, HPLC and FPLC. Any purification scheme that
does not result in substantial degradation of the protein is suitable for use
herein.
As used herein, "Bak proteins" includes funetionally equivalent variants
thereof which do not significantly affect their properties and variants which
retain
the same overall amino acid sequence but which have enhanced or decreased
activity. For instance, conservative substitutions of amino acid residues, one
or a
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few amino acid deletions or additions, and substitution of amino acid residues
by
amino acid analogs are within the scope of the invention.
Amino acid residues which can be conservatively substituted for one
another include but are not limited to: glycine/alanine;
valine/isoleucine/leucine;
asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine;
lysine/arginine; and phenylalanine/tyrosine. Any conservative amino acid
substitution which does not significantly affect the properties of Bak
proteins is
encompassed by the present invention.
Suitable antibodies for use herein are generated by using the Bak proteins
as an antigen or, preferably, peptides encompassing the Bak protein regions
that
lack substantial homology to the other gene products of the bcl family.
Antibodies to the viral proteins are also suitable for use herein. Methods of
detecting proteins using antibodies and of generating antibodies using
proteins or
synthetic peptides are known in the art and are not described in detail
herein.
Screening for therapeutically effective agents is done by exposing the Bak
protein and the viral protein to such agents which may directly or indirectly
affect
the interaction between a Bak protein and a viral protein. Suitable potential
therapeutic agents include, but are not limited to, any pharmaceutical agent
such
as cytokines, small molecule drugs, cell-permeable small molecule drugs,
hormones, combinations of interleukins, lectins and other stimulating agents,
e.g.,
PMA, LPS, bispecific antibodies, peptide mimetics, antisense oligonucleotides
and other agents which modify cellular functions or protein expression.
The proteins are added together or co-expressed, exposed to such agents at
physiologically effective concentrations, and the interaction thereof is
measured
relative to a control not exposed to such agents. Those biological modifiers
which
decrease the interaction between a Bak protein and a viral protein relative to
a
control are selected for further study of their anti-viral activity.
As previously shown, overexpressed Bak proteins protect EBV-
B cells from apoptosis following serum withdrawal or anti-Fas
transformed
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treatment. PCT/US94/13930. These results indicate that a Bak-BHRF1
interaction exists whereby BHFRI not only neutralizes the normally apoptotic
effect of Bak protein, but additionally induces a protective activity.
Alternatively,
propagation of cells transfected with the Bak cDNA might select for cells that
are
expressing high levels of BHRF1 or other EBV encoded anti-apoptotic proteins.
This could lead to an anti-apoptotic response upon subjecting the cells to an
apoptosis signal such as serum withdrawal. Example 2 shows that in vitro
translated Flag-Bak (epitope tagged) and BHRF 1 can be coprecipitated with an
antibody that recognizes the Flag epitope indicating that Bak proteins and
BHRF1
interact directly with one another.
The following examples are provided to illustrate but not limit the present
invention. Unless otherwise specified, all cloning techniques were essentially
as
described by Sambrook et al. (1989) and all reagents were used according to
the
manufacturer's instructions.
Examnle 1
Expression of Recombinant Bak Gene
In order to express the recombinant Bak gene in the baculovirus system,
the Bak cDNA generated as described in PCT/US94/13930 was used to generate a
novel Bak vector, by PCR, using primers from the 3' and 5' flanking regions of
the
gene which contain restriction sites to facilitate cloning. The plasmids were
sequenced by the dideoxy terminator method (Sanger et al., 1977) using
sequencing kits (USB, Sequenase version 2.0) and internal primers. This was to
confirm that no mutations resulted from PCR.
A clone was used to generate recombinant viruses by in vivo homologous
recombination between the overlapping sequences of the plasmid and AcNPV
wild type baculovirus. After 48 hours post-transfection in insect Spodoptera
frugiperda clone 9 (SF9) cells, the recombinant viruses were collected,
identified
by PCR and further purified. Standard procedures for selection, screening and
propagation of recombinant baculovirus were performed in accordance with the
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manufacturer's instructions (Invitrogen). The molecular mass, on sodium
dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), of the protein produced
in the baculovirus system was compared with the predicted molecular mass of
Bak
protein according to the amino-acid sequence.
