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CROSS-REFERENCE TO RELATED APPLICATION
This application is related to provisional patent application Ser. No.
60/026,978, filed Sep. 20, 1996, now abandoned from which priority is
claimed under 35 USC .sctn.119(e)(1) and which is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
The present invention relates generally to polypeptide inhibitors of gene
expression. More particularly, the invention relates to polypeptide
inhibitors of nuclear protein translocation which have gene expression
modulating activity, immunosuppressive activity, antiviral activity, and
antitumor activity.
BACKGROUND OF THE INVENTION
Nuclear transport is essential to a number of biological processes
including gene expression and cell division, as well as to viral
replication, tumorigenesis and tumor cell proliferation. The mechanism of
nuclear transport has only recently been characterized in detail and has
been shown to involve a number of discrete steps. Proteins that are
destined to be transported into the nucleus contain within their amino
acid sequence a short stretch of amino acids termed a nuclear localization
sequence ("NLS"). These sequences are generally basic in nature, however,
there has been no consensus sequence identified. Thus, there is a wide
variety of these sequences that appear to be specific for particular
proteins.
Within the cell, these NLSs may be either masked or unmasked by accessory
proteins or by conformational changes within the NLS-containing protein.
An NLS may be masked because it is buried in the core of the protein and
not exposed on the surface of the protein. Unmasking of NLSs, and nuclear
translocation of cytoplasmic proteins may be triggered by phosphorylation,
dephosphorylation, proteolytic digestion, subunit association or
dissociation of an inhibitory subunit, or the like. Accordingly, the
masking and unmasking of NLSs provides a mechanism by which the transport
of these cytoplasmic proteins into the nucleus may be regulated.
Nuclear translocation of transcription factors requires the presence of an
unmasked or activated NLS in the nucleus-targeted protein. The binding of
certain ligands to cell surface receptors activates the nuclear
translocation of cytoplasmic transcription factors. Once in the nucleus,
these transcription factors exert gene expression modulatory activity.
NF-.kappa.B is a ubiquitous transcription factor found in various levels
and states of activation in different cell types. NF-.kappa.B is composed
of several different subunits including p65, p50, c-rel, p52 and p105.
Recent studies suggest that distinct NF-.kappa.B complexes contribute to
the regulatory control of gene transcription. The function and regulation
of NF-.kappa.B has been most well-characterized in lymphocytic cells. In
these cells, there is a wide variety of target genes, e.g.,
immunoregulatory genes, that are regulated by NF-.kappa.B including
.kappa. Ig light chains. Such genes include those that encode the
interleukin-2.alpha. ("IL-2.alpha.") receptor, interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), tumor necrosis factor-.alpha. ("TNF-.alpha."), and
the like.
In unstimulated cells, a major form of NF-.kappa.B is a heterodimer of p50
and p65 (RelA) subunits. Nonactive NF-.kappa.B is retained in the
cytoplasm as an inactive complex by inhibitory proteins such as
I.kappa.B.alpha., .beta. and .gamma.. When cells are appropriately
stimulated, e.g., by a proinflammatory stimulus such as a cytokine, the
I.kappa.Bs are degraded, thereby releasing free NF-.kappa.B dimers, which
translocate to the nucleus and activate target genes, e.g., lymphokine
genes and other immunoregulatory genes. This response is transient and is
terminated through delayed NF-.kappa.B-mediated I.kappa.B.alpha.
induction.
Recently it has been demonstrated that glucocorticoids exert their
immunosuppressive activity by inhibiting NF-.kappa.B nuclear
translocation. Scheinman et al. (1995) Science 270:283-286 and Auphan et
al. (1995) Science 270:286-290 independently demonstrated that the
inhibition is mediated by an increase in the induction by glucocorticoids
of I.kappa.B inhibitory proteins. These investigators proposed that
inhibitors of NF-.kappa.B may be useful immunosuppressive and
anti-inflammatory agents. Such an NF-.kappa.B nuclear translocation
inhibitor, comprising an NLS from the p50 subunit of NF-.kappa.B attached
to a membrane-permeable polypeptide motif, was described in Lin et al.
(1995) J. Biol. Chem. 270:14255-14258.
Nuclear translocation of proteins other than endogenous transcription
factors and other cytoplasmic proteins also depends on the presence of an
activated or unmasked NLS. For example, nuclear translocation of the
retroviral preintegration complex is a crucial step in human
immunodeficiency virus type-1 ("HIV-1") replication in nondividing cells
such as monocytes and growth-arrested T cells. Such translocation is
dependent on the presence of an NLS in the N-terminal portion of HIV
matrix antigen ("MA") p17. Indeed, the HIV-1 enhancer contains tandem
binding sites for NF-.kappa.B that can be essential for virus replication
(Ross et al. (1991) J. Virol. 65:4350-4358; Parrott et al. (1991) J. Virol
65:1414-1419). Nuclear translocation of the HIV-1 preintegration complex
can be partially inhibited by an excess of the SV40 large T antigen NLS
(Gulizia et al. (1994) J. Virol. 68:2021-2025). Furthermore, Dubrovsky et
al. (1995) Molecular Med. 2:217-230 reported that a series of compounds
capable of binding to and reacting with the HIV-1 MA p17 NLS inhibit HIV-1
replication in human monocytes.
