U.S. patent application number 12/158871 was filed with the patent office on 2010-05-13 for inhibitor of the uracil-dna glycosylase enzyme and uses thereof.
This patent application is currently assigned to Consejo Superior de Investigaciones Cientificas. Invention is credited to Alicia Bravo Garcia, Margarita Salas Falgueras, Gemma Serrano De Las Heras.
Application Number | 20100120675 12/158871 |
Document ID | / |
Family ID | 38217718 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100120675 |
Kind Code |
A1 |
Serrano De Las Heras; Gemma ;
et al. |
May 13, 2010 |
INHIBITOR OF THE URACIL-DNA GLYCOSYLASE ENZYME AND USES THEREOF
Abstract
The present invention relates to a protein which has the
capacity to bind to and inhibit the viral uracil DNA glycosylase
(UDG) enzyme and its use as a therapeutic agent; in particular, as
an antiviral agent.
Inventors: |
Serrano De Las Heras; Gemma;
(Madrid, ES) ; Bravo Garcia; Alicia; (Madrid,
ES) ; Salas Falgueras; Margarita; (Madrid,
ES) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
Consejo Superior de Investigaciones
Cientificas
Madrid
ES
|
Family ID: |
38217718 |
Appl. No.: |
12/158871 |
Filed: |
December 1, 2006 |
PCT Filed: |
December 1, 2006 |
PCT NO: |
PCT/ES06/70187 |
371 Date: |
September 12, 2008 |
Current U.S.
Class: |
514/1.1 ;
435/320.1; 435/325; 435/69.1; 514/14.2; 514/9.4; 530/324;
536/23.1 |
Current CPC
Class: |
C12N 2795/10222
20130101; C07K 14/005 20130101; A61P 31/12 20180101; A61K 38/00
20130101 |
Class at
Publication: |
514/12 ; 530/324;
536/23.1; 435/320.1; 435/325; 435/69.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07H 21/04 20060101 C07H021/04; C07K 14/00 20060101
C07K014/00; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12P 21/02 20060101 C12P021/02; A61P 31/12 20060101
A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2005 |
ES |
P200503240 |
Claims
1. A protein comprising amino acid sequence SEQ ID NO: 1, or a
variant or fragment thereof, which has the capacity to inhibit the
uracil DNA glycosylase (UDG) enzyme.
2. An isolated polynucleotide that encodes a protein according to
claim 1.
3. The A polynucleotide of claim 2, comprising nucleotide sequence
SEQ ID NO: 2.
4. A gene construct comprising a polynucleotide according to claim
2.
5. A vector comprising a polynucleotide according to claim 2.
6. A cell comprising a polynucleotide according to claim 2.
7. A method of producing a protein according comprising culturing a
cell that comprises an isolated polynucleotide that encodes a
protein; wherein the protein comprises amino acid sequence SEQ ID
NO: 1, or a variant or fragment thereof; and wherein the protein
has the capacity to inhibit the uracil DNA glycosylase (UDG)
enzyme; and producing the protein.
8. A composition comprising a protein according to claim 1 and an
inert vehicle.
9. A pharmaceutical composition comprising a protein according to
claim 1, and one or more pharmaceutically acceptable
excipients.
10. (canceled)
11. A vector comprising a gene construct according to claim 3.
12. A cell comprising a gene construct according to claim 4.
13. A cell comprising a vector according to claim 5.
14. A cell comprising a vector according to claim 11.
15. The method of claim 7, further comprising the step of
recovering the protein from the culture medium.
16. A method of treating a viral infection, comprising
administering to a subject in need thereof a composition
comprising: a therapeutically effective quantity of a protein
comprising amino acid sequence SEQ ID NO: 1, or a variant or
fragment thereof; and one or more pharmaceutically acceptable
excipients.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a protein that inhibits the viral
uracil DNA glycosylase (UDG) enzyme and the use thereof as a
therapeutic agent, in particular, as an antiviral agent.
BACKGROUND OF THE INVENTION
[0002] Viral infections in persons and animals, especially in
persons, are widely spread and pose numerous problems for
healthcare workers. Pharmaceutical agents capable of effectively
and specifically fighting viruses are very limited in number and,
moreover, generally cause undesirable secondary effects. Viral
infections not only destroy host cells, but also affect the
functioning of various proteins and enzymes. Viral invasion favours
infection by other pathogenic agents, such as other viruses,
bacteria, fungi, etc. Thus, for example, due to the immune loss
which it causes, the human immunodeficiency virus (HIV) opens the
door to other viruses (herpes simplex, cytomegalovirus, hepatitis B
virus) and other pathogenic agents that invade the human body,
creating dangerous situations.
[0003] Despite intense efforts, thus far it has not been possible
to find chemotherapeutic agents which interfere, with an
essentially recognisable success, either at the origin or in terms
of the symptoms, with the pathogenic episodes caused by viral
agents. Therefore, the treatment of viral diseases by
chemotherapeutic agents is still incomplete.
[0004] Antibody conjugates, formed by a conjugated or hybrid
monoclonal antibody and a toxin, have been used to eradicate
specific colonies of target cells, directing them against
"undesired" target cells that carry surface target antigens and
destroying them. The various toxins that have been used by
different researchers may be broadly classified into two groups.
The first group consists of intact toxins, such as intact ricin.
These toxins may not be safely applied in vivo due to their lethal
toxicity. The toxins in the second group are called hemitoxins.
Hemitoxins are single-strand ribosome-inactivating proteins which
act catalytically on eukaryotic ribosomes and inactivate the 60S
subunit, leading to a dose-dependent inhibition of the synthesis of
cell proteins at the peptide elongation level.
