U.S. patent application number 10/646267 was filed with the patent office on 2004-10-28 for methods and means for inhibition of cdk4 activity.
Invention is credited to Ball, Kathryn Lindsay, Lane, David Philip.
Application Number | 20040214765 10/646267 |
Document ID | / |
Family ID | 26309279 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214765 |
Kind Code |
A1 |
Ball, Kathryn Lindsay ; et
al. |
October 28, 2004 |
Methods and means for inhibition of CDK4 activity
Abstract
p21.sup.WAF1 interacts with cyclin D1 and Cdk4. Peptide
fragments of p21 inhibit the interaction and/or affect Cdk4
activity. The peptides, derivative peptides and non-peptidyl
mimetics thereof are useful in affecting activity of Cdk4, such as
RB phosphorylation, and cellular proliferation, indicative of
therapeutic usefulness in treatment of tumours and other
hyperproliferative disorders. Assay and screening methods allow
identification of such modulators, especially inhibitors, of Cdk4
activity.
Inventors: |
Ball, Kathryn Lindsay;
(Dundee, GB) ; Lane, David Philip; (St Andrews,
GB) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
26309279 |
Appl. No.: |
10/646267 |
Filed: |
August 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646267 |
Aug 22, 2003 |
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09180269 |
Jul 8, 1999 |
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09180269 |
Jul 8, 1999 |
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PCT/GB97/01250 |
May 8, 1997 |
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Current U.S.
Class: |
435/7.1 ;
514/18.9; 514/19.3 |
Current CPC
Class: |
C07K 14/4703 20130101;
A61P 35/00 20180101; A61P 43/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
May 8, 1996 |
GB |
9609521.1 |
Oct 9, 1996 |
GB |
9621314.5 |
Claims
1. A method of inhibiting the activity of a G1 cdk, the method
including contacting said cdk with a substance that includes a
peptide of 40 amino acids or less, the peptide including the motif:
Kxxrryfzp (wherein x may be any amino acid, y and z may be
hydrophobic, and each of the underlined residues may be absent or
different).
2. A method according to claim 1 wherein y or/and z is, or are
independently, any one of: alanine, valine, leucine, isoleucine,
proline, phenylalanine, tryptophan, methionine.
3. A method according to claim 1, wherein said peptide is a
fragment of p21 or an active portion or derivative thereof.
4. A method according to claim 1, wherein said peptide consists of
residues 16-35 of the p21.sup.WAF1 amino acid sequence or an active
portion or derivative thereof.
5. A method according to claim 3 or claim 4, wherein said peptide
is a said active portion or a said derivative and said active
portion or said derivative has at least 80% identity with p21 over
a window of at least 5 amino acids.
6. A method according to claim 1 wherein said peptide is coupled to
a carrier molecule.
7. A method according to claim 6, wherein the carrier molecule has
the sequence RQIKIWFQNRRMKWKK.
8. A method according to claim 1 wherein the peptide binds to a G1
cyclin or a G1 cdk.
9. An assay method for a compound with ability to modulate
interaction or binding between a peptide as defined in claim 1 and
a G1 cyclin and/or a G1 cdk, the method including: (a) bringing
into contact said peptide, a substance including a said cyclin or
an active portion or derivative thereof, and/or a substance
including a said cdk or an active portion or derivative thereof,
and a test compound, under conditions wherein, in the absence of
the test compound being an inhibitor of interaction or binding of
said peptide and one or more of said substances, said peptide and
one or more of said substances interact or bind; and (b)
determining interaction or binding between said peptide and one or
more of said substances.
10. A method according to claim 9, wherein a compound is
additionally tested for ability to modulate a p21-mediated effect
on activity of a G1 cdk.
11. A method according to claim 1 or claim 10 wherein the cdk
activity includes Rb phosphorylation.
12. A method according to claim 1 or claim 10 wherein induction of
cell cycle arrest is tested.
Description
[0001] The present invention relates to substances and their
therapeutic use, and in particular to the identification of regions
of p.sub.21.sup.WAF1 that bind to cyclin dependent kinases,
specifically Cdk4, and/or cyclin D1, and to substances, fragments
and mimetics based on this region. The present invention also
relates to pharmaceutical compositions comprising these molecules
and their use in therapeutic applications for treating
hyperproliferative disorders, such as cancer and psoriasis, and
compositions comprising these Molecules and their use in
applications relating to growth in eukaryotic cells. The invention
also relates to assay methods and means for identifying substances
useful for interfering with p21/Cdk4/cyclin interaction, and
preferably inhibiting Cdk4 activity.
[0002] The tumour suppressor function of p53 is linked to a
DNA-damage inducible cell cycle checkpoint pathway (Kastan et al.,
1991), in which p53 can induce either growth arrest (Agarwal et
al., 1995) or apoptosis (Clarke et al., 1993; Lowe et al., 1993;
Merritt et al., 1994) in the damaged cells. The biochemical
activity of p53 most tightly associated with tumour suppression and
growth arrest involves an ionising radiation-dependent activation
of sequence-specific transcriptional activity (Kastan et al., 1991;
Lu and Lane, 1993; Pietenpol, et al., 1994). p53 induces the
transcription of a number of genes, the products of which play a
direct role in mediating growth arrest. These p53-inducible
negative regulators of cell proliferation include: the cyclin
dependent kinase inhibitor (CKI), p21.sup.WAF1 (El-Deiry et al.,
1993; Harper et al., 1993; Xiong et al., 1993; Gu et al., 1993);
ran apoptosis promoting protein, Bax (Miyashita and Reed, 1995);
the insulin growth factor binding protein IGF-BP3 (Buckbinder et
al., 1995); and Gadd45 (Kastan et al., 1992), a potent inhibitor of
cell proliferation with an as yet unclear biochemical function
(Kearsey et al., 1995).
[0003] A common event in the development of human neoplasia is the
inactivation of a DNA damage-inducible cell cycle checkpoint
pathway regulated by p53 (Hollstein et al., 1991; Lane, 1992;
Agrawal et al., 1995) A variety of mechanisms can lead to the
functional inactivation of the p53 pathway, including: missense
mutations within, or deletions of the p53 gene, inactivation of
wild type p53 protein function by interaction with the oncogenic
cellular protein mdm-2 (Momand et al., 1992), or the inability to
induce downstream effector molecules, such as p21.sup.WAF1 (Deng et
al., 1995; Waldman et al., 1995).
[0004] Our growing knowledge of the molecular mechanisms underlying
the transformation of mammalian cells offers the opportunity to
create rationally designed inhibitors of specific biochemical
processes essential to uncontrolled cell proliferation or cancer.
Recent developments have shown that the reactivation of the p53
pathway in some human tumours could in theory be achieved by: (i)
activating the biochemical function of mutant p53 protein
(Halazonetis and Kandil, 1993; Hupp et al., 1993), possibly using
small peptides as leads for drug design (Hupp et al., 1995); (ii)
disrupting the interaction of the oncogene mdm-2 and wild type p53
through the use of peptide-mimetic inhibitors of complex formation
(Picksley et al., 1994); (iii) restoring or mimicking the function
of the downstream effector molecule p21.sup.WAF1, which on its own
is capable of mediating growth suppression (El-Deiry et al., 1993;
Eastham et al., 1995).
[0005] p21.sup.WAF1 is an inhibitor of both the G1 cyclin dependent
protein kinases (CDKS; which control the progression from G1 into S
phase) (Harper et al., 1995) and proliferating cell nuclear antigen
(PCNA; an essential DNA-replication factor) (Florez-Rozas et al.,
1994; Waga et al., 1994). Thus, inhibition of the function of
either CDKs or PCNA provides, in theory, two distinct avenues for
development of drug discovery programmes which are based on the
activity of p21.sup.WAF1. The PCNA binding function of p21.sup.WAF1
can be mimicked by a 20-amino acid peptide derived from the
C-terminal domain of p21.sup.WAF1 and this peptide is sufficient to
partially inhibit SV40 replication in vitro (Warbrick et al.,
1995).
[0006] Despite its PCNA binding role, the primary function of the
p21.sup.WAF1 protein as a growth suppressor appears to be
inhibition of the G1 cyclin-CDK complexes (Chen et al., 1995;
Harper et al., 1995; Luo et al., 1995; Nakanishi et al., 1995b).
Luo et al. (1995) reported the N-terminal domain of p21, composed
of residues 1-75, to act as a CDK-inhibitor in vitro, inhibiting
cyclin E-Cdk2.
[0007] The present invention concerns (i) the elucidation of the
molecular mechanism of cyclin D1 -Cdk4 complex inhibition by
p21.sup.WAF1, and (ii) the identification of peptide mimetics of
p21.sup.WAF1 inhibitory activity, through the examination of the
binding and inhibitory properties of a series of synthetic peptides
based on the amino acid sequence of p21.sup.WAF1. Our studies found
that two peptides derived from the N-terminal domain of
p21.sup.WAF1 have biochemical activity; a peptide 4 (residues
46-65) forms a stable complex with Cdk4, but has no inhibitory
activity, while a peptide 2 (residues 16-35) binds to cyclin D1 and
inhibits Cdk4 activity with a I.sub.0.5 of 2 .mu.M.
[0008] These data define for a cyclin binding site on p21.sup.WAF1
and suggest that one mechanism involved in the CDK inhibitory
action of p21.sup.WAF1 employs binding to the cyclin subunit of the
CDK holoenzyme. This has lead us to propose that p21.sup.WAF1 can
inhibit Cdk4 activity allosterically through conformational or (ii)
interfering with the cyclin-Cdk interaction or (iii) interfering
with the cyclin-Cdk-substrate interaction changes in the structure
of cyclin D1. Furthermore, peptides based on the C-terminal
sequence of p21.sup.WAF1 interact with both cyclin D1 and Cdk4, and
are potent inhibitors of Cdk4 activity, with a peptide (peptide 10
herein) composed of residues 141-160 having an I.sub.0.5 of 0.1
.mu.M. We show that both of the inhibitory peptides bind at
physiologically relevant sites on cyclin D1 and/or Cdk4, and that
they display specificity mimicking that of full length
p21.sup.WAF1. Importantly, the potency of the C-terminal peptide is
improved by making a single amino acid substitution (D-A at
position 149). We have mapped the inhibitory component of this
peptide using alanine mutation analysis and show that it is
distinct from the PCNA interaction domain, which also resides in
the C-terminal region of the p21.sup.WAF1 protein.
[0009] Remarkably, a stretch of just five amino acids contains the
Cdk4 inhibitory motif and a single conservative mutation at either
of two hydrophobic amino acid residues completely abolishes the
inhibitory activity of the peptide. These data have exciting
implications for the mechanism of action of p21.sup.WAF1 protein
and represent a starting point for a drug design programme aimed at
producing synthetic molecules functioning as tumour suppressors
downstream of p53.
[0010] Accordingly, in one aspect, the present invention provides a
substance which has the property of inhibiting Cdk4, said substance
comprising:
[0011] (i) a peptide fragment consisting of the motif xyLzF,
wherein y and z are any amino acid and x is preferably R, or a
derivative of said peptide fragment; or,
[0012] (ii) a functional mimetic of said peptide fragment.
[0013] In a further aspect, the present invention provides the
above substance for use in a method of medical treatment.
[0014] In a further aspect, the present invention provides the use
of a substance which has the property of inhibiting Cdk4 in the
preparation of a medicament for the treatment of a
hyperproliferative disorder, said substance comprising:
[0015] (i) fragment of the C-terminal portion of p21.sup.WAF1, or
an active portion or derivative thereof; or,
[0016] (ii) a peptide fragment including the motif xyLzF, wherein y
and z are any amino acid and x is preferably R, or a derivative of
said peptide fragment; or,
[0017] (iii) a functional mimetic of (i) or (ii).
[0018] In a preferred embodiment, the C-terminal portion of
p21.sup.WAF1 consisting of the peptide motif KRRLIFSK was found to
completely inhibit cyclin-Cdk4 activity and to prevent
phosphorylation of pRb (see FIG. 6).
[0019] In a further aspect, the present invention provides a
substance which has the property of binding to Cdk4 for use in a
method of medical treatment, said substance comprising:
[0020] (i) a fragment of the p21.sup.WAF1 protein consisting of
residues 46-65 of the p21.sup.WAF1 amino acid sequence, or an
active portion or derivative thereof; or,
[0021] (ii) a functional mimetic of said fragment.
[0022] In a further aspect, the present invention provides the use
of a substance which has the property of binding Cdk4 in the
preparation of a medicament for the treatment of a
hyperproliferative disorder, said substance comprising:
[0023] (i) a fragment of the p21.sup.WAF1 protein consisting of
residues 46-65 of the p21.sup.WAF1 amino acid sequence, or an
active portion or derivative thereof; or,
[0024] (ii) a functional mimetic of said fragment.
[0025] In a further aspect, the present invention provides a
substance which has the properties of binding cyclin D and/or
inhibiting Cdk4 for use in a method of medical treatment, said
substance comprising:
[0026] (i) a fragment of the p21.sup.WAF1 protein consisting of
residues 16-35 of the p21.sup.WAF1 amino acid sequence, or an
active portion or derivative thereof; or,
[0027] (ii) a functional mimetic of said fragment.
[0028] In a further aspect, the present invention provides the use
of a substance which has the property of binding cyclin D1 and/or
inhibiting Cdk4 in the preparation of a medicament for the
treatment of a hyperproliferative disorder, said substance
comprising:
[0029] (i) a fragment of the p21.sup.WAF1 protein consisting of
residues 16-35 of the p21.sup.WAF1 amino acid sequence, or an
active portion or derivative thereof; or,
[0030] (ii) a functional mimetic of said peptide fragment.
[0031] Based on experimental evidence included below showing
residues involved in binding of peptide 2 (residues 16-35), and the
crystal structure available for p27 (related to p21), the following
general formula for peptides useful in accordance with various
aspects of the present invention is provided:
KxxRRyFzP
[0032] wherein x may be any amino acid, y and z may be hydrophobic,
and each of the underlined residues may be absent or different,
i.e. another amino acid. Hydrophobic residues may be alanine,
valine, leucine, isoleucine, proline, phenylalanine, tryptophan,
methionine. Either or both of the amino acids R may be substituted
by other basic residues, particularly lysine (K) or histidine
(H).
[0033] In the present invention, "an active portion" means a
peptide which is less than the fragment of the p21.sup.WAF1 amino
acid sequence, but which retains the relevant property mentioned
above.
[0034] In the present invention, "functional mimetic" means a
substance which may not contain an active portion of the
p21.sup.WAF1 amino acid sequence and is probably not a peptide at
all, but which has the relevant property mentioned above.