In addition, tumorigenesis and tumor cell proliferation are regulated by
the expression of oncoproteins, many of which are cytoplasmic
transcription factors that are translocated into the nucleus by virtue of
the presence of an NLS. Miller et al. (1996) J. Cell Biochemistry 60:560.
Accordingly, inhibitors of nuclear translocation of cytoplasmic proteins
would be useful as gene expression modulating agents, immunoregulatory
agents, antiviral agents, antitumor agents, and the like.
SUMMARY OF THE INVENTION
The present invention provides for a polypeptide that can be introduced
into an intact cell for the purpose of inhibiting the nuclear
translocation of a cytoplasmic protein. The polypeptide contains at least
two NLSs and an amino acid sequence that can deliver the polypeptide
through the cytoplasmic membrane into the cell. The inventors herein have
found that such a polypeptide exhibits surprisingly superior
characteristics compared to a polypeptide having only one NLS.
Accordingly, in one embodiment, the invention is directed to a polypeptide
comprising a signal sequence peptide and at least two NLSs covalently
attached thereto.
In another embodiment, the invention is directed to a method of introducing
an exogenous polypeptide comprising an NLS into an intact cell to inhibit
nuclear translocation of a cellular protein. The method includes providing
a polypeptide comprising a signal sequence peptide and at least two NLSs
as described above, and contacting the cell with the polypeptide for a
period of time effective to introduce the exogenous polypeptide into the
cell.
In still another embodiment, the invention is directed to a method of
suppressing an immune response of a subject comprising administering to
the subject an immunosuppressive amount of a polypeptide comprising a
signal sequence peptide and at least two NLSs.
In a further embodiment, the invention is directed to a method of treating
or preventing a viral infection in an individual comprising administering
to the individual an effective antiviral amount of a polypeptide inhibitor
of nuclear translocation of a cellular protein, said inhibitor comprising
a signal sequence peptide and at least two NLSs.
In yet a further embodiment, the invention is directed to a method of
transcriptionally modulating the expression of cellular genes comprising
contacting a target cell with an inhibitor of nuclear translocation of a
cellular protein, wherein the inhibitor comprises a signal sequence
peptide and at least two NLSs.
These and other embodiments of the subject invention will readily occur to
those of ordinary skill in the art in view of the disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a graphical representation of the effect of
PKKKRKVAAVALLPAVLLALLAPKKKRKVC (SEQ ID NO:1) (the "SV40MEM" polypeptide)
on lipopolysaccharide ("LPS")-stimulated surface antigen expression in
70Z/3 murine leukemia pre-B cells (solid bar: .kappa. Ig light chain;
stippled bar: IL-2.alpha. receptor; striped bar: CD45). FIG. 1B is a
graphical representation of the effect of three inhibitory polypeptides on
LPS-stimulated .kappa. Ig light chain production in 70Z/3 murine leukemia
pre-B cells (diamonds: the SV40MEM polypeptide; squares:
KKKYKAAVALLPAVLLALLAKKKYKC (SEQ ID NO:2) (the "HIV-1MEM" polypeptide);
triangles: AKRVKLAAVALLPAVLLALLAKRVKLC (SEQ ID NO:3) (the "C-MYCMEM"
polypeptide)). FIG. 1C is a graphical representation of the effect of
increasing LPS concentrations on SV40MEM-inhibited .kappa. Ig light chain
production in 70Z/3 murine leukemia pre-B cells.
FIG. 2 is a graphical representation of the effect of the SV40MEM
polypeptide on LPS-stimulated cytokine production in 70Z/3 murine leukemia
pre-B cells (squares: untreated; diamonds: the SV40MEM polypeptide (10
.mu.M); circles: the SV40MEM polypeptide (5 .mu.M); triangles: the SV40
NLS (10 .mu.M)).
FIG. 3 is a graphical representation of dose-response relationships of
three inhibitory polypeptides on LPS-stimulated .kappa. Ig light chain
production in 70Z/3 murine leukemia pre-B cells (triangles: a peptide
containing only the fibroblast growth factor ("FGF") signal sequence
AAVALLPAVLLALLAP (SEQ ID NO:4) (the "MEM" peptide); squares: a polypeptide
having the FGF signal sequence and the NLS of NF-.kappa.B p50 on the
carboxy terminus of the signal sequence, namely,
AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO:5) polypeptide (the "NF-.kappa.BMEM"
polypeptide); diamonds: the SV40MEM polypeptide comprised of L-amino
acids; circles: the SV40MEM polypeptide comprised of D-amino acids.).
FIG. 4A is a graphical representation of the effect of inhibitory
polypeptides on LPS-stimulated TNF-.alpha. production. FIG. 4B is a
graphical representation of the effect of inhibitory polypeptides on
LPS-stimulated interleukin-8 ("IL-8") production. In both FIG. 4A and FIG.