[0005] A hemitoxin of interest is the pokeweed antiviral protein
(PAP), which is isolated from Phytolacca americana. For many years,
it has been recognised that PAP has antiviral activity. It has been
demonstrated that PAP blocks the transmission of RNA-containing
viruses in plants. It has also been reported that PAP inhibits the
replication of two RNA-containing animal viruses: poliovirus and
infuenza virus, and that PAP inhibits the multiplication of simple
herpes viruses type I and type II (U.S. Pat. No. 4,672,053).
Although it has been reported that PAP monoclonal antibody
conjugates G3.7/CD7, F13/CD14 and B43/CD19 inhibit the replication
of HIV-1, these conjugates have turned out to be inconsistent in
their capacity to inhibit the replication of the viruses.
[0006] In light of the above, the need remains to provide new
antiviral compounds or drugs. Advantageously, these new antiviral
agents should exhibit an efficacy that is equal to or greater than
that of the antiviral agents disclosed in the state of the art and
should not cause undesirable secondary effects.
[0007] Most prokaryotic and eukaryotic cells encode the uracil DNA
glycosylase (UDG) enzyme. The function of this enzyme is to
eliminate the uracil residues that appear in DNA due to cytosine
deamination or to the incorrect incorporation of dUMP during the
replication process. For example, if cytosine deamination occurs
and it is not repaired, a C-to-T transition mutation will occur in
the DNA strand wherein said deamination has taken place and,
consequently, a G-to-A transition mutation will take place in the
complementary strand after the next replication round. Once the
uracil is eliminated by the UDG enzyme, an apurinic or apyrimidinic
site (AP site) is created. The mechanism in charge of repairing
these AP sites is the base-splicing repair pathway.
[0008] In human cells, up to five different enzymes with UDG
activity have been identified. Curiously, one of these enzymes,
called UNG2, is present in the particles of the type 1 human
immunodeficiency virus (HIV-1). Moreover, some DNA viruses, such as
herpesviruses and poxviruses, encode their own UDG activity. As a
result of the UDG enzyme's capacity to influence the viral
replication of different herpesviruses, said enzyme has been
associated with the virus replication mechanism in the host cell.
In the above-mentioned viruses, it is known that the UDG enzyme is
essential for the infective process. It has been proposed that this
enzyme's function in viral replication processes is associated with
those viruses' capacity to replicate in non-dividing cells, wherein
the levels of cellular UDG enzyme are considered to be low (Priet
et al. (2005) Mol. Cell. 17: 479-490) and, consequently, the
inhibition thereof is of therapeutic interest. Thus far, some
inhibitors of the UDG enzyme encoded by simple herpes virus type 1
(SHV-1) have been designed. These non-protein synthetic compounds
have been tested in in vitro systems. On the other hand, it is
well-known that the UGI protein encoded by the PBS2 bacteriophage
inhibits the UDG enzyme of the SHV-1 virus. However, one
disadvantage of this inhibitor is that it also blocks the UDG
activity of the human UNG2 enzyme.
SUMMARY OF THE INVENTION
[0009] This invention is based on the discovery of a protein that
has the capacity to inhibit the UDG enzyme. Since the UDG activity
of some viruses is essential for the infective process, said
protein could be a useful tool to design antiviral compounds.
[0010] Therefore, one aspect of the invention relates to a protein
that comprises amino acid sequence SEQ ID NO: 1, or a variant or
fragment thereof which has the capacity to inhibit the UDG
enzyme.
[0011] In another aspect, the invention relates to an isolated
polynucleotide that encodes said protein.
[0012] In another aspect, the invention relates to a gene construct
that comprises said polynucleotide.
[0013] In another aspect, the invention relates to a vector that
comprises said polynucleotide or said gene construct.
[0014] In another aspect, the invention relates to a cell that
comprises said polynucleotide, or said gene construct, or said
vector.
[0015] In another aspect, the invention relates to a method of
obtaining said protein, which comprises culturing said cell under
conditions that allow to produce said protein and, if so desired,
recover said protein from the culture medium.
[0016] In another aspect, the invention relates to a composition
that comprises said protein.
[0017] In another aspect, the invention relates to a pharmaceutical
composition that comprises said protein, jointly with one or more
pharmaceutically acceptable excipients.
[0018] In another aspect, the invention relates to the use of said
protein in the preparation of an antiviral pharmaceutical
composition.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows the UDG activity in B. subtilis extracts. The
radioactively labelled ssDNA-U.sup.16 substrate (S) (0.55 ng) was
incubated with the specified quantity of extract in the absence of
Mg.sup.2+. As an internal control, the substrate was incubated with
the UDG enzyme of E. coli. The reaction mixtures were treated with
NaOH or not. The formation of the splicing product (P) was analysed
in polyacrylamide-urea gels.
[0020] FIG. 2 shows the inhibition of the UDG activity of B.
subtilis by protein p56. The specified quantity of p56 was added to
1.6 .mu.g of extract. The reactions were treated with NaOH.
[0021] FIG. 3 shows the co-elution of the UDG of B. subtilis with
protein p56FLAG using anti-FLAG M2 columns (Sigma). The molecular
mass (kDa) of the markers used is indicated on the right.
[0022] FIG. 4 shows the interaction of protein p56 with the UDG
enzyme of E. coli analysed in a native polyacrylamide gel.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention relates, in general, to a protein with the
capacity to inhibit the uracil DNA glycosylase (UDG) enzyme, which
comprises amino acid sequence SEQ ID NO: 1, or a variant or
fragment thereof that maintains said capacity to inhibit the UDG
enzyme, and to its use as a therapeutic agent, in particular, as an
antiviral agent.
[0024] In one aspect, the invention relates to a protein that
comprises amino acid sequence SEQ ID NO: 1, or a variant or
fragment thereof with the capacity to inhibit the uracil DNA
glycosylase (UDG) enzyme.
