U.S. patent application number 10/286964 was filed with the patent office on 2004-02-12 for cyclin dependent kinase binding compounds.
This patent application is currently assigned to Cyclacel Limited. Invention is credited to Fahraeus, Robin, Lane, David Philip.
Application Number | 20040029791 10/286964 |
Document ID | / |
Family ID | 10781044 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029791 |
Kind Code |
A1 |
Fahraeus, Robin ; et
al. |
February 12, 2004 |
Cyclin dependent kinase binding compounds
Abstract
The present invention identifies substances having the property
of binding to cyclin dependent kinase (cdk) comprising: (i) a
peptide including amino acid residues 84 to 103 of full length p16
protein, or an active portion or derivative thereof; or (ii) a
functional mimetic of the fragment. active portion or derivative;
the substance excludes full length p16. p15. p18 and p19 proteins.
These substances are useful in tumour suppression by inhibiting the
phosphorylation of Rb protein. Also described herein is the
resolution of the amino acid motifs responsible for binding cdks,
an FLD motif. corresponding to amino acid residues 90 to 92 of full
length p16 protein. and an LVVL motif, corresponding to amino acid
residues 94 to 97 of full length p16 protein. The substances
disclosed herein can be used in the treatment of hyperproliferative
disorders and to screen and design molecules having the similar
properties.
Inventors: |
Fahraeus, Robin; (Dundee,
GB) ; Lane, David Philip; (St. Andrews, GB) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Cyclacel Limited
|
Family ID: |
10781044 |
Appl. No.: |
10/286964 |
Filed: |
November 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10286964 |
Nov 1, 2002 |
|
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09043560 |
Apr 7, 1999 |
|
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09043560 |
Apr 7, 1999 |
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PCT/GB96/02340 |
Sep 23, 1996 |
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Current U.S.
Class: |
514/21.4 ;
435/184 |
Current CPC
Class: |
C07K 14/4703 20130101;
A61K 38/00 20130101; A61P 17/06 20180101; A61P 35/00 20180101; C07K
14/4738 20130101; C07K 2319/02 20130101 |
Class at
Publication: |
514/12 ;
435/184 |
International
Class: |
C12N 009/99; A61K
038/55 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 21, 1995 |
GB |
9519275.3 |
Claims
1. A substance having the property of binding to cyclin dependent
kinase (cdk) comprising: (i) a peptide including amino acid
residues 84 to 103 of full length p16 protein, or an active portion
or derivative thereof; or, (ii) a functional mimetic of the
fragment, active portion or derivative; wherein the substance
excludes full length p16, p15, p18 and p19 proteins.
2. The substance of claim 1 wherein the cyclin dependent kinase is
cdk4 or cdk6.
3. The substance of claim 1 or claim 2 wherein binding of the
substance to the cyclin dependent kinase causes inhibition of pRb
phosphorylation.
4. The substance of any one of claims 1 to 3 wherein the peptide
includes amino acid residues 89 to 97 of full length p16
protein.
5. The substance of any one of the preceding claims wherein the
peptide fragment includes the amino acid motifs FLD, corresponding
to amino acid residues 90 to 92 of full length p16 protein, and/or
LVVL, corresponding to amino acid residues 94 to 97 of full length
p16 protein.
6. The substance of claim 5 wherein the peptide fragment includes
the amino acid motif FLDxLVVL, wherein x one or more amino
acids.
7. The substance of any one of the preceding claims wherein the
aspartic acid residue in the peptide fragment at the position
corresponding to position 92 of full length p16 protein is
substituted for a hydrophobic amino acid residue.
8. The substance of claim 8 wherein the hydrophobic amino acid
residue is alanine.
9. The substance of any one of the preceding claims derived from
p16, p15, p18 or p19 protein.
10. The substance of any one of the preceding claims coupled to a
carrier molecule so that the substance can be delivered to
cells.
11. The substance of claim 10 wherein the carrier molecule
comprises a peptide derived from an Ancennapedia homeodomain.
12. The substance of any one of the preceding claims wherein the
peptide fragment is coupled to a stabilising molecule.
13. A pharmaceutical composition comprising one or more of the
substances of any one of the preceding claims, in combination with
a physiologically acceptable carrier.
14. A substance of any one of claims 1 to 12 for use in a method of
medical treatment.
15. The use of a substance of any one of claims 1 to 12 in the
preparation of a medicament for the treatment of a
hyperproliferative disorder.
16. The use of claim 15 wherein the hyperproliferative disorder is
cancer, psoriasis or arteriogenesis.
17. Nucleic acid encoding the substances of any one of claims 1 to
12.
18. A vector incorporating the nucleic acid of claim 17 operably
linked to expression control sequences.
19. The use of a substance of any one of claims 1 to 12 in a method
of in screening for (i) compounds having the one or more of the
biological activities of the substances described above or (ii)
compounds which are binding partners of one of the substances.