[0035] In the present invention, "a derivative" means a peptide
modified by varying its amino acid sequence, eg by manipulation of
the nucleic acid encoding the peptide or by altering the peptide
itself. Such derivatives of the natural amino acid sequence may
involve insertion, addition, deletion or substitution of one or
more amino acids, without fundamentally altering the essential
activity of the peptides. An example of a derivative is the
p21.sup.WAF1 mutant in which A was substituted for D at position
149 of the full length protein, this mutant having enhanced cyclin
D1 -Cdk4 inhibitory activity.
[0036] Preferred substances according to certain embodiments of the
present invention do not bind PCNA, and/or do not interfere with
p21 interaction or binding with PCNA.
[0037] Cell cycle arrest may be induced by various aspects
according to the present invention in Rb negative and/or Rb
positive cells, as exemplified experimentally below.
[0038] In a further aspect, the present invention provides
pharmaceutical compositions comprising one or more of the above
substances in combination with a pharmaceutically acceptable
carrier.
[0039] In a further aspect, the present invention relates to
compositions comprising one or more of the above substances and
their use in controlling the growth of eukaryotic cells, eg as a
food preservative or as an agent to promote the growth of
plants.
[0040] In a further aspect, the present invention provides
compounds comprising any of the above substances coupled to carrier
molecules, enabling the compounds to be delivered to cells in vivo.
In one embodiment, the carrier molecule is a 16 aa peptide sequence
derived from the homeodomain of Antennapedia (e.g. as sold under
the name "Penetratin") which can be coupled to one of the above
substances via a terminal Cys residue. Alternatively, as in the
examples described below, a carrier peptide (having the sequence
RQIKIWFQNRRMKWKK) can be synthesised so it is directly attached to
peptide fragments. The "Penetratin" molecule and its properties are
described in WO91/18981.
[0041] Thus, the present invention in various aspects provides for
interfering with or interrupting interaction between p21 and cyclin
D1 and/or Cdk4 using an appropriate agent.
[0042] Such an agent may be capable of blocking binding between a
site located at amino acid residues identified herein as being
involved in and/or important for binding or interaction with cyclin
D1 and/or Cdk4.
[0043] The full sequence of the p21 protein has been elucidated and
is set out in WO95/13375, WO93/12251 and WO95/06415 which are
incorporated herein by reference.
[0044] Such agents may be identified by screening techniques which
involve determining whether an agent under test inhibits or
disrupts the binding of p21 protein or a suitable fragment,
derivative, analogue or functional mimetic thereof, with cyclin D1
and/or Cdk4, or a relevant fragment, derivative, analogue or
functional mimetic thereof.
[0045] Suitable fragments of p21 include those which include
residues as identified herein. Smaller fragments, and derivatives,
analogues and functional mimetics of this fragment may similarly be
employed, e.g. peptides identified using a technique such as
alanine scanning.
[0046] In a further aspect of the invention, whereas assays using
the peptides described herein and the use of the peptides are
described in the context of modulating the interaction of p21 with
cyclin D1 and/or Cdk4, these peptides may also be useful as p21
mimetics to inhibit the interaction of p21 and other cyclin-Cdk
interactions, particularly G1 complexes such as cyclin E-Cdk2. Thus
the various described embodiments of the invention above and below
herein with regard to cyclin D1 and/or Cdk4 is applicable mutatis
mutandis to these other cyclins and/or Cdks respectively.
[0047] Screening methods and assays are discussed in further detail
below.
[0048] One class of agents that can be used to disrupt the binding
of p21 and cyclin D1 and/or Cdk4 are peptides based on the sequence
motifs of p21 that interact with cyclin D1 and/or Cdk4. Such
peptides tend to be small molecules, and may be about 40 amino
acids in length or less, preferably about 35 amino acids in length
or less, more preferably about 30 amino acids in length, or less,
more preferably about 25 amino acids or less, more preferably about
20 amino acids or less, more preferably about 15 amino acids or
less, more preferably about 10 amino acids or less, or 9, 8, 7, 6 5
or less in length. The present invention also encompasses peptides
which are sequence variants or derivatives of a wild type p21
sequence.
[0049] Preferably, the amino acid sequence shares homology with a
fragment of the relevant p21 fragment sequence shown preferably at
least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or
85% homology, or at least about 90% or 95% homology. Thus, a
peptide fragment of p21 may include 1, 2, 3, 4, 5, greater than 5,
or greater than 10 amino acid alterations such as substitutions
with respect to the wild-type sequence.
[0050] A derivative of a peptide for which the specific sequence is
disclosed herein may be in certain embodiments the same length or
shorter than the specific peptide. In other embodiments the peptide
sequence or a variant thereof may be included in a larger peptide,
as discussed above, which may or may not include an additional
portion of p21. 1, 2, 3, 4 or 5 or more additional amino acids,
adjacent to the relevant specific peptide fragment in p21, or
heterologous thereto may be included at one end or both ends of the
peptide.
[0051] As is well-understood, homology at the amino acid level is
generally in terms of amino acid similarity or identity. Similarity
allows for "conservative variation", i.e. substitution of one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as arginine for lysine, glutamic for aspartic
acid, or glutamine for asparagine. Similarity may be as defined and
determined by the TBLASTN program, of Altschul et al. (1990) J.
Mol. Biol. 215: 403-10, which is in standard use in the art.
Homology may be over the full-length of the relevant peptide or
over a contiguous sequence of about 5, 10, 15, 20, 25, 30 or 35
amino acids, compared with the relevant wild-type amino acid
sequence.
[0052] As noted, variant peptide sequences and peptide and
non-peptide analogues and mimetics may be employed, as discussed
further below.
[0053] Various aspects of the present invention provide a
substance, which may be a single molecule or a composition
including two or more components, which includes a peptide fragment
of p21 which includes a sequence as recited above and/or disclosed
elsewhere herein, a peptide consisting essentially of such a
sequence, a peptide including a variant, derivative or analogue
sequence, or a non-peptide analogue or mimetic which has the
ability to bind cyclin D1 and/or Cdk4 and/or disrupt or interfere
with interaction between p21 and cyclin D1 and/or Cdk 4.
[0054] Variants include peptides in which individual amino acids
can be substituted by other amino acids which are closely related
as is understood in the art and indicated above.
[0055] Non-Peptide Mimetics of Peptides are Discussed Further
Below.
[0056] As noted, a peptide according to the present invention and
for use in various aspects of the present invention may include or
consist essentially of a fragment of p21 as disclosed, such as a
fragment whose sequence is given above. Where one or more
additional amino acids are included, such amino acids may be from
p21 or may be heterologous or foreign to p21. A peptide may also be
included within a larger fusion protein, particularly where the
peptide is fused to a non-p21 (i.e. heterologous or foreign)
sequence, such as a polypeptide or protein domain.
[0057] The invention also includes derivatives of the peptides,
including the peptide linked to a coupling partner, e.g. an
effector molecule, a label, a drug, a toxin and/or a carrier or
transport molecule. Techniques for coupling the peptides of the
invention to both peptidyl and non-peptidyl coupling partners are
well known in the art. In one embodiment, the carrier molecule is a
16 aa peptide sequence derived from the homeodomain of Antennapedia
(e.g. as sold under the name "Penetratin"), which can be coupled to
a peptide via a terminal Cys residue. The "Penetratin" molecule and
its properties are described in WO 91/18981.
[0058] Peptides may be generated wholly or partly by chemical
synthesis. The compounds of the present invention can be readily
prepared according to well-established, standard liquid or,
preferably, solid-phase peptide synthesis methods, general
descriptions of which are broadly available (see, for example, in
J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd
edition, Pierce Chemical Company, Rockford, Ill. (1984), in M.
Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis,
Springer Verlag, N.Y.(1984); and Applied Biosystems 430A Users
Manual, ABI Inc., Foster City, Calif.), or they may be prepared in
solution, by the liquid phase method or by any combination of
solid-phase, liquid phase and solution chemistry, e.g. by first
completing the respective peptide portion and then, if desired and
appropriate, after removal of any protecting groups being present,
by introduction of the residue X by reaction of the respective
carbonic or sulfonic acid or a reactive derivative thereof.
[0059] Another convenient way of producing a peptidyl molecule
according to the present invention (peptide or polypeptide) is to
express nucleic acid encoding it, by use of nucleic acid in an
expression system.
[0060] Accordingly the present invention also provides in various
aspects nucleic acid encoding the polypeptides and peptides of the
invention.
[0061] Generally, nucleic acid according to the present invention
is provided as an isolate, in isolated and/or purified form, or
free or substantially free of material with which it is naturally
associated, such as free or substantially free of nucleic acid
flanking the gene in the human genome, except possibly one or more
regulatory sequence(s) for expression. Nucleic acid may be wholly
or partially synthetic and may include genomic DNA, cDNA or RNA.
Where nucleic acid according to the invention includes RNA,
reference to the sequence shown should be construed as reference to
the RNA equivalent, with U substituted for T.
[0062] Nucleic acid sequences encoding a polypeptide or peptide in
accordance with the present invention can be readily prepared by
the skilled person using the information and references contained
herein and techniques known in the art (for example, see Sambrook,
Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short
Protocols in Molecular Biology, John Wiley and Sons, 1992), given
the nucleic acid sequence and clones available. These techniques
include (i) the use of the polymerase chain reaction (PCR) to
amplify samples of such nucleic acid, e.g. from genomic sources,
(ii) chemical synthesis, or (iii) preparing cDNA sequences. DNA
encoding p21 fragments may be generated and used in any suitable
way known to those of skill in the art, including by taking
encoding DNA, identifying suitable restriction enzyme recognition
sites either side of the portion to be expressed, and cutting out
said portion from the DNA. The portion may then be operably linked
to a suitable promoter in a standard commercially available
expression system. Another recombinant approach is to amplify the
relevant portion of the DNA with suitable PCR primers.
[0063] Modifications to the p21 sequences can be made, e.g. using
Site directed mutagenesis, to lead to the expression of modified
p21 peptide or to take account of codon preference in the host
cells used to express the nucleic acid.
[0064] In order to obtain expression of the nucleic acid sequences,
the sequences can be incorporated in a vector having one or more
control sequences operably linked to the nucleic acid to control
its expression. The vectors may include other sequences such as
promoters or enhancers to drive the expression of the inserted
nucleic acid, nucleic acid sequences so that the polypeptide or
peptide is produced as a fusion and/or nucleic acid encoding
secretion signals so that the polypeptide produced in the host cell
is secreted from the cell. Polypeptide can then be obtained by
transforming the vectors into host cells in which the vector is
functional, culturing the host cells so that the polypeptide is
produced and recovering the polypeptide from the host cells or the
surrounding medium. Prokaryotic and eukaryotic cells are used for
this purpose in the art, including strains of E. coli, yeast, and
eukaryotic cells such as COS or CHO cells.
[0065] Thus, the present invention also encompasses a method of
making a polypeptide or peptide (as disclosed), the method
including expression from nucleic acid encoding the polypeptide or
peptide (generally nucleic acid according to the invention). This
may conveniently be achieved by growing a host cell in culture,
containing such a vector, under appropriate conditions which cause
or allow expression of the polypeptide. Polypeptides and peptides
may also be expressed in in vitro systems, such as reticulocyte
lysate.
[0066] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, eukaryotic cells such as mammalian and yeast, and
baculovirus systems. Mammalian cell lines available in the art for
expression of a heterologous polypeptide include Chinese hamster
ovary cells, HeLa cells, baby hamster kidney cells, COS cells and
many others. A common, preferred bacterial host is E. coli.
[0067] Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator fragments, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. Vectors
may be plasmids, viral e.g. phage, or phagemid, as appropriate. For
further details see, for example, Molecular Cloning: a Laboratory
Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor
Laboratory Press. Many known techniques and protocols for
manipulation of nucleic acid, for example in preparation of nucleic
acid constructs, mutagenesis, sequencing, introduction of DNA into
cells and gene expression, and analysis of proteins, are described
in detail in Current Protocols in Molecular Biology, Ausubel et al.
eds., John Wiley & Sons, 1992.
[0068] Thus, a further aspect of the present invention provides a
host cell containing heterologous nucleic acid as disclosed
herein.
[0069] The nucleic acid of the invention may be integrated into the
genome (e.g. chromosome) of the host cell. Integration may be
promoted by inclusion of sequences which promote recombination with
the genome, in accordance with standard techniques. The nucleic
acid may be on an extra-chromosomal vector within the cell, or
otherwise identifiably heterologous or foreign to the cell.
[0070] A still further aspect provides a method which includes
introducing the nucleic acid into a host cell. The introduction,
which may (particularly for in vitro introduction) be generally
referred to without limitation as "transformation", may employ any
available technique. For eukaryotic cells, suitable techniques may
include calcium phosphate transfection, DEAE-Dextran,
electroporation, liposome-mediated transfection and transduction
using retrovirus or other virus, e.g. vaccinia or, for insect
cells, baculovirus. For bacterial cells, suitable techniques may
include calcium chloride transformation, electroporation and
transfection using bacteriophage. As an alternative, direct
injection of the nucleic acid could be employed.
[0071] Marker genes such as antibiotic resistance or sensitivity
genes may be used in identifying clones containing nucleic acid of
interest, as is well known in the art.
[0072] The introduction may be followed by causing or allowing
expression from the nucleic acid, e.g. by culturing host cells
(which may include cells actually transformed although more likely
the cells will be descendants of the transformed cells) under
conditions for expression of the gene, so that the encoded
polypeptide (or peptide) is produced. If the polypeptide is
expressed coupled to an appropriate signal leader peptide it may be
secreted from the cell into the culture medium. Following
production by expression, a polypeptide or peptide may be isolated
and/or purified from the host cell and/or culture medium, as the
case may be, and subsequently used as desired, e.g. in the
formulation of a composition which may include one or more
additional components, such as a pharmaceutical composition which
includes one or more pharmaceutically acceptable excipiencs,
vehicles or carriers (e.g. see below).
[0073] Introduction of nucleic acid encoding a peptidyl molecule
according to the present invention may take place in vivo by way of
gene therapy, to disrupt or interfere with interaction between p21
and cyclin D1 and/or ckd4.
[0074] Thus, a host cell containing nucleic acid according to the
present invention, e.g. as a result of introduction of the nucleic
acid into the cell or into an ancestor of the cell and/or genetic
alteration of the sequence endogenous to the cell or ancestor
(which introduction or alteration may take place in vivo or ex
vivo), may be comprised (e.g. in the soma) within an organism which
is an animal, particularly a mammal, which may be human or
non-human, such as rabbit, guinea pig, rat, mouse or other rodent,
cat, dog, pig, sheep, goat, cattle or horse, or which is a bird,
such as a chicken. Genetically modified or transgenic animals or
birds comprising such a cell are also provided as further aspects
of the present invention.