4B the following symbols are used: squares--media control; crossed
squares--the MEM peptide (5 .mu.M); diamonds--the NF-.kappa.BMEM
polypeptide (5 .mu.M); circles--the SV40MEM polypeptide comprised of
L-amino acids (5 .mu.M); and triangles--the SV40MEM polypeptide comprised
of D-amino acids (5 .mu.M).
FIGS. 5A and 5B are graphical representation of the effect of the SV40MEM
polypeptide on LPS-induced CD40 expression in 70Z/3 murine leukemia pre-B
cells.
FIG. 6A is a graphical representation of the effect of the HIV-1MEM
polypeptide on .sup.3 H-deoxyribothymidine uptake into peripheral blood
mononuclear cells ("PBMCs"). FIG. 6B is a graphical representation of the
effect of HIV-1MEM on viral p24 production in anti-CD3 stimulated PBMCs
infected with HIV-1 primary isolate M1. FIG. 6C is a photograph of a gel
depicting the effect of HIV-1MEM polypeptide on the expression of proviral
gag sequences in anti-CD3 activated PBMCs infected with HIV-1 primary
isolate M1.
FIG. 7 is a photograph of a gel depicting the results of a polymerase chain
reaction analysis of the effect of HIV-1MEM polypeptide on the expression
of proviral gag sequences in H9 human lymphoma T-cells or Jurkat human
leukemia T-cells infected with HIV-1 primary isolate M1.
FIG. 8A is a photograph of gels depicting the results of polymerase chain
analyses of the effect of HIV-1MEM and the NF-.kappa.BMEM polypeptide on
the expression of proviral gag sequences in Jurkat T-cells infected with
HIV.sub.LAI. FIG. 8B is a photograph of gels depicting the results of
polymerase chain analyses of the effect of HIV-1MEM and the NF-.kappa.BMEM
polypeptide on the expression of 2-long terminal repeat ("LTR") circles in
Jurkat T-cells infected with HIV.sub.LAI.
FIG. 9 is a graphical representation of the effect of SV40MEM prepared from
D-amino acids on proliferation of 70Z/3 murine leukemia pre-B cells.
FIG. 10 is a graphical representation of the effect of SV40MEM prepared
from D-amino acids on proliferation of RAJI human B-cell leukemia cell
line.
FIG. 11A is a graphical representation of the effect of an intravenous
administration of the SV40MEM polypeptide on the in vivo response of mice
to sheep red blood cells. FIG. 11B is a graphical representation of the
effect of an oral administration of the SV40MEM polypeptide on the in vivo
response of mice to sheep red blood cells.
FIG. 12 shows the effect of BMS 205820 and C-MYCMEM (BMS 214572) on the
anti-hemocyanin (KLH) response in mice.
FIG. 13A, shows the effect of the BMS 205820 polypeptide on the production
of TNF-.alpha. in vivo. FIG. 13B shows the effect of the BMS 205820
polypeptide on the production of IL-6 in vivo. FIG. 13C shows the effect
of the BMS 205820 polypeptide on the production of IL-10 in vivo.
FIG. 14 depicts the effect of BMS 205820, C-MYCMEM (BMS 214572), an SV40
NLS polypeptide containing a single NLS, a c-myc NLS alone, without a
translocation sequence (AKRVKL (SEQ ID NO:6)) and a control non-NLS
polypeptide, 377G, on lipopolysaccharide (LPS) binding to CD14.
FIG. 15A is a time line depicting the administration scheme for the
intraperitoneal injection of ovalbumin (OVA), nebulized OVA, BMS 205820
and C-MYCMEM. FIG. 15B shows the % eosinophils in lung following the
treatment outlined in FIG. 15A.
DETAILED DESCRIPTION
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of protein chemistry and biochemistry,
molecular biology, microbiology and recombinant DNA technology, which are
within the skill of the art. Such techniques are explained fully in the
literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A
Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II (D.
N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);
Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal
Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL
press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984);
the series, Methods In Enzymology (S. Colowick and N. Kaplan eds.,
Academic Press, Inc.).
All patents, patent applications and publications cited herein, whether
supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms
"a," "an" and "the" include plural references unless the content clearly
dictates otherwise.
A. Definitions
In describing the present invention, the following terms will be employed,
and are intended to be defined as indicated below.
As used herein, the term "signal sequence" or "signal sequence peptide" is
used to indicate a peptide that is capable of directing the movement of
the polypeptide of which it is a part through a cell membrane. In
particular, the term is used to indicate a peptide that directs the
movement of a polypeptide across the cytoplasmic membrane into the cell.
The term "signal sequence" is intended to encompass not only the signal
sequence of a particular polypeptide, but also fragments or derivatives
thereof that are capable of delivering a polypeptide through a cell
membrane. A "signal sequence" may be composed of L- or D-amino acids.
The terms "nuclear localization sequence" and "NLS" are used
interchangeably to indicate a peptide that directs the transport of a
protein with which it is associated from the cytoplasm of a cell across
the nuclear envelope barrier. The term "NLS" is intended to encompass not
only the nuclear localization sequence of a particular peptide, but also
derivatives thereof that are capable of directing translocation of a
cytoplasmic polypeptide across the nuclear envelope barrier. NLSs are
capable of directing nuclear translocation of a polypeptide when attached
to the N-terminus, the C-terminus, or both the N- and C-termini of the
polypeptide. In addition, a polypeptide having an NLS coupled by its N- or
C-terminus to amino acid side chains located randomly along the amino acid
sequence of the polypeptide will be translocated. Adam et al. (1990) J.