20. A method of identifying compounds which compete with a
substance of any one of claims 1 to 12, the method comprising: (a)
binding a predetermined quantity of the substance which is
detectably labelled to a cyclin dependent kinase (cdk); (b) adding
a candidate compound; and, (c) determining the amount of the
labelled compound that remains bound to thecdk or which becomes
displaced by the candidate compound.
21. A method of identifying mimetics of a substance of any one of
claims 1 to 12, the method comprising: (a) immobilising one or more
candidate compounds on a solid substrate; (b) exposing the
substrate to a labelled cyclin dependent kinase (cdk); (c)
selecting the candidate compounds that bind to cdk.
22. The method of claim 20 or claim 21 wherein the cdks are
produced in reticulocyte lysates.
23. The method of any one of claims 20 to 22 wherein the cell
dependent kinase is cdk4 or cdk6.
24. The method of any one of claims 20 to 23, further comprising
testing the candidate compound for the property of inhibiting pRb
phosphorylation and/or testing the compound for the property of
inhibiting the entry of cells into the S-phase.
25. The method of any one of claims 20 to 24 wherein the candidate
compounds are selected from a synthetic combinatorial library.
26. The use of a fragment of p16 protein including the amino acid
motifs FLD, corresponding to amino acid residues 90 to 92 of full
length p16 protein, and/or LVVL, corresponding to amino acid
residues 94 to 97 of full length p16 protein in the design of an
organic compound which is modelled to resemble the three
dimensional structure of said amino acid motifs, the organic
compound having the properties of binding to cyclin dependent
kinase and/or inhibiting pRb phosphorylation.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to substances having the
property of binding to cyclin dependent kinase (cdk), and in
particular to substances having this property derived from analysis
of fragments of p16 protein. The present invention also relates to
pharmaceutical compositions comprising these substances and their
use in methods of medical treatment, especially in the treatment of
hyperproliferative disorders. The invention also relates to methods
and uses of these substances in identifying compounds having
related activities.
BACKGROUND TO THE INVENTION
[0002] Phosphorylation of the Rb gene product (pRb) by members of
the cyclin dependent kinase (cdk) family is an important step in
the cells commitment to undergo mitosis This step is regulated in
the later part of the G1 phase of the cell cycle at what is known
as the restriction (R) point (6) The cdks are key regulatory
factors through which both positively and negatively acting cell
signal transduction factors merge. Mitogenic stimulation induces an
active complex between the D-cyclins and cdk4 or cdk6 that is
capable of phosphorylating pRb in late G1. These kinases are also
the targets for cell growth inhibitory signals arising from contact
inhibition, growth factor starvation or TGF-.beta.. The inhibitory
signals can block kinase activity by inducing the production or
activity of different members of the two rapidly enlarging families
of INK4 and p21/KIP cdk-inhibitors that either interfere directly
with the kinases or with the cyclin-kinase complexes (7). The
family of INK proteins that have been identified consists of p15,
p16, p18 and p19 (20, 22, 23).
[0003] However, unlike p21.sup.Cip1/wAF1, which is indirectly
linked to tumour suppression activity through p53 transcriptional
stimulation (8), the INK4p16 gene is itself deleted or mutated in a
large number of human tumours (9-15). Germ line mutations in
INK4p16 have been associated with an increased risk of developing
melanoma (9, 10) The 156 amino acid product of the INK4p16 gene is
known as CDKN2 or p16INK4a (referred to in this application as
"p16")
SUMMARY OF THE INVENTION
[0004] We set out to identify and study the region of p16 that
interacts with cyclin dependent kinases such as cdk4 and cdk6, and
to investigate applications of these properties, in particular the
possibility that the binding of cdks by substances comprising a
peptide based on this region of p16 could be used in tumour
suppression by inhibiting the phosphorylation of Rb protein.
[0005] Small peptides can sometimes be powerful tools to identify
regions of proteins involved in protein-protein interactions and
biological activity (16-19). In this work, we synthesised a series
of overlapping 20 amino acid (aa) peptides that spanned the p16
amino acid sequence, and tested the capacity of each biotinylated
peptide to interact with .sup.35S-labelled cdk4 and cdk6 expressed
in rabbit reticulocyte lysates
[0006] These experiments identified a 20 amino acid synthetic
peptide corresponding to residues 84 to 103 of p16 that interacts
with cdk4 and cdk6, and inhibits cdk4-cyclin D1 mediated
phosphorylation of Rb protein in vitro. An alanine substitution
series defined amino acid residues important for the, cdk4 and cdk6
interaction and for the inhibition of Rb phosphorylation. In this
application, residues 84 to 103 of p16 correspond to the sequence
set out in FIG. 1C, i.e. DAAREGFLDTLVVLHRAGAR.
[0007] Further, when coupled to a small peptide carrier molecule
and applied directly to tissue culture medium, the p16-derived
peptide blocked cell cycle entrance into S-phase in both serum
starved human HaCaT cells and other types of cells that are cycling
normally. This was associated with an inhibition of pRb
phosphorylation in vivo. These results demonstrate that a
p16-derived synthetic peptide coupled to a small carrier molecule
can mimic the G1-phase arrest associated with overexpression of
full length p16 protein. This provides a route to the restoration
of the p16 suppressor gene function in human tumours.