[0025] For simplicity reasons, the expression "protein of the
invention" includes this protein, which comprises amino acid
sequence SEQ ID NO: 1, or a variant or fragment thereof with the
capacity to inhibit the UDG enzyme. The term "protein", as used
herein, includes all forms of post-translational modifications; for
example, glycosylation, phosphorylation or acetylation.
[0026] In a particular embodiment, the protein of the invention
comprises, or is composed of, the amino acid sequence shown in SEQ
ID NO: 1 and exhibits, at least, the capacity to bind to said UDG
enzyme and inhibit the activity thereof, for which reason it may be
used as an antiviral agent. In a specific embodiment, the protein
of the invention is the so-called protein p56 of the phi29
(.phi.29) bacteriophage.
[0027] In the sense used in this description, the term "variant"
refers to a peptide that is substantially homologous and
functionally equivalent to the protein that comprises amino acid
sequence SEQ ID NO: 1. As used herein, a peptide is "substantially
homologous" to said protein when its amino acid sequence has a
degree of identity with respect to the amino acid sequence of said
protein of, at least, 60%, advantageously of, at least, 70%,
preferably of, at least, 85%, and, more preferably of, at least,
95%. Likewise, the expression "functionally equivalent", as used
herein, means that the peptide in question maintains the capacity
to inhibit UDG enzyme activity. The capacity to inhibit UDG enzyme
activity may be determined by the assay described in Example 1 (see
section 1.6 of Materials and Methods); similarly, a peptide's or
protein's capacity to bind to the UDG enzyme may be determined by
the assay described in Example 1 (see section 1.6 of Materials and
Methods).
[0028] In a particular embodiment, said variant is a mutant form of
the protein that comprises amino acid sequence SEQ ID NO: 1 which
maintains the capacity to inhibit UDG enzyme activity. This mutant
form may have insertions, deletions or modifications of one or more
amino acids with respect to the protein that comprises SEQ ID NO:
1, provided that it preserves the capacity to inhibit UDG enzyme
activity.
[0029] Illustrative, non-limiting examples of variants included
within the scope of this invention include the protein identified
in this description as protein p56FLAG, obtained by the
modification of gene 56 (which encodes protein p56 of .phi.29) such
that it encodes a p56 protein that contains amino acid sequence
DYKDDDDK (FLAG peptide) [SEQ ID NO: 9] fused to the C-terminal end,
as described in Example 1 (see section 1.3 of Materials and
Methods). By means of affinity chromatography, it has been shown
that said protein p56FLAG interacts with the UDG enzyme (see
Example 1).
[0030] Likewise, in the sense used in this description, the term
"fragment" refers to a peptide which comprises a portion of said
protein that comprises amino acid sequence SEQ ID NO: 1, that is, a
sequence of adjacent amino acids comprised within said SEQ ID NO:
1; to be used in this invention, said fragment must have the
capacity to inhibit UDG enzyme activity.
[0031] The protein of the invention may be obtained from an
organism that produces it, using a method which consists of
culturing said organism under suitable conditions for the
expression of said protein, and recovering it. In a particular
embodiment of this invention, the producing organism is the .phi.29
bacteriophage. Example 1 discloses the production, isolation and
purification of a protein that comprises amino acid sequence SEQ ID
NO: 1, specifically, protein p56 of .phi.29, as well as of a
variant thereof (p56FLAG) that has the capacity to bind to the UDG
enzyme and inhibit its enzymatic activity.
[0032] Additionally, the protein of the invention may be a part of
a fusion protein. In this regard, for illustrative, non-limiting
purposes, said fusion protein may contain a region A composed of a
first peptide that comprises the protein of the invention bound to
a region B that comprises a second peptide. Said second peptide may
be any appropriate peptide; for example, a peptide with antiviral
activity. In a particular embodiment, said second peptide may be a
protein of the invention. Said region B may be bound to the
amino-terminal region of said region A, or, alternatively, said
region B may be bound to the carboxyl-terminal region of said
region A. Both regions, A and B, may be bound directly or through a
spacer peptide (linker) between said regions A and B. The fusion
protein may be obtained by conventional methods known by those
skilled in the art; for example, by the gene expression of the
nucleotide sequence that encodes said fusion protein in appropriate
host cells.
[0033] The protein of the invention may be found, if so desired, in
a composition that comprises said protein of the invention and an
inert vehicle. Said composition constitutes an additional aspect of
this invention.
[0034] Practically any inert vehicle, that is, one that is not
harmful to the protein of the invention, may be used in the
preparation of said composition. In a particular embodiment, for
illustrative, non-limiting purposes, said composition comprises a
protein of the invention and a buffer composed of 50 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 7 mM .beta.-mercaptoethanol and 50% glycerol,
suitable to keep the protein of the invention purified at
-70.degree. C.
[0035] From the information provided by the protein of the
invention, the nucleic acid sequence that encodes said protein may
be identified and isolated using conventional techniques known by
those skilled in the art; for example, by creating genomic
libraries of genomic DNA (gDNA) or copy DNA (cDNA) from organisms
that produce said protein; designing suitable oligonucleotides to
amplify, by polymerase chain reaction (PCR), a region of the
genomic clone of the organisms producing said protein which may be
used to obtain probes designed to examine said genomic libraries;
and analysing and selecting the positive clones.
[0036] Therefore, in another aspect, the invention relates to an
isolated polynucleotide, hereinafter, polynucleotide of the
invention, that encodes said protein of the invention.
[0037] In a particular embodiment, the polynucleotide of the
invention comprises, or is composed of, the nucleotide sequence
shown in SEQ ID NO: 2. In a specific embodiment, the polynucleotide
of the invention encodes protein p56 of .phi.29. Alternatively, the
polynucleotide of the invention may exhibit variations in its
sequence with respect to nucleotide sequence SEQ ID NO: 2; for
example, substitutions, insertions and/or deletions of one or more
nucleotides, provided that the resulting polynucleotide encodes a
protein of the invention. Therefore, the scope of this invention
includes polynucleotides that are substantially homologous to the
polynucleotide of SEQ ID NO: 2 and encode a protein of the
invention.