[0075] This may have a therapeutic aim. (Gene therapy is discussed
below.) Also, the presence of a mutant, allele, derivative or
variant sequence within cells of an organism, particularly when in
place of a homologous endogenous sequence, may allow the organism
to be used as a model in testing and/or studying substances which
modulate activity of the encoded polypeptide in vitro or are
otherwise indicated to be of therapeutic potential. Conveniently,
however, assays for such substances may be carried out in vitro,
within host cells or in cell-free systems.
[0076] Suitable screening methods are conventional in the art. They
include techniques such as radioimmunosassay, scintillation
proximetry assay and ELISA methods. Suitably either the p21 protein
or fragment or cyclinD1 and/or Cdk 4 or fragment, or an analogue,
derivative, variant or functional mimetic thereof, is immobilised
whereupon the other is applied in the presence of the agents under
test. In a scintillation proximetry assay a biotinylated protein
fragment is bound to streptavidin coated scintillant--impregnated
beads (produced by Amersham). Binding of radiolabelled peptide is
then measured by determination of radioactivity induced
scintillation as the radioactive peptide binds to the immobilized
fragment. Agents which intercept this are thus inhibitors of the
interaction.
[0077] In one general aspect, the present invention provides an
assay method for a substance with ability to disrupt interaction or
binding between p21 and cyclin D1 and/or Cdk4, the method
including:
[0078] (a) bringing into contact a substance according to the
invention including a peptide fragment of p21 or a derivative,
variant or analogue thereof as disclosed, a substance including the
relevant fragment of cyclin D1 and/or Cdk4 or a variant, derivative
or analogue thereof, and a test compound, under conditions wherein,
in the absence of the test compound being an inhibitor of
interaction or binding of said substances, said substances interact
or bind; and
[0079] (b) determining interaction or binding between said
substances.
[0080] A test compound which disrupts, reduces, interferes with or
wholly or partially abolishes binding or interaction between said
substances (e.g. including a p21 fragment and including a cyclin D1
and/or Cdk 4 fragment), and which may modulate Cdk4 activity, may
thus be identified.
[0081] Another general aspect of the present invention provides an
assay method for a substance able to bind the relevant region of
p21 as the case may be, the method including:
[0082] (a) bringing into contact a substance which includes a
peptide fragment of p21 which interacts with cyclin D1 and/or Cdk 4
as disclosed, or a variant, derivative or analogue thereof as
disclosed, and a test compound; and
[0083] (b) determining binding between said substance and the test
compound.
[0084] A test compound found to bind to the relevant portion of p21
may be tested for ability to disrupt p21 interaction or binding
with cyclin D1 and/or Cdk 4 and/or ability to affect Cdk4 activity
or other activity mediated by p21 as discussed already above.
[0085] Performance of an assay method according to the present
invention may be followed by isolation and/or manufacture and/or
use of a compound, substance or molecule which tests positive for
ability to interfere with interaction between p21 and cyclin D1
and/or Cdk4 and/or inhibit p21-mediated Cdk4 activity.
[0086] The precise format of an assay of the invention may be
varied by those of skill in the art using routine skill and
knowledge. For example, interaction between substances may be
studied in vitro by labelling one with a detectable label and
bringing it into contact with the other which has been immobilised
on a solid support. Suitable detectable labels, especially for
petidyl substances include .sup.35S-methionine which may be
incorporated into recombinantly produced peptides and polypeptides.
Recombinantly produced peptides and polypeptides may also be
expressed as a fusion protein containing an epitope which can be
labelled with an antibody.
[0087] The protein which is immobilized on-a solid support may be
immobilized using an antibody against that protein bound to a solid
support or via other technologies which are known per se. A
preferred in vitro interaction may utilise a fusion protein
including glutathione-S-transferase (GST). This may be immobilized
on glutathione agarose beads. In an in vitro assay format of the
type described above a test compound can be assayed by determining
its ability to diminish the amount of labelled peptide or
polypeptide which binds to the immobilized GST-fusion polypeptide.
This may be determined by fractionating the glutathione-agarose
beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the
beads may be rinsed to remove unbound protein and the amount of
protein which has bound can be determined by counting the amount of
label present in, for example, a suitable scintillation
counter.
[0088] An assay according to the present invention may also take
the form of an in vivo assay. The in vivo assay may be performed in
a cell line such as a yeast strain or mammalian cell line in which
the relevant polypeptides or peptides are expressed from one or
more vectors introduced into the cell.
[0089] The ability of a test compound to disrupt interaction or
binding between p21 and cyclin D1 and/or Cdk4 may be determined
using a so-called two-hybid assay.
[0090] For example, a polypeptide or peptide containing a fragment
of p21 or cyclin D1/Cdk4 as the case may be, or a peptidyl analogue
or variant thereof as disclosed, may be fused to a DNA binding
domain such as that of the yeast transcription factor CAL 4. The
GAL 4 transcription factor includes two functional domains. These
domains are the DNA binding domain (GAL4 DBD) and the GAL4
transcriptional activation domain (GAL4TAD). By fusing one
polypeptide or peptide to one of those domains and another
polypeptide or peptide to the respective counterpart, a functional
GAL 4 transcription factor is restored only when two polypeptides
or peptides of interest interact. Thus, interaction of the
polypeptides or peptides may be measured by the use of a reporter
gene probably linked to a GAL 4 DNA binding site which is capable
of activating transcription of said reporter gene. This assay
format is described by Fields and Song, 1989, Nature 340; 245-246.
This type of assay format can be used in both mammalian cells and
in yeast. Other combinations of DNA binding domain and
transcriptional activation domain are available in the art and may
be preferred, such as the LexA DNA binding domain and the VP60
transcriptional activation domain.
[0091] To take a Lex/VP60 two hybrid screen by way of example for
the purpose of illustration, yeast or mammalian cells may be
transformed with a reporter gene construction which expresses a
selective marker protein (e.g. encoding .beta.-galactosidase or
luciferase). The promoter of that gene is designed such that it
contains binding site for the LexA DNA-binding protein. Gene
expression from that plasmid is usually very low. Two more
expression vectors may be transformed into the yeast containing the
selectable marker expression plasmid, one containing the coding
sequence for the full length LexA gene linked to a multiple cloning
site. This multiple cloning site is used to clone a gene of
interest, i.e. encoding a p21 or cyclinD1/Cdk4 polypeptide or
peptide in accordance with the present invention, in frame on to
the LexA coding region. The second expression vector then contains
the activation domain of the herpes simplex transactivator VP16
fused to a test peptide sequence or more preferably a library of
sequences encoding peptides with diverse e.g. random sequences.
Those two plasmids facilitate expression from the reporter
construct containing this selectable marker only when the LexA
fusion construct interacts with a polypeptide or peptide sequence
derived from the peptide library.
[0092] A modification of this when looking for peptides or other
substances which interfere with interaction between a p21
polypeptide or peptide and an cyclin D1/Cdk 4 polypeptide or
peptide, employs the p21 or cyclin D1/Cdk4 polypeptide or peptide
as a fusion with the LexA DNA binding domain, and the counterpart
cyclin D1/Cdk4 or p21 polypeptide or peptide as a fusion with VP60,
and involves a third expression cassette, which may be on a
separate expression vector, from which a peptide or a library of
peptides of diverse and/or random sequence may be expressed. A
reduction in reporter gene expression (e.g. in the case of
.beta.-galactosidase a weakening of the blue colour) results from
the presence of a peptide which disrupts the p21/cyclinD1 and/or
Cdk4 interaction, which interaction is required for transcriptional
activation of the .beta.-galactosidase gene. Where a test substance
is not peptidyl and may not be expressed from encoding nucleic acid
within a said third expression cassette, a similar system may be
employed with the test substance supplied exogenously.
[0093] As noted, instead of using LexA and VP60, other similar
combinations of proteins which together form a functional
transcriptional activator may be used, such as the GAL4 DNA binding
domain and the GAL4 transcriptional activation domain.
[0094] When performing a two hybrid assay to look for substances
which interfere with the interaction between two polypeptides or
peptides it may be preferred to use mammalian cells instead of
yeast cells. The same principles apply and appropriate methods are
well known to those skilled in the art.
[0095] The amount of test substance or compound which may be added
to an assay of the invention will normally be determined by trial
and error depending upon the type of compound used. Typically, from
about 0.01 to 100 nM concentrations of putative inhibitor compound
may be used, for example from 0.1 to 10 nM. Greater concentrations
may be used when a peptide is the test substance.
[0096] Compounds which may be used may be natural or synthetic
chemical compounds used in drug screening programmes. Extracts of
plants which contain several characterised or uncharacterised
components may also be used.
[0097] Antibodies directed to the site of interaction in either
protein form a further class of putative inhibitor compounds.
Candidate inhibitor antibodies may be characterised and their
binding regions determined to provide single chain antibodies and
fragments thereof which are responsible for disrupting the
interaction.
[0098] Antibodies may be obtained using techniques which are
standard in the art. Methods of producing antibodies include
immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or
monkey) with the protein or a fragment thereof. Antibodies may be
obtained from immunised animals using any of a variety of
techniques known in the art, and screened, preferably using binding
of antibody to antigen of interest. For instance, Western blotting
techniques or immunoprecipitation may be used (Armitage et al.,
1992, Nature 357: 80-82). Isolation of antibodies and/or
antibody-producing cells from an animal may be accompanied by a
step of sacrificing the animal.
[0099] As an alternative or supplement to immunising a mammal with
a peptide, an antibody specific for a protein may be obtained from
a recombinantly produced library of expressed immunoglobulin
variable domains, e.g. using lambda bacteriophage or filamentous
bacteriophage which display functional immunoglobulin binding
domains on their surfaces; for instance see WO92/01047. The library
may be naive, that is constructed from sequences obtained from an
organism which has not been immunised with any of the proteins (or
fragments), or may be one constructed using sequences obtained from
an organism which has been exposed to the antigen of interest.
[0100] Antibodies according to the present invention may be
modified in a number of ways. Indeed the term "antibody" should be
construed as covering any binding substance having a binding domain
with the required specificity. Thus the invention covers antibody
fragments, derivatives, functional equivalents and homologues of
antibodies, including synthetic molecules and molecules whose shape
mimicks that of an antibody enabling it to bind an antigen or
epitope.
[0101] Example antibody fragments, capable of binding an antigen or
other binding partner are the Fab fragment consisting of the VL,
VH, C1 and CH1 domains; the Fd fragment consisting of the VH and
CH1 domains; the Fv fragment consisting of the VL and VH domains of
a single arm of an antibody; the dAb fragment which consists of a
VH domain; isolated CDR regions and F(ab').sub.2 fragments, a
bivalent fragment including two Fab fragments linked by a
disulphide bridge at the hinge region. Single chain Fv fragments
are also included.
[0102] A hybridoma producing a monoclonal antibody according to the
present invention may be subject to genetic mutation or other
changes. It will further be understood by those skilled in the art
that a monoclonal antibody can be subjected to the techniques of
recombinant DNA technology to produce other antibodies or chimeric
molecules which retain the specificity of the original antibody.
Such techniques may involve introducing DNA encoding the
immunoglobulin variable region, or the complementarity determining
regions (CDRs), of an antibody to the constant regions, or constant
regions plus framework regions, of a different immunoglobulin. See,
for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and
expression of chimeric antibodies are described in EP-A-0120694 and
EP-A-0125023.
[0103] Hybridomas capable of producing antibody with desired
binding characteristics are within the scope of the present
invention, as are host cells, eukaryotic or prokaryotic, containing
nucleic acid encoding antibodies (including antibody fragments) and
capable of their expression. The invention also provides methods of
production of the antibodies including growing a cell capable of
producing the antibody under conditions in which the antibody is
produced, and preferably secreted.
[0104] The reactivities of antibodies on a sample may be determined
by any appropriate means. Tagging with individual reporter
molecules is one possibility. The reporter molecules may directly
or indirectly generate detectable, and preferably measurable,
signals. The linkage of reporter molecules may be directly or
indirectly, covalently, e.g. via a peptide bond or non-covalently.
Linkage via a peptide bond may be as a result of recombinant
expression of a gene fusion encoding antibody and reporter
molecule.
[0105] One favoured mode is by covalent linkage of each antibody
with an individual fluorochrome, phosphor or laser dye with
spectrally isolated absorption or emission characteristics.
Suitable fluorochromes include fluorescein, rhodamine,
phycoerythrin and Texas Red. Suitable chromogenic dyes include
diaminobenzidine.
[0106] Other reporters include macromolecular colloidal particles
or particulate material such as latex beads that are coloured,
magnetic or paramagnetic, and biologically or chemically active
agents that can directly or indirectly cause detectable signals to
be visually observed, electronicallv detected or otherwise
recorded. These molecules may be enzymes which catalyse reactions
that develop or change colours or cause changes in electrical
properties, for example. They may be molecularly excitable, such
that electronic transitions between energy states result in
characteristic spectral absorptions or emissions. They may include
chemical entities used in conjunction with biosensors.
Biotin/avidin or biotin/streptavidin and alkaline phosphatase
detection systems may be employed.
[0107] The mode of determining binding is not a feature of the
present invention and those skilled in the art are able to choose a
suitable mode according to their preference and general
knowledge.
[0108] Antibodies may also be used in purifying and/or isolating a
polypeptide or peptide according to the present invention, for
instance following production of the polypeptide or peptide by
expression from encoding nucleic acid therefor. Antibodies may be
useful in a therapeutic context (which may include prophylaxis) to
disrupt p21/cyclin D1/Cdk4 interaction with a view to inhibiting
Cdk4 activity and so cellular proliferation. Antibodies can for
instance be micro-injected into cells, e.g. at a tumour site.
[0109] Other candidate inhibitor compounds may be based on
modelling the 3-dimensional structure of a polypeptide or peptide
fragment and using rational drug design to provide potential
inhibitor compounds with particular molecular shape, size and
charge characteristics.
[0110] A compound found to have the ability to affect Cdk4 activity
has therapeutic potential in anti-tumour treatment, and may be used
in combination with any other anti-tumour compound. In such a case,
the assay of the invention, when conducted in vivo, need not
measure the degree of inhibition of binding or of modulation of
Cdk4 activity caused by the compound being tested. Instead the
effect on tumorigenicity and/or cell viability may be measured. It
may be that such a modified assay is run in parallel with or
subsequent to the main assay of the invention in order to confirm
that any effect on tumorigenicity or and/or cell viability is as a
result of the inhibition of binding or interaction between p21 and
cyclin D1/Cdk 4 caused by said inhibitor compound and not merely a
general toxic effect.
[0111] Following identification of a substance or agent which
modulates or affects Cdk4 activity, the substance or agent may be
investigated further. Furthermore, it may be manufactured and/or
used in preparation, i.e. manufacture or formulation, of a
composition such as a medicament, pharmaceutical composition or
drug. These may be administered to individuals.