[0008] Accordingly, in a first aspect, the present invention
provides a substance having the property of binding to cyclin
dependent kinase (cdk) comprising:
[0009] (i) a peptide including amino acid residues 84 to 103 of
full length p16 protein, or an active portion or derivative
thereof; or,
[0010] (ii) a functional mimetic of the fragment, active portion or
derivative;
[0011] wherein the substance excludes full length p16, p15, p18 and
p1 9 proteins;
[0012] Preferably, the cyclin dependent kinase (cdk) is cdk4 or
cdk6. The substance preferably also has the property of inhibiting
the phosphorylation of Rb protein which is mediated by a complex
formed between. cdks-and cyclin D. This in turn can be used to
block cellular differentiation by preventing the entry of cells
into the S-phase. As the substances bind cyclin dependent kinases,
they can also be used to prevent the formation of the complex
between cdks and cyclin D, having the additional biological effect
of increasing cyclin D levels in cells. As well as blocking cdk4
and cdk6 dependent phosphorylation of pRb, the substances described
herein could be used to target other cellular substrates, including
the pRb family members p107 and p130, or other substances that are
targets for cdk4 and cdk6 mediated regulation.
[0013] In the present invention, "an active portion" means a
portion of the peptide which is less than the full amino acid
sequence of the fragment above, but which retains the property of
binding to a cyclin dependent kinase (cdk) . Preferably, the
peptide also has the property of inhibiting pRb
phosphorylation.
[0014] In the present invention, a "derivative" is a protein
modified by varying the amino acid sequence of the protein, e.g. by
manipulation of the nucleic acid encoding the protein or by
altering the protein 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 proteins. As an example, a
derivative of peptide 6 in which aspartic acid 92 was substituted
for alanine was found to be more potent than the peptide 6
(residues 84 to 103) in binding to cdk4 and cdk6, and having a
greater inhibition of pRb phosphorylation. Other derivatives
include inserting one or more amino acid residues between amino
acid motifs FLD and LVVL.
[0015] In the present invention, "functional mimetic" means a
substance which may not contain a fragment or active portion of,
the p16 amino acid sequence, and probably is not a peptide at all,
but which has some or all of the properties of the p16 fragment, in
particular the property of binding to a cyclin dependent kinase
and/or inhibiting pRb phosphorylation.
[0016] In a preferred embodiment, the peptide includes residues 89
to 97 of full length p16 protein. More preferably, the peptide
includes the peptide motif FLD, corresponding to amino acids 90 to
92 of full length p16 protein, and/or the peptide motif LVVL,
corresponding to amino acids 94 to 97 of full length p16 protein.
We have also found that both the D and L isoforms of the peptides
share the property of binding to cdk and/or inhibiting pRb
phosphorylation.
[0017] 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. The "Penetratin" molecule
and its properties are described in WO 91/18981.
[0018] In a further aspect, a substance comprising one of the above
peptides can be stabilised by coupling to another peptide sequence.
Preferably, this allows the peptide to adopt a conformation more
closely resembling that of full length p16, typically having the
advantage of increasing the activity of the peptide relative to the
uncoupled fragment, e.g. so that the peptide fragment has an
activity more closely approaching or surpassing that of full length
p16.
[0019] In further aspects, the present invention provides
pharmaceutical compositions comprising one or more of the above
substances and the use of these compositions in methods of medical
treatment. In a preferred embodiment, the present invention relates
to the use of these substances in the preparation of medicaments
for the treatment of hyperproliferative disorders, such as cancer,
psoriasis or arteriogenesis. In particular, cancers which are p16
negative or associated with the overexpression of cdks are
especially likely to respond well to compositions comprising one or
more of the above substances.
[0020] Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may comprise, in addition to one of the above substances, 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 may depend on the route of
administration, e.g. oral, intravenous, cutaneous or subcutaneous,
nasal, intramuscular, intraperitoneal routes. For such,
administration, a parenterally acceptable aqueous solution may be
employed which is pyrogen-free and has suitable pH, isotonicity and
stability. Those skilled in the art are well able to prepare
suitable solutions. Preservatives, stabilisers, buffers,
antioxidants and/or other additives may be included, as required.
Dosage levels can be determined by the those skilled in the art,
taking into account the disorder to be treated, the condition of
the individual patient, the site of delivery, the method of
administration and other factors known to practitioners Examples of
the techniques and protocols mentioned above can be found in
Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed)
1980.
[0021] In embodiments in which the substances are proteins, the
present invention also provides nucleic acid encoding these
proteins. Those skilled in the art can readily construct such
nucleic acid sequences from the amino acid sequences disclosed
herein, taking account of factors such as codon preference in the
host used to express the nucleic acid sequences. In embodiments of
the invention in which the protein is coupled to a carrier protein,
nucleic acid encoding the carrier protein can be linked to the
sequence encoding the. peptides and the sequences expressed as a
fusion.