[0038] In the sense used in this description, a polynucleotide is
"substantially homologous" to the polynucleotide of SEQ ID NO: 2
when its nucleotide sequence has a degree of identity with respect
to nucleotide sequence SEQ ID NO: 2 of, at least, 60%,
advantageously of, at least, 70%, preferably of, at least, 85%,
and, more preferably of, at least, 95%. Typically, a polynucleotide
that is substantially homologous to the polynucleotide of SEQ ID
NO: 2 may be isolated from an organism that produces the protein of
the invention on the basis of the information contained in said SEQ
ID NO: 2, or is constructed on the basis of the DNA sequence shown
in SEQ ID NO: 2; for example, by introducing conservative or
non-conservative substitutions. Other examples of possible
modifications include the insertion of one or more nucleotides in
the sequence, the addition of one or more nucleotides at either end
of the sequence, or the deletion of one or more nucleotides at
either end or in the interior of the sequence.
[0039] In another aspect, the invention relates to a gene
construct, hereinafter, gene construct of the invention, that
comprises said polynucleotide of the invention.
[0040] The gene construct of the invention may incorporate,
operatively bound, a sequence that regulates the expression of the
polynucleotide of the invention, thus constituting an expression
cassette. As used in this description, the expression "operatively
bound" means that the protein of the invention, encoded by the
polynucleotide of the invention, is expressed within the correct
reading frame under the control of the expression control or
regulatory sequences.
[0041] Control sequences are sequences that control and regulate
the transcription and, if applicable, the translation of the
protein of the invention, and include promoter sequences,
transcriptional regulator encoding sequences, ribosome-binding
sequences (RBS) and/or transcription termination sequences. In a
particular embodiment, said expression control sequence is
functional in prokaryotic cells and organisms; for example,
bacteria, etc., whereas, in another particular embodiment, said
expression control sequence is functional in eukaryotic cells and
organisms; for example, insect cells, vegetable cells, mammal
cells, etc. Advantageously, the construct of the invention
additionally comprises a marker or gene that encodes a motif or a
phenotype which allows for the selection of the host cell
transformed by said construct.
[0042] The gene construct of the invention may be obtained using
techniques that are widely known in the state of the art .left
brkt-bot.Sambrook et al., "Molecular cloning, a Laboratory Manual",
2nd ed., Cold Spring Harbor Laboratory Press, N.Y, 1989 Vol.
1-3.left brkt-bot..
[0043] The gene construct of the invention may be inserted in an
appropriate vector. Therefore, in another aspect, the invention
relates to a recombinant vector, hereinafter vector of the
invention, that comprises the polynucleotide of the invention or
the gene construct of the invention. The choice of the vector will
depend on the host cell wherein it will be subsequently introduced.
In a particular embodiment, the vector of the invention is an
expression vector.
[0044] For illustrative purposes, the vector wherein said nucleic
acid sequence is introduced may be a plasmid, which, when
introduced in a host cell, is integrated or not in said cell's
genome. Illustrative, non-limiting examples of vectors wherein the
polynucleotide of the invention or the gene construct of the
invention may be inserted include plasmid pCR2.1-TOPO (expression
vector of E. coli), marketed by Invitrogen, or plasmid pPR53
(expression vector of Bacillus subtilis) (Bravo and Salas (1997) J.
Mol. Biol. 269: 102-112).
[0045] The vector of the invention may be obtained by conventional
methods known by those skilled in the art [Sambrook et al.,
"Molecular cloning, a Laboratory Manual", 2nd ed., Cold Spring
Harbor Laboratory Press, N.Y, 1989 Vol. 1-3.left brkt-bot.. In a
particular embodiment, said vector is a useful vector to transform
animal cells.
[0046] The vector of the invention may be used to transform,
transfect or infect cells that are susceptible to being
transformed, transfected or infected by said vector. These cells
may be prokaryotic or eukaryotic. The vector of the invention may
be used to transform eukaryotic cells, such as yeast cells, for
example, Saccharomyces cerevisiae, or prokaryotic cells, such as
bacteria, for example, Escherichia coli or Bacillus subtilis.
Illustrative, non-limiting examples of cells that are susceptible
to being transformed, transfected or infected by the vector of the
invention include E. coli TOP10 (Invitrogen), E. coli BL21 (DE3)
(Studier and Moffatt (1986) J. Mol. Biol. 189: 113-130), B.
subtilis 110NA (Moreno et al. (1974) Virology 62: 1-16) and B.
subtilis YB886 (Yasbin et al. (1980) Gene 12: 155-159).
[0047] Therefore, in another aspect, the invention relates to a
host cell, hereinafter cell of the invention, that is transformed,
transfected or infected with a vector provided by this invention.
The cell of the invention comprises, therefore, a polynucleotide of
the invention, a gene construct of the invention, an expression
cassette provided by this invention or a vector of the invention,
and is capable of expressing the protein of the invention.
[0048] The cell of the invention may be a eukaryotic cell, such as
a yeast cell, for example, S. cerevisiae, or a prokaryotic cell,
such as a bacterium, for example, E. coli or B. subtilis.
Illustrative, non-limiting examples of cells that may be used to
obtain cells of the invention include E. coli TOP10 (Invitrogen),
E. coli BL21 (DE3) (Studier and Moffatt (1986) J. Mol. Biol. 189:
113-130), B. subtilis 110NA (Moreno et al. (1974) Virology 62:
1-16) and B. subtilis YB886 (Yasbin et al. (1980) Gene 12:
155-159).
[0049] The cells of the invention may be obtained by conventional
methods known by those skilled in the art [Sambrook et al., 1989,
cited supra.left brkt-bot..