[0112] As noted, the agent may be peptidyl, e.g. a peptide which
includes a sequence as recited above, or may be a functional
analogue of such a peptide.
[0113] As used herein, the expression "functional analogue" relates
to peptide variants or organic compounds having the same functional
activity as the peptide in question, which may interfere with the
binding between p21 and cyclin D1/Cdk4. Examples of such analogues
include chemical compounds which are modelled to resemble the three
dimensional structure of the p21 or cyclin D1 /Ckd4 domain in the
contact area, and in particular the arrangement of the key amino
acid residues identified above as they appear in human p21.
[0114] In a further aspect, the present invention provides the use
of the above substances in methods of designing or screening for
mimetics of the substances.
[0115] Accordingly, the present invention or provides a method of
designing mimetics of p21.sup.WAF1 having the biological activity
of Cdk4 binding or inhibition, the activity of allosteric
inhibition of Cdk4 and/or the activity of cyclin D1 binding, said
method comprising:
[0116] (i) analysing a substance having the biological activity to
determine the amino acid residues essential and important for the
activity to define a pharmacophore; and,
[0117] (ii) modelling the pharmacophore to design and/or screen
candidate mimetics having the biological activity.
[0118] Suitable modelling techniques are known in the art. This
includes the design of so-called "mimetics">which involves the
study of the functional interactions fluorogenic oligonucleotide
the molecules and the design of compounds which contain functional
groups arranged in such a manner that they could reproduced those
interactions.
[0119] The designing of mimetics to a known pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound. This might be desirable where the
active compound is difficult or expensive to synthesise or where it
is unsuitable for a particular method of administration, e.g.
peptides are not well suited as active agents for oral compositions
as they tend to be quickly degraded by proteases in the alimentary
canal. Mimetic design, synthesis and testing may be used to avoid
randomly screening large number of molecules for a target
property.
[0120] There are several steps commonly taken in the design of a
mimetic from a compound having a given target property. Firstly,
the particular parts of the compound that are critical and/or
important in determining the target property are determined. In the
case of a peptide, this can be done by systematically varying the
amino acid residues in the peptide, e.g. by substituting each
residue in turn. These parts or residues constituting the active
region of the compound are known as its "pharmacophore".
[0121] Once the pharmacophore has been found, its structure is
modelled to according its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data from a
range of sources, e.g. spectroscopic techniques, X-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can be used in this
modelling process.
[0122] In a variant of this approach, the three-dimensional
structure of the ligand and its binding partner are modelled. This
can be especially useful where the ligand and/or binding partner
change conformation on binding, allowing the model to take account
of this the design of the mimetic.
[0123] A template molecule is then selected onto which chemical
groups which mimic the pharmacophore can be grafted. The template
molecule and the chemical groups grafted on to it can conveniently
be selected so that the mimetic is easy to synthesise, is likely to
be pharmacologically acceptable, and does not degrade in vivo,
while retaining the biological activity of the lead compound. The
mimetic or mimetics found by this approach can then be screened to
see whether they have the target property, or to what extent they
exhibit it. Further optimisation or modification can then be
carried out to arrive at one or more final mimetics for in vivo or
clinical testing.
[0124] The mimetic or mimetics found by this approach can then be
screened to see whether they have the target property, or to what
extent they exhibit it. Further optimisation or modification can
then be carried out to arrive at one or more final mimetics for in
vivo or clinical testing.
[0125] Mimetics of this type together with their use in therapy
form a further aspect of the invention.
[0126] The present invention further provides the use of a peptide
which includes a sequence as disclosed, or a derivative, active
portion, analogue, variant or mimetic, thereof able to bind Cdk4
and/or inhibit Cdk4 activity, in screening for a substance able to
bind Cdk4 and/or inhibit Cdk4 activity. Generally, an inhibitor
according to the present invention is provided in an isolated
and/or purified form, i.e. substantially pure. This may include
being in a composition where it represents at least about 90%
active ingredient, more preferably at least about 95%, more
preferably at least about 98%. Such a composition may, however,
include inert carrier materials or other pharmaceutically and
physiologicaly acceptable excipients. As noted below, a composition
according to the present invention may include in addition to an
inhibitor compound as disclosed, one or more other molecules of
therapeutic use, such as an anti-tumour agent.
[0127] The present invention extends in various aspects not only to
a substance identified as a modulator of p21 and cyclin D1/Ckd4
interaction and/or Cdk4-mediated RB phosphorylation or other
substrates of Cdk4 or other p21-mediated activity, property or
pathway in accordance with what is disclosed herein, but also a
pharmaceutical composition, medicament, drug or other composition
comprising such a substance, a method comprising administration of
such a composition to a patient, e.g. for anti-tumour or other
anti-proliferative treatment, which may include preventative
treatment, use of such a substance in manufacture of a composition
for administration, e.g. for anti-tumour or other
anti-proliferative treatment, and a method of making a
pharmaceutical composition comprising admixing such a substance
with a pharmaceutically acceptable excipient, vehicle or carrier,
and optionally other ingredients.
[0128] A substance according to the present invention such as an
inhibitor of p21 and cyclin D1 and/or Cdk4 interaction or binding
may be provided for use in a method of treatment of the human or
animal body by therapy which affects Cdk 4 activity or other
p21-mediated activity in cells, e.g. tumour cells.
[0129] Thus the invention further provides a method of modulating
Cdk4 activity, or other p2'-mediated activity in a cell, which
includes administering an agent which inhibits or blocks the
binding of p21 to cyclin D1 and/or Cdk4 protein, such a method
being useful in treatment of cancer or other diseases or disorders
including malignancies where inhibition of cellular growth and/or
proliferation is desirable.
[0130] The invention further provides a method of treating tumours
which includes administering to a patient an agent which interferes
with the binding of p21 to cyclin D1 and/or Cdk4.
[0131] Whether it is a polypeptide, antibody, peptide, nucleic acid
molecule, small molecule, mimetic or other pharmaceutically useful
compound according to the present invention that is to be given to
an individual, administration is preferably in a "prophylactically
effective amount" or a "therapeutically effective amount" (as the
case may be, although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual. The actual
amount administered, and rate and time-course of administration,
will depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practioners and other medical
doctors.
[0132] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0133] Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may include, in addition to active ingredient, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled in the art. Such materials
should be non-toxic and should not interfere with the efficacy of
the active ingredient. The precise nature of the carrier or other
material will depend on the route of administration, which may be
oral, or by injection, e.g. cutaneous, subcutaneous or
intravenous.
[0134] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may include a
solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally include a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0135] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0136] Examples of techniques and protocols mentioned above can be
found in Remington's Pharmaceutical Sciences, 16th edition, Osol,
A. (ed), 1980.
[0137] The agent may be administered in a localised manner to a
tumour site or other desired site or may be delivered in a manner
in which it targets tumour or other cells.
[0138] Targeting therapies may be used to deliver the active agent
more specifically to certain types of cell, by the use of targeting
systems such as antibody or cell specific ligands. Targeting may be
desirable for a variety of reasons, for example if the agent is
unacceptably toxic, or if it would otherwise require too high a
dosage, or if it would not otherwise be able to enter the target
cells.
[0139] Instead of administering these agents directly, they may be
produced in the target cells by expression from an encoding gene
introduced into the cells, eg in a viral vector (a variant of the
VDEPT technique--see below). The vector may targeted to the
specific cells to be treated, or it may contain regulatory elements
which are switched on more or less selectively by the target
cells.
[0140] The agent may be administered in a precursor form, for
conversion to the active form by an activating agent produced in,
or targeted to, the cells to be treated. This type of approach is
sometimes known as ADEPT or VDEPT, the former involving targeting
the activating agent to the cells by conjugation to a cell-specific
antibody, while the latter involves producing the activating agent,
e.g. an enzyme, in a vector by expression from encoding DNA in a
viral vector (see for example, EP-A-415731 and WO 90/07936).
[0141] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated, such as cancer, virus
infection or any other condition in which a p21-mediated effect is
desirable.
[0142] Nucleic acid according to the present invention, encoding a
polypeptide or peptide able to interfere with p21 and cyclin D1
and/or Cdk 4 interaction or binding and/or induce or modulate Cdk4
activity or other p21-mediated cellular pathway or function, may be
used in methods of gene therapy, for instance in treatment of
individuals with the aim of preventing or curing (wholly or
partially) a tumour e.g. in cancer, or other disorder involving
loss of proper regulation of the cell-cycle and/or cell growth, or
other disorder in which specific cell death is desirable, such as
in certain viral infections.
[0143] Vectors such as viral vectors have been used in the prior
art to introduce nucleic acid into a wide variety of different
target cells. Typically the vectors are exposed to the target cells
so that transfection can take place in a sufficient proportion of
the cells to provide a useful therapeutic or prophylactic effect
from the expression of the desired polypeptide. The transfected
nucleic acid may be permanently incorporated into the genome of
each of the targeted tumour cells, providing long lasting effect,
or alternatively the treatment may have to be repeated
periodically.
[0144] A variety of vectors, both viral vectors and plasmid
vectors, are known in the art, see U.S. Pat. No. 5,252,479 and WO
93/07282. In particular, a number of viruses have been used as gene
transfer vectors, including papovaviruses, such as SV40, vaccinia
virus, herpesviruses, including HSV and EBV, and retroviruses. Many
gene therapy protocols in the prior art have used disabled murine
retroviruses.
[0145] As an alternative to the use of viral vectors other known
methods of introducing nucleic acid into cells includes
electroporation, calcium phosphate co-precipitation, mechanical
techniques such as microinjection, transfer mediated by liposomes
and direct DNA uptake and receptor-mediated DNA transfer.
[0146] Receptor-mediated gene transfer, in which the nucleic acid
is linked to a protein ligand via polylysine, with the ligand being
specific for a receptor present on the surface of the target cells,
is an example of a technique for specifically targeting nucleic
acid to particular cells.
[0147] A polypeptide, peptide or other substance able to interfere
with the interaction of the relevant polypeptide, peptide or other
substance as disclosed herein, or a nucleic acid molecule encoding
a peptidyl such molecule, may be provided in a kit, e.g. sealed in
a suitable container which protects its contents from the external
environment. Such a kit may include instructions for use.
[0148] Various further aspects and embodiments of the present
invention will be apparent to those skilled in the art in view of
the present disclosure. Certain aspects and embodiments of the
invention will now be illustrated by way of example and with
reference to the following figures:
[0149] FIG. 1. The Ability of Peptides from p21.sup.WAF1 to
Interact with Cdk4 and Cyclin D1.
[0150] FIG. 1a: a list of the peptides 1-11 based on the sequence
of p21.sup.WAF1. FIG. 1b: The p21.sup.WAF1 peptides were bound to
streptavidin-agarose beads and added to reticulocyte lysates
containing either Cdk4 or cyclin D1 labelled with [.sup.35S]
methionine. After extensive washing bound proteins were analysed
using SDS-PAGE followed by autoradiography. The bands were
quantified using a Bio-Imager and Whole Band Analysis Software
(Millipore). The results are representative of 3 such experiments.
"x" indicates beads without peptide.
[0151] FIG. 2. Addition of p21.sup.WAF1 Based Peptides to Cyclin
D1-Cdk4 Phosphorylation Assays.
[0152] Cyclin D1-Cdk4 assays were carried out in vitro using
lysates from Sf9 insect cell following co-infection with Cdk4 and
cyclin D1 baculovirus constructs and GST-Rb as the substrate.
p.sub.21.sup.WAF1 peptides were added to the assays at a
concentration of 17 .mu.M and the effect on Cdk4 activity was
assessed by SDS-PAGE and autoradiography. The figure shows
quantification of the autoradiograph using bio-imaging, relative
binding is expressed in terms of Cdk4 activity in the absence of
peptide. The data are representative of 4 experiments. "x"
indicates no addition of peptide.
[0153] FIG. 3. Quantification of Peptide Inhibition.
[0154] Peptides 4, 8, 2 and 10 were added to cyclin D1-Cdk4 assays
at various concentrations between 0.01-34 .mu.M. The figure gives a
plot of activity (%) relative to Cdk4 activity measured in the
absence of peptide against peptide concentration and the I.sub.0.5
for each peptide. The data represent the mean of 3 experiments.
[0155] FIG. 4. Peptides 2 or 10 are Not Substrates for Cyclin
D1-Cdk4.
[0156] The figure shows the results of phosphorylation assays using
peptides 2, 4 & 10.
[0157] FIG. 5. Size Scan of Peptide 10.
[0158] The figure shows the sequences of a series of peptides based
on peptide 10 designed to find the minimal inhibitory domain. The
boxed residues represent the minimal inhibitory domain. The
peptides ware added to cyclin D1-Cdk4 assays and analysed by
SDS-PAGE and autoradiography.
[0159] FIG. 6. Alanine Scan Mutations of Peptide 10.
[0160] In order to pinpoint residues that were critical for the
inhibition of Cdk4 by peptide 10 a series of point mutations were
constructed in which each residue was sequentially changed to
alanine. The peptides were added to cyclin D1-Cdk4 assays and the
results were analysed by SDS-PAGE and autoradiography then
quantified using a Bio-Imager. The results are expressed relative
to Cdk4 activity in the absence of peptide and are representative
of 3 experiments. Having identified the critical residues we then
synthesised an untagged eight amino acid peptide which contained
the R, L and F (KRRLIFS) and determined the phosphorylation of
GST-Rb by cyclin D1-Cdk4 in the presence of increasing
concentrations of this truncated peptide.
[0161] FIG. 7. Comparison of Inhibitory Peptides with Full Length
p21.sup.WAF1 Protein.
[0162] This shows concentration curves for peptide 10, D to A
mutant peptide 10, a p16INK4 derived peptide (Fahraeus et al.,
1996) and full length his-p21.sup.WAF1 determined using the cyclin
D1-Cdk4 assay analysed by SDS-PAGE, autoradiography and bio-imaging
and for the I.sub.0.5 of each inhibitor. The results are the mean
of 3 such experiments.
[0163] FIG. 8. Binding and Inhibitory Domains of
p.sub.21.sup.WAF1.
[0164] The hatched residues show the regions of p21.sup.WAF1
identified in this study as being important for cyclin D1 and Cdk4
binding, and Cdk4 inhibition in the N-terminal domain, as well as a
novel inhibitory domain in the C-terminus of p21.sup.WAF1 The
residues found to be important for the interaction of p21.sup.WAF1
with PCNA (Warbrick et al., 1995) are shown in black. In addition,
the smallest portion of p21.sup.WAF1 that was found to inhibit CDK
activity in vitro (Luo et al., 1995) prior to the present study is
indicated.