[0022] In further aspects, the present invention provides vectors
incorporating the above nucleic acid and host cells transformed
with the vectors.
[0023] In a further aspect, the present invention provides the use
of any one of the above substances in screening for (i) compounds
having one or more of the biological activities of the substances
described above or (ii) compounds which are binding partners of one
of the substances, e.g. antibodies or complementary peptides
specific for p16 or a p16 mimetic. Preferably, the substances are
peptide fragments of p16 protein. Examples of screening procedures
for mimetics or binding partners include:
[0024] (a) immobilising the p16 fragments on a solid support and
exposing the support to a library of labelled peptides or other
candidate compounds, and detecting the binding of the peptides or
candidate compound to the p16 fragments;
[0025] (b) using labelled cdks and a library of unlabelled
candidate compound or peptides;
[0026] (c) other combinations of solid phases substrates and
binding measurements;
[0027] (d) Western blots using the fragments of p16 protein and
antibodies raised to the p16 fragments and determining the
displacement of the antibodies by candidate compounds;
[0028] (e) using yeast two hybrid screens to detect candidate
peptides which bind to the p16 peptide or to oligonucleotides
derived from the p16 fragments (for a description of yeast two
hybrid screens see our earlier application WO96/14334);
[0029] (f) using the fragments of p16 protein and/or candidate
compounds in cell systems to determine whether the fragments or
candidate compounds inhibit phosphorylation of Rb and/or prevent
the cells from cycling;
[0030] (g) using the fragments- of p16 protein and/or candidate
compounds in animal models of tumour growth to determine whether
the fragments -or candidate compounds prevent the occurrence of
tumours, reduce tumour size, inhibit tumour growth and/or inhibit
tumour cell migration.
[0031] In a further aspect, the present invention provides method
of identifying compounds which compete with one of the above
substances, the method comprising:
[0032] (a) binding a predetermined quantity of the substance which
is detectably labelled to a cyclin dependent kinase (cdk);
[0033] (b) adding a candidate compound; and,
[0034] (c) determining the amount of the labelled compound that
remains bound to the cdk or which becomes displaced by the
candidate compound
[0035] In a further aspect, the present invention provides a method
of identifying mimetics of one of the above substances, the method
comprising:
[0036] (a) immobilising one or more candidate compounds on a solid
substrate;
[0037] (b) exposing the substrate to a labelled cyclin dependent
kinase (cdk);
[0038] (c) selecting the candidate compounds that bind to cdk.
[0039] In the above aspects, preferably the cyclin dependent kinase
is cdk4 or cdk6. Preferably, the substance is a fragment of p16
protein, and more preferably the FLD and LVVL motifs disclosed
above. Conveniently, the candidate compounds can be selected from a
synthetic combinatorial library.
[0040] The present invention may further comprise testing the
candidate compound for the property of inhibiting pRb
phosphorylation and/or testing the compound for the property of
inhibiting the entry of cells into the S-phase.
[0041] In a further aspect, the present invention provides the use
of a fragment of p16 protein including the amino acid motifs FLD,
corresponding to amino acid residues 90 to 92 of full length p16
protein, and/or LVVL, corresponding to amino acid residues 94 to 97
of full length p16 protein in the design of an organic compound
which is modelled to resemble the three dimensional structure of
said amino acid motifs, the organic compound having the properties
of binding to cyclin dependent kinase and/or inhibiting pRb
phosphorylation.
[0042] 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, eg
peptides are unsuitable active agents for oral compositions as they
tend to be quickly degraded by proteases in the alimentary canal.
Mimetic design, synthesis and testing is generally used to avoid
randomly screening large number of molecules for a target
property.
[0043] 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, eg by substituting each residue
in turn. These parts or residues constituting the active region of
the compound are known as its "pharmacophore".
[0044] Once the pharmacophore has been found, its structure is
modelled to according its physical properties, eg stereochemistry,
bonding, size and/or charge, using data from a range of sources, eg
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.
[0045] 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 in the design of the mimetic.
[0046] 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.
[0047] By way of example, the present invention will now be
described in more detail with reference to the accompanying
figures. The following examples are provided to illustrate the
present invention, and should not be interpreted as limiting the
scope of the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIGS. 1A and B show the relative binding of the p16 derived
peptides to in vitro expressed cdk4 or cdk6.
[0049] FIG. 1C shows peptide 6 corresponds to amino acids 84 to 103
of the p16 protein.
[0050] FIGS. 1D and E show similar binding to in vitro translated
cdk4 and cdk6 of an alanine substitution series of peptide 6 amino
acids. The amino acid residues substituted by alanine are indicated
and the relative amount of cdk4 and cdk6 precipitated by each
peptide is shown. Substituting the hydrophobic residues
corresponding to amino acids 89-90 and 94-97 to alanine decreases
the binding of peptide 6 to both kinases, while substitution of
Asp92 significantly increases the interaction.