[0050] In another aspect, the invention relates to a method of
obtaining a protein of the invention, which comprises culturing a
cell of the invention under conditions that allow to produce said
protein and, if so desired, recover said protein from the culture
medium. The conditions to optimise the culture of said cell will
depend on the cell used. The method of producing the protein of the
invention includes, optionally, isolating and purifying said
protein of the invention.
[0051] As mentioned above, the protein of the invention has the
capacity to inhibit the UDG enzyme, for which reason it may be used
as a therapeutic agent; in particular, as an antiviral agent.
[0052] Therefore, in another aspect, the invention relates to the
protein of the invention as a therapeutic agent. In a particular
embodiment, the invention relates to the protein of the invention
as an antiviral agent.
[0053] In general, in order to be administered to a subject, the
protein of the invention will be formulated in a pharmaceutical
composition. Therefore, in another aspect, the invention relates to
a pharmaceutical composition, hereinafter pharmaceutical
composition of the invention, that comprises a protein of the
invention, jointly with one or more pharmaceutically acceptable
excipients.
[0054] The term "subject", as used herein, refers to a member of a
mammal species, and includes, but is not limited thereto, pets,
primates and humans; preferably, the subject is a human being, male
or female, of any age or race.
[0055] More specifically, in order to be administered to a subject,
the protein of the invention will be formulated in a pharmaceutical
form suitable to be administered to a subject by any administration
route. To this end, the pharmaceutical composition of the invention
will include the necessary pharmaceutically acceptable vehicles and
excipients to prepare the pharmaceutical form for the selected
administration.
[0056] The pharmaceutical composition of the invention comprises,
at least, a protein of the invention in a therapeutically effective
quantity. In the sense used in this description, the expression
"therapeutically effective quantity" refers to the quantity of the
protein of the invention calculated to produce the desired effect
and, in general, will be determined, amongst other causes, by the
protein's characteristics and the therapeutic effect to be
achieved.
[0057] For illustrative purposes, the dose of the protein of the
invention to be administered to a subject will be a therapeutically
effective quantity and may vary within a broad range. The
pharmaceutical composition of the invention may be administered one
or more times a day for preventive or therapeutic purposes. The
dose of the protein of the invention to be administered will depend
on numerous factors, which include the characteristics of the
protein of the invention used, such as, for example, its activity
and biological half-life, the concentration of the protein of the
invention in the pharmaceutical composition, the subject's clinical
condition, the severity of the infection or pathology, the
pharmaceutical form for the selected administration, etc. For this
reason, the doses mentioned in this invention should be considered
to be only guides for the person skilled in the art, who must
adjust the doses on the basis of the above-mentioned variables.
[0058] The pharmaceutical composition of the invention may be
formulated in a solid pharmaceutical form, for example (tablets,
capsules, pills, granules, suppositories, etc.), or in liquid form
(solutions, suspensions, emulsions, etc.) to be administered by any
suitable administration route. In a particular embodiment, the
pharmaceutical composition of the invention is administered by
oral, rectal, topical or parenteral route (e.g., intramuscular,
subcutaneous, intravenous, etc.). In each case, the suitable
pharmaceutically acceptable excipients for the chosen
pharmaceutical form and administration route will be selected.
[0059] In a particular embodiment, the protein of the invention
will be formulated in a pharmaceutical form suitable for topical
administration. Illustrative, non-limiting examples of said
pharmaceutical forms include aerosols, solutions, suspensions,
emulsions, gels, ointments, creams, dressings, patches,
collutories, etc. To this end, the pharmaceutical composition of
the invention will include the necessary pharmaceutically
acceptable vehicles and excipients to prepare the pharmaceutical
form. Information about said vehicles and excipients, as well as
about said pharmaceutical forms to administer the protein of the
invention may be found in gallenic pharmacy treatises. A review of
the different pharmaceutical forms of administering drugs, in
general, and of the methods of preparing them may be found in the
book "Tratado de Farmacia Galenica", by C. Fauli i Trillo, 1st
Edition, 1993, Luzan 5, S. A. de Ediciones.
[0060] In another particular embodiment, the protein of the
invention will be formulated in a pharmaceutical form suitable for
oral administration. In a particular embodiment, said
pharmaceutical form for oral administration of the protein of the
invention may be solid or liquid. Illustrative, non-limiting
examples of suitable pharmaceutical forms to administer the protein
of the invention by oral route include tablets, capsules, syrups
and solutions, and may contain conventional excipients known in the
state of the art, such as binding agents, for example, syrup,
acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.;
loads, for example, lactose, sugar, corn starch, calcium phosphate,
sorbitol, glycine, etc.; lubricants to prepare tablets, for
example, magnesium stearate, etc.; disaggregating agents, for
example, starch, polyvinylpyrrolidone, starch sodium glycolate,
microcrystalline cellulose, etc.; pharmaceutically acceptable
wetting agents, such as sodium lauryl sulfate, etc. Information
about said vehicles or excipients, as well as about said
pharmaceutical forms to administer the protein of the invention may
be found in the book "Tratado de Farmacia Galenica", by C. Fauli i
Trillo, 1st Edition, 1993, Luzan 5, S. A. de Ediciones, cited
supra.
[0061] In another particular embodiment, the protein of the
invention will be formulated in a pharmaceutical form suitable for
parenteral administration (e.g., intramuscular, subcutaneous,
intravenous, etc); for example, in the form of sterile solutions,
suspensions or lyophilised products in a suitable unit
pharmaceutical form. Suitable excipients may be used, such as
buffering agents, surfactants, preservatives, etc. Information
about said vehicles or excipients, as well as about said
pharmaceutical forms to administer the protein of the invention may
be found in the book "Tratado de Farmacia Galenica", by C. Fauli i
Trillo, 1st Edition, 1993, Luzan 5, S. A. de Ediciones, cited
supra.