Cell. Biol. 111:807-818. "Nuclear localization sequences" may be composed
of D- or L-amino acids.
By "interchangeably flanked at its amino- and carboxy-termini by a first
and a second NLS" is intended to mean that the first or second NLS may be
located at either the amino- or carboxy-terminus of the signal sequence
polypeptide.
An "inhibitor of nuclear translocation" is a polypeptide composed of a
signal sequence peptide and at least two NLSs which inhibits, e.g., either
decreases or halts, nuclear localization of a cytoplasmic protein.
Preferably, the polypeptide comprises a signal sequence peptide
interchangeably flanked at its amino- and carboxy-termini by a first and a
second NLS. The NLSs at the N- and C-termini may be the same or different.
In one preferred embodiment, the signal sequence peptide and the NLSs are
each composed of L-amino acids. In another preferred embodiment, the
signal sequence peptide and the NLSs are each composed of D-amino acids.
A "derivative" of a polypeptide is intended to include homologous
polypeptides in which conservative amino acid substitutions have been
made, as well as to include other amino acid substitutions that result in
a polypeptide that retains its function, e.g., as a signal sequence
peptide, an NLS, or an inhibitor of nuclear localization. A "derivative"
of a peptide may be a peptide mimetic.
"Peptide mimetics" are structures which serve as substitutes for peptides
in interactions with acceptor molecules (see Morgan et al. (1989) Ann.
Reports Med. Chem. 24:243-252 for a review of peptide mimetics). Peptide
mimetics, as used herein, include synthetic structures which may or may
not contain amino acids and/or peptide bonds, but retain the structural
and functional features of a peptide ligand. The term, "peptide mimetics"
also includes peptoids and oligopeptoids, which are peptides or oligomers
of N-substituted amino acids (Simon et al. (1972) Proc. Natl. Acad. Sci.
USA 89:9367-9371). Further included as peptide mimetics are peptide
libraries, which are collections of peptides designed to be of a given
amino acid length and representing all conceivable sequences of amino
acids corresponding thereto. Methods for the production of peptide
mimetics are described more fully below.
Two polypeptide sequences are "substantially homologous" when at least
about 85% (preferably at least about 85% to 90%, and most preferably at
least about 95%) of the nucleotides or amino acids match over a defined
length of the molecule. As used herein, substantially homologous also
refers to sequences showing identity to the specified polypeptide
sequence.
The terms "polypeptide", "peptide" and "protein" are used interchangeably
and refer to any polymer of amino acids (dipeptide or greater) linked
through peptide bonds. Thus, the terms "polypeptide", "peptide" and
"protein" include oligopeptides, protein fragments, analogues, muteins,
fusion proteins and the like.
The following single-letter amino acid abbreviations are used throughout
the text:
______________________________________
Alanine A Arginine R
Asparagine N Aspartic acid
D
Cysteine C Glutamine Q
Glutamic acid
E Glycine G
Histidine H Isoleucine I
Leucine L Lysine K
Methionine M Phenylalanine
F
Proline P Serine S
Threonine T Tryptophan W
Tyrosine Y Valine V
______________________________________
By an "isolated polypeptide" is meant a polypeptide which is devoid of, in
whole or part, tissue or cellular components with which the protein is
normally associated in nature. Thus, a polypeptide contained in a tissue
extract would constitute an "isolated" polypeptide, as would a polypeptide
synthetically or recombinantly produced.
By "mammalian subject" is meant any member of the class Mammalia,
including, without limitation, humans and non-human primates, such as
chimpanzees and other apes and monkey species; farm animals such as
cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and
cats; and laboratory animals including rodents such as mice, rats and
guinea pigs. The term does not denote a particular age. Thus, adult,
newborn and fetal mammals are intended to be covered.
The term "treatment" as used herein refers to either (i) the prevention of
infection or reinfection (prophylaxis), or (ii) the reduction or
elimination of symptoms of the disease of interest (therapy).
B. General Methods
Central to the present invention is the discovery of polypeptide molecules
that inhibit nuclear localization of cytoplasmic proteins. These molecules
comprise a signal sequence peptide and at least two NLSs. The polypeptide
inhibitors, and derivatives thereof, provide useful tools for introducing
an exogenous polypeptide comprising an NLS into an intact cell to inhibit
nuclear translocation of a cellular protein, for studying the role of
nuclear translocation in the regulation of cellular processes.
In addition, since the nuclear translocation of certain cellular peptides
is required for the host organism to mount an immune response, the
polypeptide inhibitors are useful as immunosuppression agents. Immune
responses are typically manifested by the expression of antibodies, the
production of a number of cytokines, and/or the expression of cell surface
receptors. Thus, inhibition of immune responses by the inhibitory peptides
can take the form of: inhibition of antibody production, including the
production of antibody component peptides such as a .kappa. light chain
polypeptide; inhibition of cytokine production, including such cytokines
as interleukin-1, interleukin-2, interleukin-4, interleukin-6,
interleukin-10, tumor necrosis factor, or granulocyte-macrophage
colony-stimulating factor; and/or the inhibition of the expression of
cell-surface receptors such as an interleukin-2 receptor, gp39, CD40,
CD45, CD80, CD86, ICAM, ELAM, major histocompatibility complex ("MHC")
class II, or VCAM. Clark et al. (1994) Nature 367:425.