[0051] FIG. 1F shows results from an experiment in which Sf9 insect
cell lysates containing cdk4 were incubated with the following
biotinylated peptides: peptide 6 (lanes 1 and 4), peptide 1 (lanes
2 and 5) and peptide 10 (lanes 3 and 8). The complexes were
precipitated with streptavidin-coated agarose beads. The lower of
the cdk4 bands is related to the extraction method (see Materials
and Methods). These results show that if cyclin Di containing
insect cell lysates were added to cdk4 prior to the addition of
peptide, the extracts containing the peptide can not bind the cdk4.
However, if the peptides are added to the cdk4 prior to cyclin D1,
the peptide binds cdk4, suggesting that the peptides can be sued to
interfere in the binding between cyclin D and cdks.
[0052] FIG. 2A shows the inhibition of phosphorylation of E. coli
expressed and purified full length Rb protein. p16 derived wild
type peptides (peptides 1, 6 and 10) or the peptide 6 alanine
substitution series were tested for their capacity to interfere
with pRb phosphorylation by lysates from Sf9 insect cells
containing cdk4. Peptides 1 and 10 of the p16 series do not
precipitate cdk4 or cdk6 and do not affect pRb phosphorylation.
Peptide 6 binds to cdk4 and cdk6 and significantly reduces the
level of pRb phosphorylation. The amino acid residues of the
peptide 6 substituted with alanine are indicated and the levels of
pRb phosphorylation in the presence of each peptide are shown. The
Ala-Asp substitution at residue 92 (Ala92) inhibits phosphorylation
of the full length Rb protein even more efficiently than the wild
type peptide 6, reflecting its more potent binding to cdk4 and
cdk6. Alanine substitution of the hydrophobic residues at positions
90 and 95-97 or closely adjacent residues reduced the inhibitory
capacity to background levels.
[0053] FIG. 2B shows the effect of increasing amounts of peptides
6, Ala92 and Ala94 on pRb phosphorylation. At 50 .mu.M the block of
cdk4-cyclin D dependent pRb phosphorylation by peptide Ala-92 is
nearly complete.
[0054] FIG. 3 shows the inhibition of S-phase entry, of human
keratinocyte derived HaCaT cells with peptide 6 coupled to a small
peptide carrier. Cells were synchronised in G0 by serum starvation
for 72 hours before serum and 10 .mu.M BrdU were added. FIG. 3A
shows the indicated time points after serum stimulation when 100 nM
peptide 6 coupled to the Penetratin carrier molecule was added to
the tissue culture medium. The data presented show % inhibition of
cells entering S-phase after incubation with peptide 6 coupled to
Penetratin in relation to cells incubated with serum only
[0055] FIG. 3B sets out panels A, C, E and G which show cells
synthesising DNA by BrdU labelling, and panels B, D, F and H show
the same fields of cells stained with Hoescht. The percentage of
cells that incorporated BrdU after serum stimulation was 71% at 24
hours (E and F) and 14% (G and H) at 3 hours. The number of cells
incorporating BrdU at 24 hours was significantly reduced when
peptide 6 coupled to the Penetratin carrier molecule was added at
12 hours (panel A and B) compared to at 14 hours (panel C and D).
No effect on DNA synthesis could be observed with Penetratin only
(not shown).
[0056] FIG. 4A shows the phosphorylation of pRb in vivo.
Hyperphosphorylated pRb has a lower affinity for the nuclear
compartment compared to the hypophosphorylated subtypes and can be
extracted from the nucleus by using a hypotonic buffer containing
Triton X-100(26). Panels A, C and E show staining with anti-pRb
monoclonal antibody and B, D and F show the same field of cells
stained with Hoescht. HaCaT cells were serum starved for 72 hours
before serum addition. Peptide 6 coupled to Penetratin (panel C) or
Penetratin by itself (panel A) was added at 8 hours after serum
addition at 100 nM and the amount of phosphorylated pRb was
estimated at 23 hours by determining pRb extractability (panels A
and C) compared with a non-extracted staining (panel E).
[0057] FIG. 4B shows the status of pRb phosphorylation in HaCaT
whole cell extracts as determined by Western blot analysis. Cells
were starved for 72 hours before serum was added and harvested at
the indicated time points. Peptide 6 coupled to Penetratin or
Penetratin by itself was added at 10 hours as indicated.
[0058] FIG. 5A shows the relative inhibition of pRb phosphorylation
by p16, related peptides from corresponding regions of other INK
family members and fragments of p16. In particular, it shows the
effect of reducing the size of the p16 fragment on pRb
phosphorylation. FIGS. 5B and 5C show the corresponding
determination of the binding of the INK family members and
fragments to cdk4 and cdk6.
[0059] FIGS. 6A and 6B show the graphs indicating the effect of
increasing concentrations of peptides 20 and 21 (FIG. 6A) and wt
p16 and V95,96A p16 (FIG. 6B) on the inhibition of cdk-cyclin D
kinase activity. Peptide 20 is a 36 aa long synthetic peptide that
carries the D92A mutation and peptide 21 carries the VV95,96AA
mutation.