[0062] The production of said pharmaceutical forms to administer
the protein of the invention by any of the selected routes may be
performed by conventional methods known by those skilled in the
art, such as the habitual methods described or mentioned in the
Spanish and US Pharmacopeias and in similar reference texts. For
illustrative purposes, information about the methods of producing
said pharmaceutical forms to administer the protein of the
invention may be found in the book "Tratado de Farmacia Galenica",
by C. Fauli i Trillo, 1st Edition, 1993, Luzan 5, S. A. de
Ediciones, cited supra.
[0063] In a particular embodiment, the pharmaceutical composition
of the invention is used as an antiviral agent, that is, in the
treatment and/or prevention of viral infections, such as infections
caused by herpesviruses and poxviruses.
[0064] The pharmaceutical composition of the invention may be used
with other drugs to provide a combination therapy. The other drugs
may be a part of the same composition or be supplied as a separate
pharmaceutical composition to be administered at the same time
(simultaneous administration) as the pharmaceutical composition of
the invention or at a different time (sequential administration).
In a particular embodiment, said drugs used in combination therapy
include antiviral agents.
[0065] In another aspect, the invention relates to the use of the
protein of the invention in the preparation of an antiviral
pharmaceutical composition, that is, in the preparation of a
pharmaceutical composition for the treatment and/or prevention of
viral infections.
[0066] Said antiviral pharmaceutical composition a pharmaceutical
composition of the invention and comprises, at least, one protein
of the invention, jointly with one or more pharmaceutically
acceptable vehicles or excipients.
[0067] In a particular embodiment, said antiviral pharmaceutical
composition may contain, in addition to a protein of the invention,
one or more additional antiviral compounds or drugs, of protein
origin or not, in order to increase the effectiveness of the
protein of the invention as an antiviral agent, thereby generating
a combination therapy. Said additional drugs may be a part of the
same pharmaceutical composition or, alternatively, may be supplied
in the form of a separate composition to be administered
simultaneously or successively (time-sequenced) with respect to the
administration of the pharmaceutical composition of the
invention.
[0068] The following example illustrates the invention and should
not be considered to limit the scope thereof.
Example 1
Protein p56 Inhibits UDG Enzyme Activity and, Moreover, Binds
Thereto
I. Materials and Methods
[0069] 1.1 Construction of plasmid pCR2.1-TOPO.p56
[0070] The TOPO TA cloning system developed by Invitrogen was used.
Briefly, a DNA region of .phi.29, containing gene 56, was amplified
by the polymerase chain reaction technique (PCR), using the
following oligonucleotides as primers:
TABLE-US-00001 [SEQ ID NO: 3] 5'-CGCATTGTATGAGCTTTCTAGGATGG-3' [SEQ
ID NO: 4] 5'-GCAGGGAATTCTGCAGTCAAAGGACTTTATC-3'
[0071] In this amplification, Taq DNA polymerase was used, which
has the peculiarity of adding a deoxyadenosine residue to the 3'
ends of the amplified fragment (267 pb), thereby generating
protruding ends. As an inducible expression vector, the linear form
of plasmid pCR2.1-TOPO, marketed by Invitrogen, was used; its
protruding 3' ends have a deoxythymidine residue and are covalently
bound to the DNA topoisomerase I enzyme of the Vaccinia virus.
Using this cloning system, digestion with restriction enzymes and
the use of DNA ligase are not necessary, since DNA topoisomerase I
is responsible for the ligation reaction. Subsequently, the
ligation mixture was used to transform the E. coli TOP10 strain
(Invitrogen). Transformants resistant to kanamycin (50 .mu.g/ml)
were selected and the plasmid content was analysed by digestion
with restriction enzymes. The recombinant plasmid was called
pCR2.1-TOPO.p56. The integrity of gene 56 was confirmed by
sequencing.
1.2 Construction of Plasmid pPR53.p56
[0072] The 272-pb PstI fragment of plasmid pCR2.1-TOPO.p56, which
carries gene 56, was cloned in the PstI site of expression vector
pPR53 (Bravo and Salas (1997) J. Mol. Biol. 269: 102-112). For the
cloning, the B. subtilis YB886 strain was used (Yasbin et al.
(1980) Gene 12: 151-159). Transformants resistant to phleomycin
(0.8 .mu.g/ml) were selected. The recombinant plasmid was called
pPR53.p56. The YB886[pPR53.p56] strain constitutively produces
protein p56.
1.3. Construction of Plasmid pPR53.p56Flag
[0073] The sequence of gene 56 was modified by directed mutagenesis
so that it would encode a p56 protein carrying the DYKDDDDK peptide
fused to the C-terminal end (protein p56FLAG). The mutagenesis was
performed in two steps. First, gene 56 was amplified by PCR using
plasmid pPR53.p56 as the template and the following
oligonucleotides as primers:
TABLE-US-00002 [SEQ ID NO: 5] 5'-CCTCTAGAGTCGACCTGCAG-3' [SEQ ID
NO: 6] 5'-GTCATCGTCATCCTTATAGTCAGGACTTTATCCAACCTTAG-3'
[0074] In the second step, the 298-pb amplified fragment was used
as the template and the oligonucleotides identified as SEQ ID NO: 5
and SEQ ID NO: 7
[5'-CCCTCAGGGCTGCAGTTATTACTTGTCATCGTCATCCTTATAGTC-3'] were used as
primers.
[0075] Primer oligonucleotides SEQ ID NO: 5 and SEQ ID NO: 7 carry
a recognition sequence for the PstI enzyme. Subsequently, the
amplified fragment (322 pb) was digested with the PstI enzyme, and
the 293-pb digestion product was cloned in the PstI site of
expression vector pPR53 (Bravo and Salas (1997) J. Mol. Biol. 269:
102-112). The ligation mixture was used to transform the B.
subtilis YB886 strain (Yasbin et al. (1980) Gene 12: 151-159).