By virtue of their immunosuppressive activities, the polypeptide inhibitors
of the present invention are useful in the treatment of a wide variety of
immune disorders, including but not limited to, the treatment of
autoimmune diseases such as rheumatoid arthritis, multiple sclerosis,
juvenile-onset diabetes, systemic lupus erythematosus (SLE), autoimmune
uveoretinitis, autoimmune vasculitis, bullous pemphigus, myasthenia
gravis, autoimmune thyroiditis or Hashimoto's disease, Sjogren's syndrome,
granulomatous orchitis, autoimmune oophoritis, Crohn's disease,
sarcoidosis, rheumatic carditis, ankylosing spondylitis, Grave's disease,
and autoimmune thrombocytopenic purpura (see e.g., Paul, W. E. (1993)
Fundamental Immunology, Third Edition, Raven Press, New York, Chapter 30,
pp. 1033-1097; and Cohen et al. (1994) Autoimmune Disease Models, A
Guidebook, Academic Press, 1994).
Similarly, the polypeptide inhibitors of the present invention are useful
for treating physical symptoms manifested by responses to allergens which
can initiate a state of hypersensitivity, or which can provoke a
hypersensitivity reaction in a subject already sensitized with the
allergen. Such physical symptoms include asthma, joint swelling,
urticaria, and the like. Additionally, due to their immunosuppressive
properties, the polypeptide inhibitors of the present invention are useful
in the treatment of sepsis and in the prevention of septic shock, a
potentially lethal condition caused by the uncontrolled production of
certain cytokines due to the presence of endotoxins, such as
lipopolysaccharide (LPS), from extracellular bacteria.
Furthermore, since many viruses, e.g., herpes virus, cytomegalovirus,
retroviruses, and the like, make use of the host cell's nuclear
translocation machinery, the inhibitory polypeptides are useful as
antiviral agents. In addition, since tumorigenesis and tumor cell
proliferation appear to be mediated by the expression of oncogenes to make
oncoproteins, many of which are transcription factors that are
translocated into the nucleus, Miller et al. (1996), supra, the
polypeptide inhibitors, or derivatives thereof, can be used to suppress
tumor growth.
The claimed inhibitors include sequences of amino acids that comprise
signal sequences from such polypeptides as the antennapedia homeodomain,
FGF, HIV Tat, or Hsc70, and derivatives or mimetics thereof capable of
delivering the inhibitor through the cytoplasmic membrane into the cell.
Preferred signal sequences include RQIKIWFQNRRMKWKK (SEQ ID NO:7),
AAVALLPAVLLALLA (SEQ ID NO:8), AAVALLPAVLLALLAP (SEQ ID NO:4),
CFITKALGISYGRKKRRQRRRPPQGSQTH (SEQ ID NO:9), and the like, or derivatives
or mimetics thereof capable of delivering the inhibitor through the
cytoplasmic membrane into the cell.
Candidate signal sequences can be tested for their ability to direct the
translocation of proteins across cell membranes, for example, by
monitoring the localization of exogenous detectably labeled proteins into
the cell cytoplasm. Lin et al. (1995), supra, describe the use of
radiolabeled proteins. In vitro nuclear peptide import can be measured
using NLS peptides coupled to a fluorescent protein by methods described
in Adam et al. (1990), supra.
The polypeptide inhibitor further comprises at least two NLSs. The NLSs can
be covalently bonded to the N-terminus, to the C-terminus, to both the N-
and C-termini of the signal sequence polypeptide, to amino acid side
chains located along the amino acid sequence of the signal sequence
polypeptide, or any combination thereof. Preferably, the signal sequence
polypeptide is interchangeably flanked at its amino- and carboxy-termini
by a first and a second NLS. The first and second NLSs may be the same or
different. A discussion of NLSs and a list of NLSs can be found in
Boulikas (1993) Crit. Rev. Eukaryotic Gene Expression 3:193-227, and
references cited therein.
Approaches for identifying NLSs include: (1) gene fusion experiments
between a candidate NLS-coding DNA segment and the gene coding for a
cytoplasmic protein (see, e.g., Silver et al. (1984) Proc. Natl. Acad.
Sci. U.S.A. 81:5951; Moreland et al. (1987) Mol. Cell. Biol. 7:4048; and
Picard et al. (1987) EMBO J. 6:3333); (2) nuclear import of nonnuclear
proteins conjugated to synthetic NLS peptides (see, e.g., Goldfarb et al.
(1986) Nature 322:641; Markland et al. (1987) Mol. Cell. Biol. 7:4255; and
Chelsky et al. (1989) Mol. Cell. Biol. 9:2487); and (3) site-directed
mutagenesis of a specific segment of a nuclear protein, resulting in its
cytoplasmic retention (see, e.g., Greenspan et al. (1988) J. Virol.