[0060] FIG. 7 shows the results from FACS analysis of HaCaT cells
treated with three different peptides 36 aa peptide consisting of a
16 aa Penetratin sequence coupled to a 20 aa peptide (peptide 6,
the V95,96A and the D92A mutant p16 peptide) so that the Penetratin
sequence is at the N-terminus of the 20 aa peptides (i.e.
N-Penetratin-p16-C). In these experiments, the cells were starved
for 72 hours before 10% FCS and the peptides were added and
analysed 2 hours later.
DETAILED DESCRIPTION
Materials and Methods
[0061] Peptide Precipitation
[0062] A 20 aa peptide library with a 5 aa overlap of p16 (apart
from the first 8 N-terminal residues) was synthesised adding a SGSG
linker to the N-terminus to which a biotin group was coupled. The
alanine substitution series of peptide 6 was synthesised in the
same way. The peptides were coupled to streptavidin immobilised on
agarose beads and washed 4 times in PBS before incubating for 1
hour on ice with rabbit reticulocyte lysate (Promega) containing
.sup.35S-methionine labelled cdk4 or-cdk6. The beads were washed 4
times in 1.2.times.PBS with 0.2% Triton X-100 before addition of
SDS loading buffer and applied to 12% SDS polyacrylamide gels. The
gels were exposed to an autoradiography film and the bands
corresponding to cdk4 and cdk6 were analysed by densitometry.
[0063] pRb Phosphorylation in vitro
[0064] Peptides were incubated at a concentration of 25 mM in a
buffer 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 and 1 mM
Na.sub.3V0.sub.4 and 3 ml of extract from Sf9 insect cells infected
with human cdk4-expressing baculovirus lysed in 10 mM Hepes pH 7.4,
10 mM NaCl, 1 mM EDTA and 0.5 mM PMSF. The mixture was incubated
for 60 minutes on ice. Human cyclin D containing Sf9 lysate (3 ml)
prepared as above was added together with 0.6 mg of purified
recombinant full length Rb protein and 2.5 m .sup.32P ATP in a
final concentration of 50 mM ATP and incubated for 10 minutes at
+30.degree. C., the reaction was terminated by addition of SDS
loading buffer and loaded onto 8%; SDS polyacrylamide gels. The
gels were either exposed to autoradiographic film (FIG. 2A) or the
levels of pRb phosphorylation were estimated by a Phosphoimager
(FIG. 2B).
[0065] Cell Cycle Inhibition
[0066] A cysteine residue was added to the C-terminus of peptide 6
and used for coupling to the 16 amino acids long Penetratin peptide
(amino acid sequence RQIKIWFQNRRMKWKK) of the Antennapedia
homeodomain (24) (Appligen) by means of a disulphide bond. Cells
were seeded on cover slips prior to starvation for 72 hours in DMEM
medium without FCS. The medium was substituted by DMEM containing
10% FCS and BrdU. The coupled peptides were added at different time
points after serum stimulation. The number of cells entering
S-phase was determined by estimating the numbers of cells
incorporating BrdU at 24 hours by fixing the cells on cover slips
in acetone/methanol (1:1), incubating in 1M HCl for 30 minutes,
washing 6 times in PBS and then incubating with anti-BrdU
monoclonal antibody and Texas Red conjugated secondary antibody and
mounted in Mowiol containing Hoescht. At least six different areas
on three different cover slips were counted for each single
experiment which was repeated at least two times. The values
presented in FIG. 3A show one representative experiment.
[0067] FACS Analysis
[0068] Twenty minutes before harvest, cells were incubated with 10
.mu.M BrdU. Tryptinised cells were then washed in PBS and
resuspended in 1 ml of PBS and carefully mixed with 3 ml of 96%
EtOH and incubated for 1 hour at 4.degree. C. The cells were then
incubated in 2 ml of 30 mM HCl containing 1 mg/ml of Pepstatin for
30 mins at 37.degree. C. before incubation in 2M HCl for 15
minutes. After careful washing 6 times in PBS, the cells were
incubated in 200 .mu.l (1:50) of anti-BrdU antibodies (Becton
Dickinson) for 1 hour at room temperature. After washing in PBS,
the cells were incubated in FITC conjugated anti-mouse IgG (1:80)
(Sigma) for 30 mins. After washing, the cells were resuspended in
PBS containing 25 .mu.g/ml of Propidium Iodine and analysed on
FACS.
[0069] pRb Phosphorylation in vivo
[0070] Hyperphosphorylated pRb was extracted from cells cultured on
cover slips by treating the cells with hypotonic buffer containing
0.1% Triton X-100 prior to fixation in acetone/methanol (1:1) (26).
Fixed cells were incubated for 1 hour with anti-pRb monoclonal
antibody IF8, washed 3 times in PBS and incubated for 45 minutes
with TexasRed conjugated secondary antibody before the cover slips
were mounted in Mowiol containing Hoescht.