Transformants resistant to phleomycin (0.8 .mu.g/ml) were selected.
The recombinant plasmid was called pPR53.p56FLAG. The
YB886[pPR53.p56FLAG] strain constitutively produces protein
p56FLAG. Subsequently, in order to perform in vivo protein-protein
interaction studies, plasmid pPR53.p56FLAG was introduced into the
B. subtilis 110NA strain (Moreno et al. (1974) Virology
62:1-16).
1.4 Purification of Protein p56
[0076] Plasmid pCR2.1-TOPO.p56 was introduced into the E. coli BL21
(DE3) strain by electroporation techniques. The
BL21(DE3)[pCR2.1-TOPO.p56] strain was grown in kanamycin-containing
LB medium (50 .mu.g/ml) at 34.degree. C. When the culture reached
an optical density of 0.9 at 560 nm (OD.sub.560), IPTG was added at
a final concentration of 0.5 mM. After 30 minutes, rifampycin was
added (120 .mu.g/ml) and incubation of the culture was continued
for 75 minutes. The pellet of cells was kept at -70.degree. C.
until it was to be used. In order to purify p56, the cells were
lysed with alumina in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM
ethylene diamine tetra-acetic acid (EDTA), 7 mM
.beta.-mercaptoethanol, 5% glycerol) containing 0.65 M NaCl. After
eliminating the alumina and the cell residues by centrifugation,
the lysate was incubated with polyethyleneimine (0.3%) for 20
minutes and, subsequently, it was centrifuged at 12,000 rpm in the
Sorvall-GSA rotor for 10 minutes. The pellet was washed with buffer
A containing 0.7 M of NaCl. Following centrifugation (12,000 rpm in
the Sorvall-SS34 rotor for 20 minutes), successive precipitation
steps with ammonium sulfate were performed (65%, 45% and 30%
saturations, respectively). The supernatant of the last
precipitation was taken to a final saturation of 50%. Following
centrifugation, the pellet was re-suspended in buffer A, the salt
concentration being 55 mM (estimated by conductivity). The protein
preparation was loaded in a Mono column equilibrated with buffer A
containing 55 mM NaCl. Protein p56 was eluted from the column with
0.3 M NaCl. Finally, the protein preparation was loaded in a
glycerol gradient (from 15% to 30%) and centrifuged at 62,000 rpm
in a Beckman-SW.65 rotor for 20 hours. The fractions containing
protein p56 were collected and precipitated with 70% ammonium
sulfate. Protein p56 was re-suspended in buffer A containing 50%
glycerol and stored at -70.degree. C.
1.5 Inhibition of UDG Activity Mediated by Protein p56
Detection of UDG Activity in B. Subtilis Extracts
[0077] In order to prepare the cell extracts, the B. subtilis 110NA
strain (Moreno et al. (1974) Virology 62: 1-16) was grown in LB
medium at 30.degree. C. to an OD.sub.560 equivalent to 10.sup.8
viable cells per ml of culture. Then, the culture was concentrated
10-fold in buffer U (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 12 mM
.beta.-mercaptoethanol, 1 mM EDTA) containing a mixture of protease
inhibitors obtained from Roche Applied Science (a tablet of
"Complete, Mini, EDTA-free" for 10 ml). The culture was lysed using
the French press (20,000 psi) and, subsequently, the lysate was
centrifuged at 7,000 rpm and 4.degree. C. in the Sorvall-SS.34
rotor for 10 minutes. The supernatant (cell extract) was kept at
4.degree. C. for a maximum period of two weeks. The total protein
concentration in the extract (1.35 mg/ml) was calculated by Lowry's
method.
[0078] Enzymes with UDG activity that belong to Family-1 eliminate
uracil residues from both single-chain DNA and double-chain DNA.
The elimination of the uracil residue generates a baseless site (AP
site) in the DNA, which in B. subtilis is recognised and processed
by protein ExoA. In the absence of an AP endonuclease activity, DNA
chain splicing may be achieved in the AP site by treatment with
NaOH and heat. In order to measure the UDG activity in the cell
extract, a single-chain DNA (34 nucleotides) carrying a uracil
residue in position 16 (ssDNA-U.sup.16 substrate) was used. The
nucleotide sequence of the ssDNA-U.sup.16 polynucleotide is the
following: 5'-CTGCAGGTGATGCGCUGTACCGATCCCCGGGTAG-3' [SEQ ID NO: 8].
This DNA was radioactively labelled on the 5' end using
[.beta.-32P] ATP (3,000 Ci/mmol) (Amersham Pharmacia). The reaction
mixture (20 .mu.l) contained 0.55 ng of the ssDNA-U.sup.16
substrate and the specified quantity of the cell extract (from 0.05
.mu.g to 3.2 .mu.g) in buffer U. The mixture was incubated at
37.degree. C. for 10 minutes. Subsequently, it was treated with
NaOH (0.2 M) and incubated at 90.degree. C. for 30 minutes. The
sample was analysed by electrophoresis in urea-containing (8 M)
polyacrylamide gels (20%). The results obtained are shown in FIG.
1A.
[0079] In order to analyse whether protein p56 is an inhibitor of
UDG activity, the ssDNA-U.sup.16 substrate (0.55 ng) was incubated
with 1.6 .mu.g of the cell extract (a sufficient quantity to obtain
total splicing of the substrate) and with different quantities of
protein p56 (between 0.5 ng and 16 ng). As described above, the
reaction mixtures were incubated at 37.degree. C. for 10 minutes,
treated with NaOH (0.2 M) and incubated at 90.degree. C. for 30
minutes.
1.6 Interaction of Protein p56 with the UDG Enzyme
[0080] In order to analyse whether the UDG enzyme is a cellular
target of protein p56, affinity chromatography assays were
performed. Briefly, the B. subtilis 110NA [pPR53.p56FLAG] strain
was grown in LB medium with phleomycin (0.8 .mu.g/ml) at 30.degree.