[0165] FIG. 9. Introduction of p.sub.21.sup.WAF1 Based Peptides
Into Cells
[0166] A series of synthetic peptides based on the sequence of
peptide 10 (Peptides I, II and III, shown in FIG. 9a) were
synthesised with carrier peptide (shaded sequences). The underlined
residue in Peptide-1 is the M to A mutation which prevents PCNA
binding. The peptides were added to proliferating HaCaT cells,
grown in DMEM plus 10% FCS. The cells were incubated for 24 hours
pulse labelled during with 15 .mu.M BrdU, fixed and then analysed
by FACS. The G.sub.1-, S-- and G.sub.2-phase distributions for
untreated cells, Peptide-I at 25 .mu.M, Peptide-II at 50 .mu.M and
Peptide-III at 25 .mu.M, were determined. FIG. 9b shows the data
represented as the % of cells in each phase compared to the total
number of cells counted. The results of similar experiments using
MCF7 and MRCS cells are shown in FIG. 9c, which shows the
percentage of cells in each phase of the cell cycle in the absence
and presence of peptide I.
[0167] In a separate experiment DMEM+10%, FCS alone or DMEM+10% FCS
containing either 25 .mu.M Peptide-I or 50 .mu.M Peptide-II, was
added to HaCaT cells than had been starved for 72 hours. Samples
were taken at the times shown and analysed by SDS-PAGE/Western blot
stained for pRb. pRb represents hypophosphorylated Rb protein and
pRb* refers to hyperphosphorylated Rb protein. It should be pointed
out that equal amounts of total protein were loaded per lane and
that the antibody appears to preferentially recognise
phosphorylated forms of the Rb protein.
[0168] FIG. 10. Inhibition of Cyclin-Cdk4 Activity Using
Derivatives of Peptide 2.
[0169] The figure shows the degree of inhibition of cyclin D1-Cdk4
activity using pRb as a substrate, by peptide 2 (2 on the figure)
and alanine scan mutations of the peptide (each residue being
sequentially mutated to alanine) Activity is given relative to
uninhibited activity. (No is short for no added peptide.) Peptides
were present at a concentration of 10 .mu.M. A similar pattern is
seen when binding of peptide 2 mutants to cyclin D1 expressed in
reticulocyte lysates.
[0170] Important residues for the binding and inhibition are the
two arginine residues (R) and the phenylalanine (F) with the lysine
(K) and proline (P) also contributing. This is different from
residues identified as being critical for interaction of full
length protein with cyclin D1, as these studies pick out the LFG
motif as being most important for activity.
[0171] Experimental Procedures
[0172] Peptides
[0173] All peptides were synthesised by Chiron Mimotopes, Peptide
Systems (Clayton, Australia). Each peptide had a Biotin-SGSG spacer
at the C-terminus and a free N-terminus. The peptides were
dissolved in DMSO at approximately 5 mg/ml and we then determined
their concentration precisely by amino acid analysis (Smythe et
al., 1988). In addition the purity of the peptides was estimated
using mass spectrometry. Positive ion electrospray mass
spectrometry was performed on a triple-quadruple mass spectrometer
(V. G. Quattro) in (50/50/0.1) water/acetonitrile/formi- c
acid.
[0174] Proteins
[0175] Cyclins and CDKs--Cdk4 and cyclin D1, Cdk2 and cyclin E and
Cdc2 and cyclin B were co-expressed in Sf9 insect cells infected
with the appropriate baculovirus constructs. The cells were
harvested two days after infection by low speed centrifugation and
the pellet was lysed in an equal volume of 10 mM Hepes, pH 7.4
containing: 10 mM NaCl, 1 mM EDTA, and 0.1 mM phenylmethane
sulphonyl fluoride, 2 mM DTT and centrifuged at 14000.times.g for
15 min. The supernatant was removed, aliquoted and immediately
frozen in liquid nitrogen. Thawed lysate was used only once and was
never refrozen. Labelled Cdk4 and cyclin D1 were produced by
translation in the presence of [.sup.35S] methionine using a rabbit
reticulocyte lysate in vitro translation kit (Promega) His-tagged
p21.sup.WAF1 Human p21.sup.WAF1 was expressed in E. coli using a
PET expression vector. The soluble p21.sup.WAF1 protein fraction
was purified using a nickel chelating column, following the
manufacturers instructions (Pharmacia). The eluted protein peak was
dialysed against 25 mM Hepes, pH 7.4, containing: 0.1 mM EDTA, 1 mm
benzamidine, 0.01% Triton X-100, and 0.1 mM phenylmethane sulphonyl
fluoride, concentrated and applied to a Superose 12 gel-filtration
column (Phamacia) equilibrated in the above buffer. Fractions
containing p21.sup.WAF1 were detected by Western blot using the
p21.sup.WAF1 specific monoclonal antibody Ab-1 (oncogene Sciences),
concentrated to 200 .mu.g/ml and stored at -70.degree. C.
[0176] GST-Rb--An E. coli expression construct containing the
hyperphosphorylation domain of pRb (amino acids 773-924) was
purified on a glutathione-Sepharose column according to the
manufacturers instructions (Pharmacia).
[0177] Peptide Precipitation of Cdk4 and Cyclin D1
[0178] A 20 amino acid peptide library, that spanned the entire
sequence of p21.sup.WAF1 (FIG. 1), was screened for Cdk4/cyclin D1
interacting peptides. Peptide (1.5 .mu.g) was diluted in 100 .mu.l
of PBS and incubated with 10 .mu.l of packed streptavidin-agarose
beads (Sigma) for 1 h at room temperature. Unbound peptide was
removed by extensive washing with PBS and the beads, plus bound
peptide, were incubated for 1 h at 4.degree. C. with reticulocyte
lysate containing either Cdk4 or cyclin D1 labelled with [.sup.35S]
methionine. The beads were washer three times with 1.25.times.PBS
containing 0.2% Triton X-100 and boiled in the presence of 0.125 M
Tris-HCl, pH 6.8 containing: 4% (w/v) SDS, 20% (v/v) glycerol and
200 mM DTT. The bound protein was analysed by SDS-PAGE followed by
auto-radiography and quantification of the .sup.35S-labelled
protein using a Bio-Imager and Whole Band Analysis Software
(Millipore).
[0179] Enzyme Assays
[0180] Phosphorylation of GST-Rb--Cdk4 activity was measured using
the cyclin D1-Cdk4 containing insect cell lysate described above.
Extract (1 .mu.l) was added to a final reaction volume of 10/1,
containing: 50 mM Hepes, pH 7.4, 10 mM MgCl2, 2.5 mM EGTA, 1 mM
DTT, 10 mM .beta.-glycerophosphate, 1 mM NaF, 10 mM PKI, 50 .mu.M
ATP containing [.sup.32P] ATP (1 000 cpm/pMol) and 0.5 .mu.g
GST-Rb. The assays were started by the addition of the GST-Rb
substrate, incubated at 30.degree. C. for 10 min (the incorporation
of .sup.32 P into GST-Rb was linear over 1S-20 min) and terminated
by adding SDS-PAGE sample buffer and heating at 95.degree. C. for 4
min. The samples were analysed by SDS-PAGE on 12% gels followed by
auto-radiography and quantification using a Bio-Imager.
[0181] Peptide Phosphorylation
[0182] The biotinylated peptides (1 .mu.g) were incubated for 30
min at 30.degree. C. in a final volume of 20 .mu.l containing: 50
mM Hepes, pH 7.4, 10 mM MgCl.sub.2, 2.5 mM EGTA, 1 mM DTT, 10 mM
.beta.-glycerophosphate, 1 mM NaF, 10 .mu.M PKI, 50 .mu.M ATP
containing [.sup.32P]ATP (6000 cpm/pMol) and either 1 .mu.l of
cyclin D1-Cdk4 insect cell lysate, 1 .mu.l of uninfected insect
cell lysate or 0.02 mU of protein kinase C plus 0.5 mM CaCl.sub.2,
100 mg/ml phosphatidyl serine and 20 mg/ml diacylglycerol. The
reactions were stopped by heating at 60.degree. C. for 5 min and
streptavidin agarose beads were added (10 .mu.l packed cell volume
washed with 3.times.PBS) and incubated with shaking at 4.degree. C.
for 30 min. The beads were washed extensively with PBS containing
3% (v/v) Tween-20 and the incorporation of radioactivity into the
peptides was determined by Cerenkov counting.
[0183] Cell Cycle Measurements
[0184] Carrier linked peptides were designed for delivery into
proliferating HaCaT cells (see FIG. 9). Cells were seeded on 30 mm
culture plates and grown to 50's confluency in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10' (v/v) foetal calf serum
(FCS). Peptides were added to the medium and the cells were
incubated for 24 hours. During the last 30 minutes of the
incubation the cells were pulse labelled in the presence of 15
.mu.M BrdU. The cells were trypsinised, fixed in absolute alcohol
and prepared for FACS analysis using a single laser flow cytometer
(Becton-Dickinson, FACScan) as previously described (Renzing et al,
1996).
[0185] pRb Phosphorylation in HaCaT Cells
[0186] HaCaT cells were seeded on 30 mm culture plates at 25%
confluency in DMEM with 10% FCS. The FCS was withdrawn after 24
hours and the cells were starved for 72 hours. At the end of this
period the medium was supplemented with 10% FCS and carrier linked
peptides. Samples were taken over a 24 hour time course and the
cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, containing
150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) DOC, 0.1%. (w/v) SDS, 1
mM PMSF, 0.1 mg/ml aprotinin and 0.5 mg/ml leupeptin) for 30
minutes at 4.degree. C. The phosphorylation statues of pRb was
determined by Western blot analysis, as previously described
(Fahraeus et al, 1996) except that the blot was probed with a pRb
polyclonal antibody (C-15, Santa Cruz).
[0187] Results
[0188] Peptide-Binding Assay for Cyclin D1 and Cdk1
[0189] Using a series of synthetic peptides that span the entire
sequence of p21.sup.WAF1 (FIG. 1), we determined whether these
peptides could mimic full length p21.sup.WAF1 protein by forming a
stable complex with either cyclin D1 or Cdk4. If peptide-binding
mimetics of p21.sup.WAF1 protein could be identified, then this
would assist in identifying the minimal binding motif of
p21.sup.WAF1 protein required for cyclin D1-Cdk4 holoenzyme
inhibition and whether p21.sup.WAF1 was targeting the cyclin or the
kinase subunit. This would also define a system for using small
peptides to study p21.sup.WAF1 protein reaction mechanism and to
design mimetic drugs.
[0190] The peptide-binding assay involved quantifying the amount of
.sup.35S-labelled cyclin D1 or Cdk4 which bound specifically to
biotinylated peptides that were captured on streptavidin coated
agarose beads. The peptide-coated beads were added to extracts
containing either .sup.35S-labelled cyclin D1 or Cdk4 translated in
vitro, the beads were washed extensively to remove unbound protein,
and the bound cyclin D1 or Cdk4 was quantified by SDS-PAGE followed
by auto-radiography and bio-imaging. This is referred to below as a
peptide precipitation assay and has been used previously to
demonstrate evolutionary conservation of p21.sup.WAF1 binding to
PCNA (Ball and Lane, 1996).
[0191] A Small Peptide Derived from Amino Acids 46-65 in the
N-Terminal Domain of p21.sup.WAF1 Binds Directly to Cdk4
[0192] Using the peptide-precipitation assay, peptide 4 (from the
N-terminal domain of p21.sup.WAF1) bound specifically to Cdk4, but
not to cyclin D1 (FIG. 1). This interaction is physiologically
important, since the CDK interacting domain of the p21.sup.WAF1
protein has previously been proposed to localise to the N-terminal
domain of the molecule (Chen et al., 1995; Harper et al., 1995; Luo
et al., 1995). More specifically, deletions (Nakanishi et al.,
1995a) or mutations (Goubin and Ducommun, 1995) in the region of
amino acids 45-71 compromise the ability of full length
p21.sup.WAF1 to interact with Cdk2. Whether this loss of
p21.sup.WAF1 binding function is due to, (i) mutation/deletion of
residues directly involved in CDK binding, or (ii)
mutations/deletion induced conformational alterations in
p21.sup.WAF1 that prevent stable binding to CDK, has not been
demonstrated. Here we show unequivocally that residues 46-65 are
directly involved in the binding of p21 to Cdk4 and that alone they
are capable of forming a stable complex with Cdk4, in the absence
of cyclin D1 . Thus, providing direct evidence that the N-terminus
of p21.sup.WAF1 does contain a kinase binding domain.
[0193] A Small Peptide Derived from Amino Acids 16-35 in the
N-Terminus of p.sub.21.sup.WAF1 Binds Directly to Cyclin D1
[0194] We were also able to define a second and distinct N-terminal
interaction site on the p21.sup.WAF1 protein; in this case a region
of p21.sup.WAF1 which is capable of binding to cyclin D1, but not
to Cdk4 (FIG. 1). Peptide 2 comprises amino acids 16-35 of
p21.sup.WAF1 and lies within the Eminimum region required for DNA
synthesis inhibition in vivo, which is located between residues
17-71 (Nakanishi et al., 1995a). Our results might explain an
apparent contradiction encountered by Nakanishi et al. (1995a) who
found that N-terminal mutations in p21.sup.WAF1 protein which are
outside the CDK interacting domain, although insufficient to
prevent binding to the kinase, were sufficient to prevent
p21.sup.WAF1 from acting as a growth suppressor when transfected
into proliferating cells. Specifically, the direct peptide binding
data (FIG. 1) leads us to suggest that an N-terminal motif in the
p.sub.21.sup.WAF1 protein, that mediates cyclin D1 binding, could
be an essential step in the mechanism through which p21.sup.WAF1
protein functions as a growth suppressor.
[0195] A Novel Cyclin D1-Cdk4 Binding Motif Resides in the
C-Terminus of the p21.sup.WAF1 Protein
[0196] The specificity of the peptide-precipitation assay in
defining the domain of p21.sup.WAF1 protein required for binding to
either the cyclin D1 or Cdk4 (FIG. 1), indicated that using
peptides to study potential interactions between p.sub.21.sup.WAF1
and cyclin-CDK complexes would prove to be very informative. We
were intrigued however, by the finding that peptides from the
C-terminus of the p.sub.21.sup.WAF1 protein (peptides 10 and 11)
could form stable complexes with both Cdk4 and cyclin D1 (FIG. 1),
as peptide 10 is equivalent to the p21PBP peptide described by
Warbrick et al. (1995) as representing the region of p21.sup.WAF1
which binds to the replication/repair protein PCNA. We can not rule
out the possibility that endogenous cyclin or CDK present in the
reticulocyte lysate could bind to the labelled human protein
forming a bridge to the peptide. However, as peptide 2 and peptide
4 precipitate either cyclin D1 or Cdk4, respectively, this seems
unlikely. These results suggest that the p.sub.21.sup.WAF1 protein
may interact with both PCNA and cyclin-CDK complexes through the
same binding motif. Peptide 11 however, binds to both Cdk4 and
cyclin D1 but not to PCNA (FIG. 1) (Warbrick et al., 1995; Ball and
Lane, 1996); uncoupling the PCNA binding site from the cyclin/CDK
binding motif in the C-terminus of p21.sup.WAF1.