[0071] For Western blot analysis, cells were lysed in RIPA buffer
containing 50 mM Tris pH 8.0, 150 mM NaCl, 1.0% NP-40, 0:5% DOC,
0.1% SDS and 0.1 mM PMSF for 30 minutes at +4.degree. C. The
protein concentrations were determined before-the samples were
boiled in SDS loading buffer, run on 8% SDS polyacrylamide gels and
transferred to a nitrocellulose membrane. The filters were first
incubated with anti-pRb monoclonal antibody IF8 before being
incubated with a horse (DAKO) and developed with ECL
(Amersham).
[0072] Site Directed Mutagenesis
[0073] The VV95,96AA mutations were introduced into wild type his
-tagged p16 protein using the transformation site-directed
mutagenesis kit from Promega, according to manufacturer's
instructions. The mutations were introduced by changing the
corresponding codons from GTG GTG to GCG GCG and then confirming
the sequence by DNA sequencing.
[0074] Results
[0075] Identification of a region of p16 that binds to cdks
[0076] FIGS. 1A and B show that a peptide corresponding to aa 84 to
103 of p16 (peptide 6) when coupled to streptavidin agarose beads
can be used to extract both cdk4 and cdk6 from the reticulocyte
lysates. An alanine substitution series of this peptide (FIG. 1C)
revealed that substitution of hydrophobic amino acids in the region
between residues 89 to 96 decreased the capacity of the peptide to
bind both cdk4 and cdk6. Interestingly, substituion of aspartic
acid 92 with alanine significantly increased the binding of the
peptide to both kinases (FIGS. 1D and E).
[0077] p16 Fragments and the Inhibition of pRb Phosphorylation
[0078] In order to study the functional significance of p16 peptide
interactions with the cyclin dependent kinases we asked whether the
p16 derived peptides, as well as the alanine-substitution series of
peptide 6, affected cdk4-cyclin D ability to phosphorylate pRb in
an in vitro assay (FIG. 2). Only peptide 6 of the p16-derived
series significantly decreased pRb phosphorylation (only results
from peptides 1, 6 and 10 are shown) A correlation was observed
between the capacity of the various peptides in the
alanine-substitution series to bind cdk4 and cdk6 and the level of
inhibition of cdk4-cyclin D1 kinase activity.
[0079] The Effect of Mutations in the p16 Binding Domain
[0080] Most significantly, substitution of amino acids in, or
adjacent to, the two hydrophobic regions located between residues
corresponding to aa 89 to 96 of the full length p16 protein
resulted in a decrease of the peptide 6 induced inhibition of pRb
phosphorylation. Interestingly, the change of aspartic acid 92 to
alanine resulted in a peptide more potent than peptide 6 in
inhibition of the cdk4 kinase activity. A dilution series revealed
that at 50 AM peptide concentration, pRb phosphorylation was almost
completely blocked by the Ala92 peptide and peptide 6. In contrast,
the single substitution Ala94 completely inactivates the function
of peptide 6 in this assay (FIG. 2B). The correlation between the
enhanced binding of the Ala92 peptide and its greater efficiency as
a kinase inhibitor is provocative and suggests that further
variants of the peptide 6 sequence might possess greater activity,
perhaps by encouraging the peptide to adopt a conformation more
similar to that of the active inhibitory site on the native p16
protein. A comparison of the work described here in identifying the
inhibitory region of p16 represented by peptide 6 and other related
proteins shows that an identical motif is present in the
corresponding domain of the kinase inhibitor p15 (20,21) and is
conserved in the closely related p18 (21) and, p19 (22,23)
inhibitors, although prior to this work this similarity and its
significance was not realised by those skilled in the art.
[0081] Point mutations in the p16 gene have been found in tumours
from familial and primary melanomas as well as in tumours from the
oesophagus and the bladder (9, 10, 14, 15). Some encompassed by
peptide 6, and have been shown to have lost their ability to
inhibit cell proliferation and pRb phosphorylation(3,4,12). This
further supports the importance of this region for p16 protein
function.
[0082] Mutations in the p16 protein that result in its inactivation
and are outside of the region suggested above that mediates the
binding to cdk have been shown to induce global conformation
changes of the p16 protein or are temperature sensitive, suggesting
that these mutations might affect the structure of the domain we
suggest mediates binding. Furthermore, deletion of the p16 N- or
C-terminus both result in p16 inactivation, supporting the
deduction that the structure of p16 is sensitive.
Effect of p16 on Cell Proliferation and the Use of Carrier
Peptides
[0083] Since overexpression of p16 in cultured cells can block
S-phase entry (1-4), we wanted to see if peptide 6 could affect
cell proliferation. A 16 aa region of the Antennapedia homeodomain
that has been shown to translocate through biological membranes in
a rapid and energy independent fashion (24) was coupled as carrier
to peptide 6 and added to the tissue culture medium of
serum-starved human keratinocyte derived HaCaT cells.