5 In addition, similar clones can be expressed in any expression system
known in the art including, but not limited to, bacterial, yeast, insect and
mammalian. A suitable yeast intracellular expression system is described by
Barr
et al. (1992) Transgenesis ed. JAH Murray, (Wiley and Sons) pp. 55-79.
The Bak gene coding sequence was excised and introduced into plasmids
10 pCEP7, pREP7 and pcDNA3 (Invitrogen) at compatible restriction enzyme
sites.
pCEP7 was generated by removing the RSV 3'-LTR of pREP7 with XbaI/Asp718,
and substituting the CMV promoter from pCEP4 (Invitrogen). 25 g of each
Bak-containing plasmid was electroporated into the B lymphoblastoid cell line
WIL-2, and stable hygromycin resistant transformants or G418 resistant
transformants (pcDNA3 constructs) expressing Bak were selected.
Examnle 2
Bak proteins interact with Fpstein-Barr Virus encoded BHRF1 protein
BHRF 1 cDNA was amplified by RT-PCR from WI-L2 mRNA using
standard PCR protocol according to the instructions of the manufacturers of
the
PCR kit and thermal cycler (Perkin Elmer Cetus). The Flag-Bak and Flag-Bak-2
cDNAs were generated by RT-PCR as above from Bak and Bak-2/pcDNA3
plasmid templates but included the 24 base Flag encoding sequence 5'-GAC TAC
AAG GAC GAC GAT GAC AAG-3' in the sense primer. This resulted in a
cDNA encoding N-terminal Flag-Bak and Flag-Bak-2 fusion proteins that could
be recognized and purified by the anti-Flag M2 antibody (Kodak-IBI). The
cDNAs were ligated into the pcDNA3 vector which is under the control of the
CMV and T7 RNA polymerase promoter. The Flag-Bak and BHRF1 plasmids or
Flag-Bak-2 and BHRF1 plasmids were then cotranscribed and cotranslated using
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the TnT coupled reticulocyte lysate system according to manufacturer's
instructions (Promega).
Briefly, 0.5-1.0 g of the two circular plasmids were simultaneously
transcribed and translated in 50 L of TnT lysate for 90 minutes at 32 C.
After
translation, 20 L of lysate was mixed with 20 L of 2X PBS plus 40 L of anti-
Flag M2 affinity gel (Kodak) and incubated with gentle rocking overnight at 4
C.
Immunoprecipitates were collected by centrifugation in an Eppendorf microfuge
at 1500 rpm for 15 minutes at 4 C. Pellets were washed 4 times with 1.5 mL PBS
and after the fmal wash were resuspended in 30 L of SDS-PAGE sample buffer.
The samples were then analyzed by SDS-PAGE on a 18% polyacrylamide gel.
Gels were fixed with 10% glacial acetic acid, dried and exposed to X-ray film
overnight at room temperature.
As shown in Figure 3, Flag-Bak and BHRF1 as well as Flag-Bak-2 and
BHRF 1 were efficiently cotranscribed and cotranslated (lanes 1). Clearly, the
anti-Flag M2 antibody effectively coprecipitates Flag-Bak and BHRF1 or Flag-
Bak-2 and BHRF1 (lanes 2). This demonstrates that BHRF1 interacts with both
Bak and Bak-2 proteins in vitro and suggests that such interactions occur in
vivo
resulting in the modulation of apoptosis. Interactions of Bak proteins with
viral
proteins are likely to have evolved to allow viral replication or latency to
proceed
in the absence of apoptotic death of the host cell. Interference, therefore,
in these
interactions represents an important new strategy for the design of novel
antiviral
agents. Similarly, malignant cells derived from transformation by viruses such
as
EBV would also be amenable to diagnosis or therapy with these agents.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be
apparent to those skilled in the art that certain changes and modifications
may be
practiced. Therefore, the description and examples should not be construed as
limiting the scope of the invention, which is delineated by the appended
claims.