[0197] Given that we had identified three distinct motifs from the
p21.sup.WAF1 protein which bind specifically to cyclin D1 and/or
Cdk4, we then examined whether they mimicked p21.sup.WAF1 protein
by inhibiting kinase activity.
[0198] The Cyclin D1 Binding Peptide from the N-Terminal Domain of
p21.sup.WAF1 nd the Cyclin/CDK Binding Peptide from the C-Terminus
of p21.sup.WAF1 Inhibits the Activity of Cdk4
[0199] In order to determine if any of the p21.sup.WAF1 peptides
possessed Cdk4 inhibitory activity we tested, independently, their
ability to prevent pRb phosphorylation during cyclin D1-Cdk4 assays
in vitro (FIG. 2). Peptides 2, 8, 10, and 11 inhibited cyclin
D1-Cdk4 activity when added to the assay at 17 .mu.M, whereas
buffer alone and the remaining peptides had no dramatic affect on
Cdk4 activity. The cyclin D1 binding peptide (peptide 2) inhibited
the kinase activity by approximately 80% and peptides 10 and 11,
which bound both Cdk4 and cyclin D1, completely inhibited enzyme
activity at this concentration. Thus, there is a correlation
between the ability of the peptides to bind to Cdk4 and/or cyclin
D1 and to inhibit Cdk 4 kinase activity.
[0200] However, this correlation breaks down in the case of
kinase-binding peptide 4. This peptide maps to the CDK interaction
site (FIG. 8; Goubin and Ducommun, 1995; Nakanishi et al., 1995a)
and there has been speculation that a peptide from this domain,
capable of interacting with CDK, would mimic full-length
p21.sup.WAF1 inhibitory activity, and would therefore provide a
model for the design of novel molecules that could arrest cell
cycle progression by inhibiting the G1 cyclin-CDKs. Although of
high affinity for Cdk4, peptide 4 had no inhibitory activity when
added to cyclin D1-Cdk4 assays at concentrations of up to 35 .mu.M.
Our data from both p21.sup.WAF1-peptide binding data and inhibitory
properties, therefore pinpoints two novel small domains of the
p21.sup.WAF1 protein as potential candidates for small molecular
weight mimetics; an N-terminal motif from amino acids 16-35
(peptide 2) and a C-terminal motif from amino acids 141-10 (peptide
10).
[0201] It is thus possible that peptide 4 could block p21 binding,
preventing its activity as an inhibitor. Thus cells treated with
peptide 4 may be expected to continue to proliferate even in the
presence of competing signals which would normally mediate cell
cycle arrest or apoptosis. Thus peptide 4 may be used to reversibly
immortalise cells, by supplying to the peptide to the cells. This
provides a further tool in investigating the cellular mechanisms
for control of the cell cycle and may also be useful in combatting
cell loss in conditions associated with loss of cells, such as in
AIDS or degenerative conditions including MS, dementia, or in
muscle degenerative conditions such as muscular dystropy (MD)
including Duchenne MD.
[0202] The C-Terminal p21.sup.WAF1 Peptide is a More Potent
Inhibitor of Cdk4 Kinase Activity than the N-Terminal Cyclin
D1-Binding Peptide
[0203] We carried out more detailed studies to determine the
I.sub.0.5 for peptides 2, 8, and 10, using peptide 4 as a negative
control (FIG. 3). We found that peptide 10 (and peptide 11; data
not shown) was a potent inhibitor of Cdk4 activity with an
I.sub.0.5 of 0.1 .mu.M, peptide 2, was also a good inhibitor with
an I.sub.0.5 of 2 .mu.M. Peptide 8 gave only weak inhibition and
relatively high concentrations of peptide were required to approach
50% inhibition. These data support the possibility of using peptide
2 or peptide 10 to mimic the CDK inhibitory activity of the full
length p21.sup.WAF1 protein.
[0204] p21.sup.WAF1 Protein and Inhibitory Peptides Compete for the
Same Binding Site on Cdk4 Kinase
[0205] In order to determine if the Cdk4 inhibitory peptides, 2 and
10, were acting at sites on Cdk4 and cyclin D1 that were also
employed by p21.sup.WAF1, we carried out peptide precipitation
assays in the presence and absence of full length purified
his-p21.sup.WAF1 to find out if it competed with the peptides for
binding.
[0206] The ability of p21.sup.WAF1 to interfere with peptide 2 (A)
and peptide 10 (B & C) binding to Cdk4 and/or cyclin D1 was
determined by carrying out the peptide precipitation assay from
reticulocyte lysates in the presence of 0, 0.5, 2 .mu.g of
p21.sup.WAF1.
[0207] The data suggest that binding of p21.sup.WAF1 protein to
cyclin D1 and Cdk4 prevents binding of both peptide 2 and peptide
10. These data are open to two interpretations, (i) the peptides
could be competing for binding at the same site as p21.sup.WAF1, or
(ii) binding of either p21.sup.WAF1 or peptide could cause a
conformational change in the cyclin or CDK preventing further
binding. It is not clear from these experiments whether peptides 2
and 10 are acting at the same site(s). However the difference in
the peptide precipitation data, indicates that at least one of the
sites is unique, as peptide 10 can precipitate both Cdk4 and cyclin
D1, whereas, peptide 2 can only precipitate cyclin D1.
[0208] Data to support the hypothesis that peptide 10 and
p.sub.21.sup.WAF1 protein compete for the same binding site, during
kinase inhibition, employs the use of a peptide 10 mutant
(containing a point mutation resulting in a change of R-A at
residue 15 of peptide 10 which is equivalent to residue 155 of the
full length protein) which loses >60% of its inhibitory activity
(see below), but retains its binding function.
[0209] To determine if the inhibition of Cdk4 by p.sub.21.sup.WAF1
could be relieved by the addition of a peptide 10 mutant, the R to
A mutant (residue 15 of peptide 10) that was no longer an efficient
inhibitor but still displayed partial binding activity, increasing
concentrations of peptide (1, 5, 17 & 34 .mu.M) were added to
cyclin D1-Cdk4 GST-Rb phosphorylation assay in the presence or a
fixed concentration of p21.sup.WAF1 (50 .mu.M).
[0210] The experiment showed that increasing concentrations of
mutant peptide 10 were able to block the inhibitory activity of
full length p21.sup.WAF1 suggesting that peptide 10 is binding at a
site(s) which blocks subsequent binding of p21.sup.WAF1 and is
therefore functioning through a similar mechanism to the full
length protein.
[0211] The Inhibitory Peptides are not Cyclin D1-Cdk4
Substrates
[0212] Unlike the p107 protein, which appears to inhibit Cdk4s
ability to phosphorylate pRb by acting as an alternative substrate
(Zhu et al., 1995), p21.sup.WAF1 has not been reported to act as a
substrate for the cyclin D1-Cdk4 complexes (and we confirm these
observations FIG. 4). However, it is possible that by using
p21.sup.WAF1 based peptides, instead of full length protein, we
have inadvertently generated phosphorylation sites which would not
normally be exposed on the surface of the protein. Thus the
peptides could be acting as competitive substrates as opposed to
inhibitors of catalytic activity. Both peptide 2 and peptide 10
contain a number of possible phosphorylation sites, and we have
been able to demonstrate that peptide 10 is a potential substrate
for a number of protein kinases (data not shown), including protein
kinase C (PKC) which was used as a control kinase (FIG. 4). In
fact, neither peptide 2 nor peptide 10 were substrates for cyclin
D1-Cdk4 under conditions where 2.4 nMol of .sup.32P were
incorporated per nMol of GST-Rb. However, under the same conditions
peptide 10 was an extremely good substrate for PKC with 0.82 nMol
of .sup.32P being incorporated per nMol of peptide (FIG. 4). There
was a low level of incorporation into peptide 2, but as this was
also present in assays using lysate from uninfected insect cells it
must be attributed to low levels of endogenous protein kinase(s).
Thus, it appears that the peptide inhibitors are not competitive
substrates, but, are acting to block catalytic activity in a
mechanism similar to p21.sup.WAF1.
[0213] The Peptides are not Efficient Inhibitors of Cyclin B-Cdc2
Kinase Activity
[0214] Harper et al. (1995) have shown that p21.sup.WAF1 is not a
universal CDK inhibitor, but that it displays selectivity for the
G1 and S-phase cyclin-CDK complexes. When they compared the ability
of p21.sup.WAF1 to inhibit Cyclin B-Cdc2, which acts at the G2/M
transition, and cyclin D2-Cdk4, which functions during G1, they
found that the I.sub.0.5 for inhibition of cyclin B-Cdc2 was
>600-fold higher than the I.sub.0.5 for inhibition of cyclin
D2-Cdk4 using purified recombinant proteins. We looked at the
effect of adding our two cyclin D1-Cdk4 inhibitory peptides to
cyclin B-Cdc2 and Cdk2-cyclin E assays at concentrations up to 20
.mu.M and found that neither peptide 2 nor peptide 10 had a
significant effect on Cdc2-cyclin B histone H1 kinase activity.
However, Cdk2-cyclin E was inhibited by peptide 10, showing that
peptide 10 can inhibit other G.sub.1 cyclin-Cdk complexes. Thus,
the p21.sup.WAF1 based peptide inhibitors appear to have equivalent
specificity to the full length protein.
[0215] To determine if peptides 2 and 10 could inhibit cyclin
B-Cdc2 and cyclin E-Cdk2 kinase activity assays were performed
using Sf9 cell lysates which were co-expressing human cyclin B and
Cdc2. The conditions were identical to those described in the
Experimental Procedures for cyclin D1-Cdk4 except that histone H1
(0.5 .mu.g/assay) was used as the substrate for cyclin B-Cdc2.
Cyclin D1-Cdk4 cyclin B-Cdc2 and cyclin E-Cdk2 were assayed in the
presence of increasing concentrations of peptide 2 (0.25, 3, 10 and
40 .mu.M) and peptide 10 (0.1, 0.5, 5, 20 .mu.M).
[0216] The Kinase Inhibitory Motif of Peptide 10 is Distinct from
the PCNA Binding Site
[0217] We have shown that peptide 10 is an extremely potent
inhibitor of cyclin D1-Cdk4 activity, with an I.sub.0.5 of 0.1 mM
which is 20-fold more potent than peptide 2, a peptide derived from
the region of p.sub.21.sup.WAF1 previously associated with growth
arrest (Chen et al., 1995; Nakanishi et al., 1995a). We have also
shown that a peptide (peptide 4) which spans the CDK interaction
site of p21.sup.WAF1 (Goubin and Ducommun, 1995; Nakanishi et al.,
1995a), although capable of binding to Cdk4 to form a stable
complex, has no detectable activity as a cyclin D1-Cdk4 inhibitor.
Peptide 10 therefore looks like the best candidate for the
development of a small peptide mimetic with high efficacy. Peptide
10 has previously been shown to form a specific high-affinity and
reversible interaction with PCNA (Ball and Lane, 1996) and this
peptide is sufficient to partially inhibit the function of PCNA
during SV40 replication giving 50% inhibition at a concentration of
approximately 7 mM (Warbrick et al., 1995). The PCNA interaction
domain of p21.sup.WAF1 has been mapped and the important residues
were found to be amino acids 144-151 (QTSMTDFY; Warbrick et al.,
1995; Ball and Lane, 1996). Although the extreme C-terminal peptide
(peptide 11) has amino acid residues important for binding to and
inhibiting Cdk4 (see FIGS. 1 and 2), it cannot bind PCNA (Warbrick
et al., 1995; Ball and Lane, 1996). These results indicate that the
kinase inhibitory and PCNA binding motif in the C-terminus of
p21.sup.WAF1 are distinct, but it does not rule out the possibility
that an interaction between p21.sup.WAF1 and PCNA or cyclin/kinase
may require some common amino acids. It is therefore important to
identify the precise inhibitory motif within the C-terminus of
p21.sup.WAF1 and establish if it overlaps, or is distinct from, the
PCNA interaction domain. To investigate this question we took two
approaches; we synthesised, (i) a series of peptides that had been
shifted by 4 amino acids in either direction along peptide 10 (size
scan; FIG. 7), and (ii) a series of peptides based on peptide 10
where each residue was sequentially mutated to alanine (alanine
scan; FIG. 6). The ability of the peptides, in each of these two
series, to inhibit Cdk4 activity in vitro was then determined.
Using the size scan, we found that the peptide inhibition activity
required amino acids 156-160, while amino acids 148-155 were
dispensable. This uncouples the kinase inhibitory motif from the
PCNA binding motif.
[0218] With the alanine scan we defined the critical residues for
inhibition showing that a stretch of just S amino acids were
essential for activity, with a single conservative point mutation
at either of two hydrophobic residues completely abolishing peptide
10 inhibitory activity (FIG. 6). The essential amino acids are
RRLIF (amino acids 155-160) where the bold characters are essential
for activity and the underlined residue contributes significantly
go inhibitory activity.
[0219] When tested in the peptide precipitation assay, mutation of
the first R of this motif to A (aa 155 of full length p21.sup.WAF1)
partially retained its ability to bind both Cdk4 and cyclin D1 ,
whereas mutations of L or F to A significantly decreased the
affinity for Cdk4 and cyclin D1 , and mutations of the second R or
the I had no effect on binding (data not shown). This is why the
R-A mutant was used in competition assays. The fact that a single
point mutation in either of two hydrophobic residues (the L or F
residues) completely abolishes inhibitory activity, suggested that
inhibition was due to a specific interaction at key hydrophobic
residues. The mapping data also explains why both peptide 10 and
peptide 11 are good inhibitors of cyclin D1-Cdk4 activity (FIG. 2)
as they both contain the inhibitory motif. Thus, it appears that
the inhibitory portion of peptide 10 does not overlap with the PCNA
binding site as they have no amino acid residues in common.
[0220] A Single Amino Acid Substitution in Peptide 10 Makes it a
More Potent Inhibitor thus Approaching the Specific Activity of
Full Length p21.sup.WAF1 Protein
[0221] Whilst carrying out the alanine scan experiments we noticed
that one of the mutant peptides (D-A at position 9 of peptide 10 or
149 of the full length protein; FIG. 6) appeared to make the
peptide a better inhibitor of cyclin D1-Cdk4 activity. We
determined the I.sub.0.5 for this peptide and compared it with
peptide 10, full length purified his-p21.sup.WAF1, and a peptide
derived from the tumour suppressor protein p16 INK4 which has
recently been reported to inhibit cyclin D1-Cdk4 activity in vitro
and to prevent cell cycle progression (Fahraeus et al, 1996). The
D-A
[0222] mutation decreases the I.sub.0.5 from 100 nM to 46 nM (FIG.