62:3020; van Etten et al. (1989) Cell 58:669; and Boulukos et al. (1989)
Mol. Cell. Biol.9:5718).
Preferred NLSs include PKKKRKV (SEQ ID NO:10) and KKKRKVC (SEQ ID NO:11)
from the SV40 large T antigen (see, Kalderon et al. (1984) Cell 39:499),
GKKRSKA (SEQ ID NO:12) from yeast histone H2B (see, Moreland et al. (1987)
Mol. Cell. Biol. 7:4048), KRPRP (SEQ ID NO:13) from adenovirus E1A (see,
Lyons et al. (1987) Mol. Cell. Biol. 7:2451), GNKAKRQRST (SEQ ID NO:14)
from the v-rel oncogene of the avian reticuloendotheliosis retrovirus
strain T (see, Gilmore et al. (1988) J. Virol. 62:703), GGAAKRVKLD (SEQ ID
NO:15) from the human c-myc oncoprotein (see, Chelsky et al. (1989) Mol.
Cell. Biol. 9:2487), SALIKKKKKMAP (SEQ ID NO:16) from the murine c-abl
(IV) gene product (see, Van Etten et al. (1989) Cell 58:669), RKLKKLGN
(SEQ ID NO:17) from the human or rat androgen receptor (see,
Guiochon-Mantel et al. (1989) Cell 57:1147), PQPKKKP (SEQ ID NO:18) from
protein p53 (see, Dang et al. (1989) J. Biol. Chem. 264:18019)), ASKSRKRKL
(SEQ ID NO:19) from viral Jun, a transcription factor of the AP-1 complex
(see, Chida et al. (1992) Proc. Natl. Acad. Sci. USA 89:4290), KKKYK (SEQ
ID NO:20) and KKKYKC (SEQ ID NO:21), both of which are from the human
immunodeficiency virus matrix protein (see, Bukrinsky et al. (1993) Nature
365:666), KSKKK (SEQ ID NO:22) from the human immunodeficiency virus
matrix 2 protein (see, Bukrinsky et al. (1993), supra), AKRVKL (SEQ ID
NO:6) and KRVKLC (SEQ ID NO:23) both of which are from the human c-myc
oncoprotein (see, Chelsky et al. (1989), supra), and derivatives and
mimetics thereof that are effective as an NLS.
The polypeptide inhibitors of the present invention may be synthesized by
conventional techniques known in the art, for example, by chemical
synthesis such as solid phase peptide synthesis. Such methods are known to
those skilled in the art. In general, these methods employ either solid or
solution phase synthesis methods, well known in the art. See, e.g., J. M.
Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce
Chemical Co., Rockford, Ill. (1984) and G. Barany and R. B. Merrifield,
The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J.
Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid
phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide
Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer,
Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for
classical solution synthesis.
In general, these methods comprise the sequential addition of one or more
amino acids or suitably protected amino acids to a growing peptide chain.
Normally, either the amino or carboxyl group of the first amino acid is
protected by a suitable protecting group. The protected or derivatized
amino acid can then be either attached to an inert solid support or
utilized in solution by adding the next amino acid in the sequence having
the complementary (amino or carboxyl) group suitably protected, under
conditions suitable for forming the amide linkage. The protecting group is
then removed from this newly added amino acid residue and the next amino
acid (suitably protected) is then added, and so forth. After all the
desired amino acids have been linked in the proper sequence, any remaining
protecting groups and any solid support are removed either sequentially or
concurrently to afford the final polypeptide. By simple modification of
this general procedure, it is possible to add more than one amino acid at
a time to a growing chain, for example, by coupling (under condition that
do not racemize chiral centers) a protected tripeptide with a properly
protected dipeptide to form, after deprotection, a pentapeptide.
Typical protecting groups include t-butyloxycarbonyl (Boc),
9-fluorenylmethoxycarbonyl (Fmoc), benxyloxycarbonyl (Cbz),
p-toluenesulfonyl (Tos); 2,4-dinitrophenyl, benzyl (Bzl),
biphenylisopropyloxy-carboxycarbonyl, cyclohexyl, isopropyl, acetyl,
o-nitrophenylsulfonyl, and the like. Of these, Boc and Fmoc are preferred.
Typical solid supports are generally cross-linked polymeric materials.
These include divinylbenzene cross-linked styrene-based polymers, for
example, divinylbenzene-hydroxymethylstyrene copolymers,
divinylbenzene-chloromethylstyrene copolymers, and
divinylbenzene-benzhydrylaminopolystyrene copolymers. The
Divinylbenzene-benzhydrylaminopolystyrene copolymers, as illustrated
herein using p-methyl-benzhydrylamine resin, offers the advantage of
directly introducing a terminal amide functional group into the peptide
chain, which function is retained by the chain when the chain is cleaved
from the support.
In one preferred method, the polypeptides are prepared by conventional
solid phase chemical synthesis on, for example, an Applied Biosystems,
Inc. (ABI) 430A peptide synthesizer using a resin that permits the
synthesis of the amide peptide form and using t-Boc amino acid derivatives
(Peninsula Laboratories, Inc.) with standard solvents and reagents.