C. to an OD.sub.560 equivalent to 10.sup.8 viable cells per ml. The
culture was concentrated 10-fold in TBS buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl) and was lysed using the French press (20,000
psi). The lysate was centrifuged at 7,000 rpm and 4.degree. C. in
the Sorvall-SS.34 rotor for 10 minutes. The supernatant was loaded
in an anti-FLAG M2 column (Sigma). The proteins bound to the column
were eluted with the TBS buffer containing the FLAG peptide (500
.mu.g/ml) (Sigma). The eluted proteins were precipitated with
acetone, re-suspended in the loading buffer (60 mM Tris-HCl, pH
6.8, 2% SDS, 5% .beta.-mercaptoethanol, 30% glycerol) and separated
by electrophoresis in polyacrylamide/Tricine/SDS gels (Schagger and
von Jagow (1987) Anal. Biochem. 166: 368-379). The gel was stained
with SyproRuby (Molecular Probes). As a negative control, an
extract from the 110NA[pPR53] strain was used. In a second
experiment, protein p56 (2 .mu.g) was incubated with the UDG enzyme
of E. coli (0.2 .mu.g, New England Biolabs). After 15 minutes at
ambient temperature, the reaction was analysed by electrophoresis
in native polyacrylamide gels (16%). The gel was stained with
SpyroRuby.
II. Results
[0081] 2.1 Protein p56 Interacts with the UDG Enzyme
[0082] Incubation of the substrate (ssDNA-U.sup.16) with 0.2 .mu.g
of the cell extract of B. subtilis generated a splicing product.
Moreover, the results show (FIG. 1A) that said substrate was
completely spliced when 1.6 .mu.g of the cell extract were used.
The same splicing product was detected when the substrate was
incubated with the UDG enzyme of E. coli (New England Biolabs).
Therefore, the results show that the extract of B. subtilis was
capable of eliminating the uracil residue from the substrate,
generating an AP site, that is, said extract has UDG activity.
However, under the conditions assayed (absence of Mg.sup.2+), the
extract of B. subtilis lacked AP endonuclease activity, since no
splicing of the substrate was detected when the reaction mixtures
were not treated with NaOH (FIG. 1A).
2.2. Protein p56 Inhibits UDG Activity
[0083] In order to prove that protein p56. is an inhibitor of UDG
activity, the ssDNA-U.sup.16 substrate (0.55 ng) was incubated with
1.6 .mu.g of the cell extract (a sufficient quantity to obtain
total splicing of the substrate) and with different quantities of
protein p56 (between 0.5 ng and 16 ng). As already described in
section 1.5 of Materials and Methods, the reaction mixtures were
incubated at 37.degree. C. for 10 minutes, treated with NaOH (0.2
M) and incubated at 90.degree. C. for 30 minutes. FIG. 1B shows the
results obtained. In the presence of 2 ng of protein p56, a
decrease in the quantity of splicing product was detected.
Moreover, the substrate remained intact when 8 ng of protein p56
were added. That is, the results show that protein p56 inhibits UDG
activity.
[0084] As shown in FIG. 2A, five proteins (A-E) co-eluted with
p56FLAG. Using peptide mass fingerprinting and the MASCOT programme
(Perkins et al. (1999) Electrophoresis 20: 3551-3567), protein D
was identified to be the UDG enzyme (26 kDa). A band that moved
faster than free UDG was detected (FIG. 2B). Using Western blot and
peptide mass fingerprinting, it was confirmed that said band
contained p56 and UDG.
Sequence CWU 1
1
9156PRTArtificial sequencesynthetic protein 1Met Val Gln Asn Asp
Phe Val Asp Ser Tyr Asp Val Thr Met Leu Leu1 5 10 15Gln Asp Asp Asp
Gly Lys Gln Tyr Tyr Glu Tyr His Lys Gly Leu Ser 20 25 30Leu Ser Asp
Phe Glu Val Leu Tyr Gly Asn Thr Ala Asp Glu Ile Ile 35 40 45Lys Leu
Arg Leu Asp Lys Val Leu 50 552171DNAArtificial sequencesynthetic
polynucleotide 2atggtgcaaa atgattttgt tgactcatac gatgtgacaa
tgttgcttca agatgatgac 60ggtaaacagt attatgagta ccacaaggga ctgagtttgt
cagactttga ggttctatac 120ggtaacactg ctgatgaaat tataaaacta
aggttggata aagtactatg a 171326DNAArtificial sequencePrimer
oligonucleotide used for amplifying p56 gene of bacteriophage
phi-29 3cgcattgtat gagctttcta ggatgg 26431DNAArtificial
sequencePrimer oligonucleotide used for amplifying p56 gene of
bacteriophage phi-29 4gcagggaatt ctgcagtcaa aggactttat c
31520DNAArtificial sequencePrimer oligonucleotide used for
amplifying p56 gene by PCR using pPR53.p56 plasmid as template
5cctctagagt cgacctgcag 20641DNAArtificial sequencePrimer
oligonucleotide used for amplifying p56 gene by PCR using pPR53.p56
plasmid as template 6gtcatcgtca tccttatagt caggacttta tccaacctta g
41745DNAArtificial sequencePrimer oligonucleotide used for
amplifying p56 gene by PCR using 298 bp fragment of pPR53.p56
plasmid as template 7ccctcagggc tgcagttatt acttgtcatc gtcatcctta
tagtc 45834DNAArtificial sequencesynthetic ssDNA-U substrate
8ctgcaggtga tgcgctgtac cgatccccgg gtag 3498PRTArtificial
sequencesynthetic FLAG peptide 9Asp Tyr Lys Asp Asp Asp Asp Lys1
5
* * * * *