7). Comparing this with the p16INK4-based peptide, which has an
I.sub.0.5 of 16.3 .mu.M (FIG. 7), we have now produced a peptide
which is approximately 350-fold more active as a Cdk4 inhibitory
compound. In fact, we now begin to approach the potency of
p21.sup.WAF1 itself, which has an I.sub.0.5 of 11 nM in the insect
cell lysate assay (FIG. 7). This value is in the same range as the
Ki of 40 nM for p21.sup.WAF1 obtained for the inhibition of cyclin
D1-Cdk4 in Sf9 cell lysates by Harper et al. (1995). Compared to
full length protein, the mutant peptide 10 has only a 3.5-fold
lower specific activity as a kinase inhibitor in crude lysates. Why
mutating the D-A in this position, which is well out side the
domain shown to be essential for activity, reduces the I.sub.0.5 is
not known. It seems likely that it involves the presentation of the
inhibitory motif, rather than a direct role for this residue in
inhibition, as this mutation does not appear to increase the
affinity of the peptide for either Cdk4 or cyclin D1 (data not
shown).
[0223] The results indicate that peptide 10 could be used as a
model on which to base small peptide mimetics of p21.sup.WAF1 and
we have provided evidence that alterations in the peptides
structure or presentation of the active residues may lead to the
generation of a peptide inhibitor which approaches the potency of
full length p21.sup.WAF1 as a cyclin D-Cdk4 inhibitor.
[0224] Results for the C-terminal peptide (Peptide 10)
[0225] An Eight Amino Acid Peptide is Sufficient to Inhibit Cyclin
D-Cdk4 Activity
[0226] Having identified residues which appeared to be critical for
the inhibition of cyclin D1-Cdk4 by peptide 10, we determined if
these residues were sufficient for inhibition, or if they had to be
presented within the context of a larger peptide. Strikingly, the
eight amino acid peptide, KRRLIFSK, retained the ability to
completely inhibit cyclin D1-Cdk4 activity and prevent
phosphorylation of pRb (FIG. 6). However, the 15 for the truncated
peptide was approximately 1000-fold higher than that of the full
length peptide (I.sub.0.5 for the truncated peptide was
approximately 100 .mu.M). This was not an unexpected result as
other studies have shown loss of potency upon reducing the length
of bio-active peptides. However, it may be possible to improve the
peptide inhibitory activity by manipulating the non-essential
residues in a manner defined by Lin et al (1995) in an elegant
series of experiments aimed at minimising the atrial natriuretic
peptide.
[0227] Peptide 10 Works in Cell Systems
[0228] The introduction of p21.sup.WAF1 cDNA into human brain, lung
and colon cancer cell lines leads to a suppression of cell growth
(El-Deiry et al, 1993). In addition, during a radiation-induced
G.sub.1 arrest in human fibroblasts p21.sup.WAF1 protein levels
increase, in a p53-dependent manner, leading to potent inhibition
of the G.sub.1 cyclin-CDKs and failure of the cells to enter
S-phase (Dulic et al, 1994; Harper et al, 1995). In order for
peptide 10 to function as a realistic template for the design of
novel anti-proliferative drugs it must be able to mimic
p21.sup.WAF1's CKI activity as a growth suppressor in a cellular
background. We and others have recently shown that a 16 amino acid
sequence from the homeodomain of the Antennapedia protein can act
as a carrier for peptides with biological activity, translocating
them across the plasma membrane and allowing them to interact with
their target molecules (F{dot over (a)}hraeus et al, 1996; Hall et
al, 1996). To determine if peptide 10 retained its biological
activity when introduced into tissue culture cells, we synthesised
it directly onto the carrier peptide and added it to a culture of
proliferating asynchronous human kerotinocyte-derived HaCaT cells.
The linked peptide (designated Peptide-I; FIG. 9) contained a
mutation of M to A at position 7, thus abolishing its activity as a
PCNA binding peptide (Warbrick et al, 1995; Ball and Lane, 1996),
and allowed us to study PCNA-independent affects of the peptide on
normal cell cycle.
[0229] Peptide-I was added to the culture media at a concentration
of 25 .mu.M, the cells were fixed 24 hours later, and then analysed
by fluorescence-activated cell sorting (FACS). G.sub.1, S- and
G.sub.2-phase distribution of untreated and Peptide-I treated cells
was assayed using bromodeoxyuracil (BrdU). The number of cells
entering S-phase in the presence of Peptide-I was dramatically
reduced and the G.sub.1 population showed a concomitant increase.
This suggests that Peptide-1 mimics the ability of full length
p21.sup.WAF1 to act as a growth suppressor by inducing a.
G.sub.1-cell cycle arrest.
[0230] In order to ascertain if Peptide-I was functioning as a
growth inhibitor by preventing the phosphorylation of pRb in a
manner analogues to p21.sup.WAF1, we used serum starvation to
produce a synchronous population of HaCaT cells. Peptide-I was
added to the cells at the same time as they were released from
serum starvation and samples from treated and untreated cells were
taken over a 24 hour period. The phosphorylation status of pRb was
monitored by a gel mobility shift assay. When serum was added to
starved cells, pRb became hyperphosphylated between 12 and 15
hours, but in the presence of Peptide-I pRb remained
hypophosphorylated. Thus, Peptide I causes a G.sub.1-arrest in
human HaCaT cells by preventing the phosphorylation of pRb.
[0231] We took an identical experimental approach to introduce, (i)
the bio-active truncated peptide 10 and (ii) a control peptide 10
which lacked essential residues for CDK inhibition, into HaCaT
cells. We found that Peptide-II effectively promoted a G1-phase
arrest and totally prevented the phosphorylation of pRb when added
at 50M (FIG. 9b). However Peptide-III, which lacked the last 4
amino acids of peptide 10 (LIFS) had no detectable effect on the
ability of HaCaT cells to enter S-phase. It is interesting that the
truncated peptide 10 when coupled to carrier peptide (Peptide-II)
and introduced into cells is only 2-fold less active as a growth
suppressor than Peptide-1 (see above for in vitro data). Linking
the truncated peptide 10 to the carrier peptide may promote a more
favourable inhibitory conformation, as the 10.5 for carrier linked
truncated peptide 10 (Peptide-II in vitro is approximately 50-fold
less than that of the free peptide 10 (data not shown).
[0232] Peptide 10 was added to Rb negative cells, and the results
support its mimicry of the full length protein, i.e. it can mimic
its biolgical activity as a cell cycle inhibitor. Peptide 10 was
found to cause cell cycle arrestin pRb negative, as well as pRb
positive cells. Using Soas2 cells the introduction of peptide I
(peptide 10 linked to penetratin and mutated to prevent PCNA
binding) leads to an increase in the population of cells in G1
phase.
[0233] Discussion
[0234] Synthetic peptides or peptido-mimetics are proving to be
useful in studying the biochemical regulation of enzymes and
proteins, and also in providing models for the design of novel
anti-proliferative agents targeted to the enzymatic pathways
amplified or proteins activated in human tumours (Powis, 1992;
Gibbs and Oliff, 1994). Peptides which have been shown to
effectively target components of the cell cycle machinery include:
FTI, which inhibit farnesyl protein transferase preventing the
activation of Ras (Gibbs et al., 1994); Ras effector domain
peptides, which can inhibit its biological function (Moodie and
Wolfman, 1994; Rodriguez-Viciana et al., 1994); SH2/SH3
domain-harbouring polypeptides, which in theory should inhibit the
growth of tumours with activated tyrosine kinases (Pawson and
Schlessinger, 1993; Yu et al., 1994), and p16INK4-derived peptides,
which inhibit cyclin D-CDK complex activity and thereby activate
pRb-dependent cell cycle arrest (F{dot over (a)}hraeus et al.,
1996).
[0235] Inactivation of the tumour suppressor protein p53 is a
common event in the development of human neoplasia (Hollstein et
al., 1991). The p53 protein is a key player in an inducible cell
cycle checkpoint pathway activated in response to DNA-damage and
nucleotide pool perturbation (Lane, 1992; Agarwal et al., 1995).
Reactivation of this pathway could therefore provide a route to the
discovery of novel anti-proliferative drugs. A variety of
mechanisms could lead to the functional inactivation of the p53
pathway, including the inactivation of downstream effector
molecules of p53, such as the cyclin-kinase inhibitor p21.sup.WAF1
(Deng et al., 1995; Waldman et al., 1995). Recent developments have
shown that reactivation of the p53 pathway in some human tumours
may be possible by activating the biochemical function of the
endogenous mutant p53 protein (Halazonetis and Kandil, 1993; Hupp
et al., 1993), possibly using small peptides as leads for drug
design (Hupp et al., 1995) or by reintroducing the wild type p53
gene using adenovirus vectors (Eastham et al., 1995). However, in
general, the pharmacological restoration of biochemical function to
a protein that has lost its normal-activity through mutation of its
amino acid sequence is more difficult than the inhibition of a
biochemical function (Gibbs and Oliff, 1994). Thus, it may prove
more productive to take alternative approaches to restore activity
to the p53 pathway such as mimicking the inhibitory activity of the
downstream effector molecule p21.sup.WAF1, which can by itself
mediate growth arrest primarily through its interaction with the G1
cyclin-CDKs (El-Deiry et al., 1993; Eastham et al., 1995; Harper et
al.; 1995).
[0236] Determining the minimal domain of p21.sup.WAF1 that can
inhibit CDK function and whether such a domain can function in
isolation with high efficiency are two important goals which must
be achieved in order to determine whether p21.sup.WAF1 will prove
to be a realistic template for use in anti-proliferative drug
design research. Prior to our studies, the minimal sequence of
p21.sup.WAF1 shown to inhibit CDK function in vitro was the
N-terminal domain (residues 1-75) (Luo et al., 1995) Whilst
peptides derived from this N-terminal domain have recently been
shown to antagonise the ability of p21.sup.WAF1 to inhibit cyclin
E-Cdk2 complex activity suggesting that this domain interacts with
the kinase (Chen et al., 1996), no data on the direct interaction
of small peptides with either cyclin or CDK has previously been
presented. In addition, no evidence existed to suggest that a small
peptide derived from p21.sup.WAF1 would in fact be biologically
active as a CDK inhibitor. As the cyclin D1 -Cdk4 complexes and
related isoforms are essential for progression through G1-phase, we
have used a series of small synthetic peptides based on the
sequence of p21.sup.WAF1 to, (i) determine whether Cdk4 inhibitory
peptide-mimetics exist and if they are of high efficacy, and (ii)
probe the mechanism by which the p21.sup.WAF1 protein inhibits
cyclin D1-Cdk4 activity.
[0237] A Model for the Inhibition of Cyclin D1-Cdk4 by
p21.sup.WAF1
[0238] Two distinct peptides from the N-terminal domain of
p21.sup.WAF1 interacted with either Cdk4 or cyclin D1 to form
stable complexes. One peptide bound to Cdk4 but did not inhibit its
activity, while the second bound specifically to cyclin D1 and had
potent inhibitory effects on cyclin D1-Cdk4 activity. The Cdk4
binding peptide 4 (residues 46-65) corresponded to a putative Cdk2
binding domain of p21.sup.WAF1 previously defined using
p21.sup.WAF1 deletion constructs (Nakanishi et al., 1995a) and
alanine mutation analysis (Goubin and Ducommun, 1995). We have
established that this region of p21.sup.WAF1 is, in fact, directly
involved in CDK binding, yet it has no Cdk4 inhibitory activity
(FIGS. 2 and 3). These data explain why certain N-terminally
deleted p21.sup.WAF1 constructs, which still contain the CDK
binding site, fail to efficiently inhibit cell growth (Nakanishi et
al., 1995a).
[0239] The second N-terminal peptide, which bound to cyclin D1,
potently inhibited cyclin D1-Cdk4 activity through a novel
mechanism (see below). The mechanism of p21.sup.WAF1 inhibition of
cyclin-CDK complexes is poorly understood, as it has not been clear
whether p21.sup.WAF1 protein inhibits by cyclin and/or kinase
subunit binding. Cdk2 binds very weakly to p21.sup.WAF1 in the
absence of cyclin, the affinity of the G1-CDKs for p21.sup.WAF1
being greatly increased if the CDK is associated with a cyclin
(Harper et al., 1995), suggesting that cyclins play an important
role in p21.sup.WAF1 inhibition of CDK activity. However, whether a
CKI, such as p21.sup.WAF1 and p27KIP1, can interact directly with
cyclin is in dispute (Toyoshima and Hunter, 1994; Harper et al.,
1995). A recent study however, suggested that p21.sup.WAF1 can
interact directly with a number of cyclins in the absence of CDK
(Fotedar et al., 1996). We show here that a small peptide composed
of residues 16-35 (peptide 2) forms a stable complex with cyclin D1
and that this peptide alone is a potent inhibitor of Cdk4 activity,
with an I.sub.0.5 of 2 mM. This peptide falls within the growth
suppressor region (residues 17-71), described by Nakanishi et al.
(1995a) This is the first time that a putative cyclin binding site
on p21.sup.WAF1 has been identified and that a small synthetic
peptide representing this domain has been shown to be sufficient to
mimic the full length p21.sup.WAF1 protein as a CDK inhibitor.
[0240] The fact that cyclin D1-Cdk4 activity can be inhibited by
interaction with the cyclin subunit alone, suggests either (i) that
conformational changes in cyclin D1 can lead to the inhibition of
Cdk4 catalytic activity, (ii) that peptide 2 interferes with the
interaction of cyclin D1 with Cdk4 or (iii) that peptide 2
interferes with the interaction of cyclin D1-Cdk4 with its
substrate pRb.
[0241] Prospects for the design of small molecular mimetics of
p21.sup.WAF1 are more viable given that the cyclin D1-binding
peptide alone can inhibit kinase function, indicating that the
prior presence of one p21.sup.WAF1 protein binding to the kinase
subunit is not required for inhibition of kinase function. In
addition, the amino acid residues that are conserved between
p21.sup.WAF1 and its close relative p27KIP1 (Polyak et al., 1994;
Toyoshima and Hunter, 1994) are clustered within the N-terminal
domain, with the regions corresponding to peptides 2 (65%
identical) and peptide 4 (50 identical) containing the majority of
the conserved amino acids. This suggests that inhibition of Cdk4
activity by interaction with the cyclin D subunit may be a common
mechanism employed by both p21.sup.WAF1 and p27KIP1.