Polypeptides containing either L- or D-amino acids may be synthesized in
this manner. Polypeptide composition is confirmed by quantitative amino
acid analysis and the specific sequence of each peptide may be determined
by sequence analysis.
Alternatively, the polypeptides can be produced by recombinant DNA
techniques by synthesizing DNA encoding the desired polypeptide, along
with an ATG initiation codon. Once coding sequences for the desired
polypeptides have been synthesized or isolated, they can be cloned into
any suitable vector for expression. Numerous cloning vectors are known to
those of skill in the art, and the selection of an appropriate cloning
vector is a matter of choice. Examples of recombinant DNA vectors for
cloning and host cells which they can transform include the bacteriophage
.lambda. (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230
(gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1
(gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria),
pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61
(Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19
(Saccharomyces) and bovine papilloma virus (mammalian cells). See,
generally, DNA Cloning: Vols. I & II, supra; Sambrook et al., supra; B.
Perbal, supra. Insect cell expression systems, such as baculovirus
systems, can also be used and are known to those of skill in the art and
described in, e.g., Summers and Smith, Texas Agricultural Experiment
Station Bulletin No. 1555 (1987). Materials and methods for
baculovirus/insect cell expression systems are commercially available in
kit form from, inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit).
The gene can be placed under the control of a promoter, ribosome binding
site (for bacterial expression) and, optionally, an operator (collectively
referred to herein as "control" elements), so that the DNA sequence
encoding the desired polypeptide is transcribed into RNA in the host cell
transformed by a vector containing this expression construction. The
coding sequence may or may not contain a signal peptide or leader
sequence. Heterologous leader sequences can be added to the coding
sequence which cause the secretion of the expressed polypeptide from the
host organism. Leader sequences can be removed by the host in
post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;
4,425,437; 4,338,397.
Other regulatory sequences may also be desirable which allow for regulation
of expression of the protein sequences relative to the growth of the host
cell. Such regulatory sequences are known to those of skill in the art,
and examples include those which cause the expression of a gene to be
turned on or off in response to a chemical or physical stimulus, including
the presence of a regulatory compound. Other types of regulatory elements
may also be present in the vector, for example, enhancer sequences.
The control sequences and other regulatory sequences may be ligated to the
coding sequence prior to insertion into a vector, such as the cloning
vectors described above. Alternatively, the coding sequence can be cloned
directly into an expression vector which already contains the control
sequences and an appropriate restriction site.
In some cases it may be necessary to modify the coding sequence so that it
may be attached to the control sequences with the appropriate orientation;
i.e., to maintain the proper reading frame. It may also be desirable to
produce mutants or analogs of the polypeptide of interest. Mutants or
analogs may be prepared by the deletion of a portion of the sequence
encoding the protein, by insertion of a sequence, and/or by substitution
of one or more nucleotides within the sequence. Techniques for modifying
nucleotide sequences, such as site-directed mutagenesis, are well known to
those skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning,
Vols. I and II, supra; Nucleic Acid Hybridization, supra.
The expression vector is then used to transform an appropriate host cell. A
number of mammalian cell lines are known in the art and include
immortalized cell lines available from the American Type Culture
Collection (ATCC), such as, but not limited to, Chinese hamster ovary
(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney
cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),
Madin-Darby bovine kidney ("MDBK") cells, as well as others. Similarly,
bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus
spp., will find use with the present expression constructs. Yeast hosts
useful in the present invention include inter alia, Saccharomyces
cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,
Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,
Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect
cells for use with baculovirus expression vectors include, inter alia,
Aedes aegypti, Autographa californica, Bombyx mori, Drosophila
melanogaster, Spodoptera frugiperda, and Trichoplusia ni. The proteins may
also be expressed in Trypanosomes.
Depending on the expression system and host selected, the proteins of the
present invention are produced by growing host cells transformed by an
expression vector described above under conditions whereby the protein of
interest is expressed. The protein is then isolated from the host cells
and purified. If the expression system secretes the protein into growth
media, the protein can be purified directly from the media. If the protein
is not secreted, it is isolated from cell lysates. The selection of the
appropriate growth conditions and recovery methods are within the skill of
the art. Once purified, the amino acid sequences of the proteins can be
determined, i.e., by repetitive cycles of Edman degradation, followed by
amino acid analysis by HPLC. Other methods of amino acid sequencing are
also known in the art.
As explained above, peptide mimetics which structurally and functionally
mimic the peptide inhibitors described above will also find use herein and
may be generated using the following strategies and procedures. Generally,
mimetics are designed based on information obtained by systematic
replacement of L-amino acids by D-amino acids or, in the case of a
polypeptide inhibitor that is made of D-amino acids, the systematic
replacement of D-amino acids by L-amino acids, replacement of side chain
moieties by a methyl group or pseudoisosteric groups with different
electronic properties (see Hruby et al. (1990) Biochem. J. 268:249-262),
and by systematic replacement of peptide bonds in the above described
polypeptide inhibitors with amide bond replacements. For example,
analogues containing amide bond surrogates may be used to investigate
aspects of peptide structure and function, such as rotational freedom in
the backbone, intra- and intermolecular hydrogen-bond patterns,
modifications of local and total polarity and hydrophobicity, and oral
bioavailability.
Local conformational constraints can also be introduced to determine
conformational requirements for activity of a candidate polypeptide
mimetic inhibitor of nuclear translocation. For example,
.beta.,.beta.-disubstituted amino acids may be used to examine the effects
of conformational constraints on peptide activity (see, e.g., Manning et
al. (1982) J. Med. Chem. 25:408-414; Mosberg et al. (1983) Proc. Natl.
Acad. Sci. USA 106:506-512; Pelton et al. (1985) Proc. Natl. Acad. Sci.
USA 82:236-239).
The mimetics can include isosteric amide bonds such as .psi.›CH.sub.2 S!,
.psi.›CH.sub.2 NH!, .psi.›CSNH.sub.2 !, .psi.›NHCO!, .psi.›COCH.sub.2 !
and .psi.›(E) or (Z) CH.dbd.CH! (see, for review, Spatola (1983) in
"Chemistry and Biochemistry of Amino Acids, Peptides and Proteins," Volume
VII, (Weinstein, ed.), Marcel Dekker, N.Y., 267-357). Structures which
mimic the tetrahedral transition state associated with hydrolysis of a
substrate bond can also be present and include hydroxymethylene,
fluoroketone moieties and phosphoramidate transition state mimics
(Buhlmayer et al. (1988) J. Med. Chem. 31:1839; Sham et al. (1988) FEBS
Lett. 220:299; Matthews (1988) Acc. Chem. Res. 21:333). Cyclic amino acid
analogues may be used to constrain amino acid residues to particular
conformational states, e.g., .alpha..alpha.'- and
.beta..beta.'-substituted cyclic amino acids such as
1-aminocyclopentanecarboxylic acid (cycloleucine) and
.beta.,.beta.-cyclopentamethylene-.beta.-mercaptopropionic acid (see Hruby
et al. (1990), supra).
The mimetics can also include mimics of inhibitor peptide secondary
structure--structures which can model the 3-dimensional orientation of
amino acid residues into the known secondary conformations of
proteins--including .beta.-turn mimetics, such as phenoxathin ring system,
and .beta.-sheet mimics, such as epindolidione structures. Design,
synthesis and conformational analysis of an .alpha.-helix inducing
template has been described (Kemp et al. (1988) Tetrahedron Lett. 29:4931;
Kemp et al. (1988) Tetrahedron Lett. 29:4935).
Similarly, peptoids will find use herein. Peptoids are oligomers of
N-substituted amino acids (Simon et al. (1972), supra), and can be used as
motifs for the generation of chemically diverse libraries of novel
molecules, which can then be tested for nuclear translocation inhibitory
activity. The monomers may incorporate t-butyl-based side-chain and 9-
fluorenylmethoxy-carbonyl .alpha.-amine protection. Oligomerization of the
peptoid monomers may be performed by, for example, in situ activation by
either benzotriazol-1-yloxytris (pyrrolidino) phosphonium
hexafluorphosphate or bromotris (pyrrolidino) phosphonium
hexafluorophosphate. Other steps are identical to conventional peptide
synthesis using .alpha.-(9-fluorenylmethoxycarbonyl) amino acids.
Oligopeptoids may be identified which have activities comparable to the
corresponding inhibitory polypeptides and, thus, are useful as inhibitors
of nuclear translocation (see Simon et al. (1992), supra).
Peptide ligands that exhibit nuclear translocation inhibitory activity can
be developed by using a biological expression system (see Christian et al.
(1992) J. Mol. Biol. 227:711-8; Devlin et al. (1990) Science 249:404-406;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382). The use of
such systems allows the production of large libraries of random peptide
sequences and the screening of these libraries for peptide sequences that
have desired biochemical activity. The libraries may be produced by
cloning synthetic DNA that encodes random peptide sequences into
Escherichia coli expression vectors. In the filamentous phage system,
foreign peptide sequences can be expressed on the surface of the
infectious phage (see Smith (1985) Science 228:1315-1317; Parmley et al.
(1988) Gene 73:305-318)
For example, a library may be made by ligating into an appropriate phage, a
synthetic DNA fragment containing a degenerate coding sequence
(NNK).sub.n, where N stands for an equal mixture of the deoxynucleotides
G, A, T, and C, K stands for an equimolar mixture of G and T, and n stands
for the number of amino acid residues desired in the product peptide.
Phage are screened for expression of inhibitory activity. Those that
express inhibitory activity can be cloned and propagated, their DNAs
sequenced to determine the amino acid sequences of their expressed
polypeptide which can be assessed for their ability to inhibit nuclear
translocation.
Large libraries of polypeptide inhibitors can also be constructed by
concurrent synthesis of overlapping peptides as described in U.S. Pat. No.
4,708,871 to Geysen. The solid support is generally a polyethylene or
polypropylene rod onto which is graft polymerized a vinyl monomer
containing at least one functional group to produce polymeric chains on
the carrier. The functional groups are reacted to provide primary or
secondary amine groups w | | |