[0242] A Novel p21.sup.WAF1 C-Terminal Cyclin D1-Cdk4 Inhibitory
Domain During the course of our studies we also found that a
peptide (peptide 10) from the C-terminal domain of p21.sup.WAF1 was
a potent inhibitor of cyclin D1-Cdk4 activity in vitro. The
inhibitory motif was identified and was distinct from the PCNA
interacting site, which also resides in the C-terminal domain of
p21.sup.WAF1 (Chen et al., 1995; Luo et al., 1995; Warbrick et al.,
1995; Ball and Lane, 1996). Our results are in contrast to previous
studies which have found that cyclin-Cdk2 inhibitory activity is
confined solely to the N-terminal domain of p21.sup.WAF1, when each
half is expressed separately (Chen et al., 1995; Luo et al., 1995).
The reasons for this discrepancy may include: (i) the use of
C-terminally his-tagged p21.sup.WAF1 in expression vectors for
purifying p21.sup.WAF1 constructs (Luo et al., 1995), which may
have affected the local structure at the C-terminus of
p21.sup.WAF1; (ii) the transfection of constructs containing only
the C-terminal half of p21.sup.WAF1 (Chen et al., 1995; Luo et al.,
1995) this may make folding into the correct native conformation
difficult precluding identification of the novel inhibitory domain;
(iii) by using peptides, rather that the C-terminal constructs or
full length p21.sup.WAF1 protein, we may have exposed sites which
would not be solvent exposed in native full length p21.sup.WAF1
protein; (iv) it is possible that there may be subtle differences
in the mechanism(s) used by p21.sup.WAF1 to inhibit cyclin-Cdk2
complexes and cyclin D1-Cdk4. Whether, the C-terminal inhibitory
motif defines a novel physiologically relevant regulatory site on
p21.sup.WAF1 is currently being addressed. However, the potency of
peptide 10 (I.sub.0.5=0.1 mM, only 10-fold lower than full length
p21.sup.WAF1 protein in these assays) and its ability to completely
inhibit cyclin D1-Cdk4, suggests to us that further studies on this
region of full length p21.sup.WAF1 will be well worth pursuing.
[0243] Peptide 10 represents a potentially exciting lead for drug
design as it is by far the most potent peptide inhibitor of CDK
activity discovered to date, being >150-fold better than the
recently identified peptide mimetic of p16INK4 (Fahraeus et al.,
1996) and 20-fold better than the N-terminal inhibitory
p21.sup.WAF1-derived-peptide which we have described. The fact that
the residues important for inhibitory activity are confined to a
stretch of just five amino acids, suggests that contact at a single
interface is sufficient to produce a highly potent inhibitor of the
cyclin D1-Cdk4 activity, making this a realistic template for the
design of small molecules which mimic p21.sup.WAF1 activity.
[0244] The fact that peptide 10 retains inhibitory activity when
reduced to just eight amino acids (KRRLIFSK) improves its appeal as
a template for rational drug design. In general protein-protein
interfaces are relatively large relying on the participation of
between 10-30 contact side chains on each interface, with each
region of contact often being composed of residues which are
dispersed throughout the primary amino acid sequence (Davies et al,
1990; de Vos et al, 1992) However, there is evidence that in some
cases only a small subset of these side chains need to be contacted
for efficient binding to occur (Kelley and O'connel, 1993,
Cunningham and Wells, 1994; Clackson and Wells, 1995). The
discovery that a single eight amino acid peptide is alone
sufficient to inhibit the activity of a critical G.sub.1-cyclin-CDK
preventing pRb phosphorylation and producing a G.sub.1-cell cycle
arrest in tissue culture cell systems, suggests that interaction at
only a small subset of contact side chains is necessary for potent
inhibition of cyclin D1-Cdk4 activity at the G1-S phase boundary.
This makes cyclin D1-Cdk4 a realistic and exciting target for the
design of small synthetic compounds which can at act as
anti-proliferative agents.
REFERENCES
[0245] All references mentioned anywhere in this document are
hereby incorporated by reference.
[0246] Agarwal, et al. (1995) Proc. Natl. Acad. Sci. U.S.A., 92:
8493-8497.
[0247] Baldin, et al. (1993). Genes Dev., 7: 8-12-821.
[0248] Ball, K. L. and Lane, D. P. (1996). Biochem., 237:
854-861.
[0249] Buckbinder, et al. (1995). Nature, 377: 646-649.
[0250] Chen, et al. (1996). Oncogene, 12: 595-607.
[0251] Chen, et al. (1995). Nature, 374: 386-388.
[0252] Clackson, T. and Wells J. A. (1995). Science, 267:
383-386.
[0253] Clarke, et al. (1993). Nature, 362: 849-852.
[0254] Cunningham, B. C. and Wells, J. A. (1994). J. Mol. Biol.,
234: 554-563,
[0255] Davies, et al. (1990). Rev. Biochem., 59: 439-473.
[0256] Deng, et al. (1995). Cell, 82: 675-684.
[0257] de Vos, et al. (1992). Science, 255: 306-312.
[0258] Dulic, et al. (1994). Cell, 76: 1013-1023.
[0259] Eastham, et al. (1995). Cancer Res., 55: 5151-5155.
[0260] El-Deiry, et al. (1993). Cell, 75: 817-825.
[0261] Fahraeus, et al. (1996). Curr. Biol., 6: 84-91.
[0262] Flores-Rozas, et al. (1994). Proc. Natl. Acad. Sci. U.S.A.,
91: 8655-8659.
[0263] Gibbs, J. B. and Oliff, A. (1994). Cell, 79: 193-198.
[0264] Gibbs, et al. (1994). Cell, 77: 175-178.
[0265] Goubin, F. and Ducommun, B. (1995). Oncogene, 10:
2281-2287.
[0266] Gu, et al. (1993). Nature, 371: 257-261.
[0267] Halazonetis, T. D. and Kandil, A. N. (1993). EMBO J., 12:
5057-5064.
[0268] Hall, et al. (1996). Curr. Biol., 6: 580-587.
[0269] Harper, et al. (1993). Cell, 75: 805-816.
[0270] Harper, et al. Tsai, L-H., Zhang, P., Dobrowolski, C.
B.,
[0271] Connell-Crowley, et al. (1995) Cell, 6: 387-400.
[0272] Hollstein, et al. (1991). Science, 253: 49-53.
[0273] Hupp, et al. (1993). Nucl. Acids Res., 21: 3167-3174.
[0274] Hupp, et al. (1995). Cell, 83: 337-345.
[0275] Kastan, et al. (1991). Cancer Res., 51: 6304-6311.
[0276] Kastan, et al. (1992). Cell, 71: 587-597.
[0277] Kearsey, et al. (1995). Science, 270: 1004-1005.
[0278] Kelley, R. F. and O'Connell, M. P. (1993). Biochemistry, 32:
6828-6835.
[0279] Lane, D. P. (1992). Nature, 358: 15-16.
[0280] Lowe, et al. (1993). Nature, 362: 84.7-849.
[0281] Luo, et al. (1995). Nature, 375: 159-161.
[0282] Lu, X. and Lane, D. P. (1993). Cell, 75: 765-778.
[0283] Merritt, et al. (1994). Cancer Res., 54: 614-617.
[0284] Miyashita, T. and Reed, J. C. (1995). Cell, 80: 293-299.
[0285] Momand, et al. (1992). Cell, 69: 1237-1245.
[0286] Moodie, S. A. and Wolfman, A. (1994). Trends Genet., 10:
44-48.
[0287] Nakanishi, et al. (1995a). EMBO J., 14: 555-563.
[0288] Nakanishi, et al. (1995b). J. Biol. Chem., 270:
17060-17063.
[0289] Pawson, T. and Schlessinger, J. (1993). Current Biology, 3:
434-432.
[0290] Picksley, et al. (1994). Oncogene, 9: 2523-2529.
[0291] Pietenpol, et al. (1994). Proc. Natl. Acad. Sci. U.S.A., 91:
1998-2002.
[0292] Pines, J. (1995). Nature, 376: 294-295.
[0293] Polyak, et al. (1994). Cell, 78: 59-66.
[0294] Powis, G. (1992). Trends Pharmacol. Sci., 12: 188-194.
[0295] Rodriguez-Viciana, et al. (1994). Nature, 370: 527-532.
[0296] Sherr, C. (1994). Cell, 79: 551-555.
[0297] Smythe, et al. (1988). EMBO J., 7: 2681-2686.
[0298] Toyoshima, H. and Hunter, T. (1994). Cell, 78: 67-74.
[0299] Waldman, et al. (1995). Cancer Research, 55: 5187-5190.
[0300] Warbrick, et al. (1995). Curr. Biol., 5: 275-282.
[0301] Waga, et al. (1994). Nature, 369: 574-578.
[0302] Yu, et al. (1994). Cell, 76: 933-945.
[0303] Xiong, et al. (1993). Nature, 366: 701-704.
[0304] Zhu, et al. (1995) Genes & Development, 9: 1740-1752.
Sequence CWU 1
1
28 1 20 PRT Artificial Sequence Description of Artificial Sequence
Synthesised 1 Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys
Gly Ser Lys 1 5 10 15 Ala Cys Arg Arg 20 2 20 PRT Artificial
Sequence Description of Artificial Sequence Synthesised 2 Lys Ala
Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser 1 5 10 15
Arg Asp Cys Asp 20 3 20 PRT Artificial Sequence Description of
Artificial Sequence Synthesised 3 Ser Arg Asp Cys Asp Ala Leu Met
Ala Gly Cys Ile Gln Glu Ala Arg 1 5 10 15 Glu Arg Trp Asn 20 4 20
PRT Artificial Sequence Description of Artificial Sequence
Synthesised 4 Arg Glu Arg Trp Asn Phe Asp Phe Val Thr Glu Thr Pro
Leu Glu Gly 1 5 10 15 Asp Phe Ala Trp 20 5 20 PRT Artificial
Sequence Description of Artificial Sequence Synthesised 5 Gly Asp
Phe Ala Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu 1 5 10 15
Tyr Leu Pro Thr 20 6 20 PRT Artificial Sequence Description of
Artificial Sequence Synthesised 6 Leu Tyr Leu Pro Thr Gly Pro Arg
Arg Gly Arg Asp Glu Leu Gly Gly 1 5 10 15 Gly Arg Arg Pro 20 7 20
PRT Artificial Sequence Description of Artificial Sequence
Synthesised 7 Gly Gly Arg Arg Pro Gly Thr Ser Pro Ala Leu Leu Gln
Gly Thr Ala 1 5 10 15 Glu Glu Asp His 20 8 20 PRT Artificial
Sequence Description of Artificial Sequence Synthesised 8 Ala Glu
Glu Asp His Val Asp Leu Ser Leu Ser Cys Thr Leu Val Pro 1 5 10 15
Arg Ser Gly Glu 20 9 20 PRT Artificial Sequence Description of
Artificial Sequence Synthesised 9 Pro Arg Ser Gly Glu Gln Ala Glu
Gly Ser Pro Gly Gly Pro Gly Asp 1 5 10 15 Ser Gln Gly Arg 20 10 20
PRT Artificial Sequence Description of Artificial Sequence
Synthesised 10 Lys Arg Arg Gln Thr Ser Met Thr Asp Phe Tyr His Ser
Lys Arg Arg 1 5 10 15 Leu Ile Phe Ser 20 11 20 PRT Artificial
Sequence Description of Artificial Sequence Synthesised 11 Thr Ser
Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 15
Lys Arg Lys Pro 20 12 5 PRT Artificial Sequence Description of
Artificial Sequence Motif 12 Arg Arg Leu Ile Phe 1 5 13 8 PRT
Artificial Sequence Description of Artificial Sequence Motif 13 Lys
Arg Arg Leu Ile Phe Ser Lys 1 5 14 9 PRT Artificial Sequence SITE
(2)..(3) Xaa may be any amino acid 14 Xaa Xaa Xaa Arg Arg Xaa Phe
Xaa Xaa 1 5 15 16 PRT Artificial Sequence Description of Artificial
Sequence Carrier peptide 15 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
Arg Met Lys Trp Lys Lys 1 5 10 15 16 20 PRT Artificial Sequence
Description of Artificial Sequence Synthesised 16 Pro Arg Ser Gly
Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly Asp 1 5 10 15 Ser Gln
Gly Arg 20 17 20 PRT Artificial Sequence Description of Artificial
Sequence Synthesised 17 Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly
Asp Ser Gln Gly Arg 1 5 10 15 Lys Arg Arg Gln 20 18 20 PRT
Artificial Sequence Description of Artificial Sequence Synthesised
18 Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln
1 5 10 15 Thr Ser Met Thr 20 19 20 PRT Artificial Sequence
Description of Artificial Sequence Synthesised 19 Gly Pro Gly Asp
Ser Gln Gly Arg Lys Arg Arg Gln Thr Ser Met Thr 1 5 10 15 Asp Phe
Tyr His 20 20 20 PRT Artificial Sequence Description of Artificial
Sequence Synthesised 20 Ser Gln Gly Arg Lys Arg Arg Gln Thr Ser Met
Thr Asp Phe Tyr His 1 5 10 15 Ser Lys Arg Arg 20 21 20 PRT
Artificial Sequence Description of Artificial Sequence Synthesised
21 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser
1 5 10 15 Lys Arg Lys Pro 20 22 16 PRT Artificial Sequence
Description of Artificial Sequence Synthesised 22 Asp Phe Tyr His
Ser Lys Arg Arg Leu Ile Phe Ser Lys Arg Lys Pro 1 5 10 15 23 8 PRT
Artificial Sequence Description of Artificial Sequence Truncated
peptide 23 Lys Arg Arg Leu Ile Phe Ser Lys 1 5 24 36 PRT Artificial
Sequence Description of Artificial Sequence Synthesised 24 Lys Arg
Arg Gln Thr Ser Ala Thr Asp Phe Tyr His Ser Lys Arg Arg 1 5 10 15
Leu Ile Phe Ser Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met 20
25 30 Lys Trp Lys Lys 35 25 24 PRT Artificial Sequence Description
of Artificial Sequence Synthesised 25 Lys Arg Arg Leu Ile Phe Ser
Lys Arg Gln Ile Lys Ile Trp Phe Gln 1 5 10 15 Asn Arg Arg Met Lys
Trp Lys Lys 20 26 30 PRT Artificial Sequence Description of
Artificial Sequence Synthesised 26 Arg Gln Thr Ser Met Thr Asp Phe
Tyr His Ser Lys Arg Arg Arg Gln 1 5 10 15 Ile Lys Ile Trp Phe Gln
Asn Arg Arg Met Lys Trp Lys Lys 20 25 30 27 8 PRT Artificial
Sequence Description of Artificial Sequence Synthesised 27 Gln Thr
Ser Met Thr Asp Phe Tyr 1 5 28 20 PRT Artificial Sequence
Description of Artificial Sequence Synthesised 28 Lys Arg Arg Gln
Thr Ser Ala Thr Asp Phe Tyr His Ser Lys Arg Arg 1 5 10 15 Leu Ile
Phe Ser 20
* * * * *