[0084] FIG. 3 shows that when 0.1 .mu.M of the p16 peptide coupled
to the carrier molecule was added at the same time or up to 12
hours after the addition of serum, the number of cells entering
S-phase were reduced dramatically according to BrdU incorporation
measured at 24 hours after serum addition. However, when the
coupled peptide was added to the medium 14 hours after addition of
serum, the number of cells entering S-phase was the same as that
seen in cells not treated with peptide. This suggests that the
effect of the peptide is limited to a rather narrow window in the
cell cycle that corresponds to the later part of G1. This includes
the restriction (R) point at which serum stimulation and protein
synthesis are no longer required to ensure entry into S-phase and
which has been suggested to be the critical time of pRb
phosphorylation (6,25).
[0085] When starved cells were incubated with the p16 peptide
coupled to the carrier molecule or the carrier molecule alone at 10
hours post serum addition a difference in pRb extractability could
be observed when assayed at 23 hours(26). Approximately 60%; of the
cells incubated with peptide 6 stained with an anti-pRb monoclonal
antibody compared to only 14% incubated with the carrier only (FIG.
4A). This observation was confirmed by Western blot analysis of
whole HaCaT cell extracts treated in a similar fashion (FIG. 4B).
The results suggest that the number of cells carrying
hypophosphorylated pRb increased significantly at 23 hours after
serum stimulation when peptide 6 coupled to the carrier peptide was
added before 12 hours. It also implies that the inhibition of
cdk4-cyclin D activity observed in baculovirus infected Sf9 cell
extracts (FIG. 2) is taking place in vivo.
[0086] The consistent inhibition of S-phase entry after adding the
coupled peptides between 0 and 12 hours suggests that the effect of
the peptide is persistent and that the carrier linked peptide is
not rapidly degraded in the cells. This is consistent with reports
suggesting that the Antennapedia homeodomain carrier peptide is
protected from proteolytic degradation in the cell (24).
[0087] Refinement of the cdk Binding Motifs of p16 and Comparison
with other Proteins
[0088] The results above show that a 20 aa peptide derived from the
third ankyrin like repeat of p16 has similar features as the full
length protein, e.g. binding to the cyclin-cell dependent kinases
cdk4 and cdk6 and to inhibit cdk4-cyclin D1 kinase activity in
vitro as well as to block cell cycle progression. This region
included the peptide sequence that corresponds to aa 84 to 103 of
the full length p16 protein and is identical to the corresponding
region of p15 and highly conserved in p18 and p19 as well as in the
mouse p16.
[0089] Since members of the INK family of kinase inhibitors inhibit
CDK-cyclin D kinase activity specifically by direct interaction
with cdk4-and cdk6, we wanted to see whether this activity can be
determined to one highly conserved domain shared between these
proteins. Since it is mainly p16, and to a lesser extent p15, of
the INK family that is associated with tumour suppressor activity
it will be important to know if these different proteins inhibit
the CDK-cyclin D kinase complex in a similar fashion through the
same domain suggesting that the regulation of expression of these
different proteins determines their role as tumour suppressors
rather than their mean of action. Thus, we carried out experiments
to see if this peptide domain could be further minimized and
substituted with modified amino acid residues that are insensitive
to protease degradation in order to improve its potential as a
model for a synthetic tumour suppressor peptide.
[0090] To study the binding of peptides to cdk4 and cdk6 we
expressed the proteins in a coupled in vitro reticulocyte
translation system in the presence of .sup.35S labelled methionine.
A biotin group coupled to a Ser-Gly-Ser-Gly-linker at the
N-terminus of the peptides was coupled to streptavidin coated
agarose beads and incubated with the cell lysates. FIG. 5A shows
the results measuring pRb phosphorylation, with FIGS. 5B and 5C
showing the determination of the binding of the INK family members
and the p16 peptide fragments to cdk4 and cdk6.
[0091] FIGS. 5A-C show that peptides, corresponding to the 84-103
region of p16, derived from p18 and mouse p16 inhibit pRb
phosphorylation, as reflected in their capacity to inhibit pRb
phosphorylation by Sf9 insect cell lysates overexpressing cdk4 and
cyclin D1, and binds to both cdk4 and cdk6 in a similar way.
[0092] We then tested a p16 peptide deletion series which was made
by deleting two residues at the same time from either the N- or
C-terminus. We found that removing 2 residues-at the N-terminus
(peptide 6 in FIGS. 5A-C) severely reduced, both the cdk binding
and the kinase inhibitory effect and that the activity could be
restored when another two residues were deleted at either terminus.
Peptide 10 only includes the 10 residues that correspond to a motif
that is conserved among ankyrin like repeats and is predicted to
form a tight secondary helical structure. This peptide has lost
some of its binding capacity but is still a good kinase inhibitor
demonstrating that the original 20 amino acid. p16 peptide can be
reduced with at least 10 residues and still inhibit CDK-cyclin D1
kinase activity. This deletion series and the alanine scan shows
that in the peptides we examined there was a strong correlation
between binding and kinase inhibit on.
[0093] It is interesting to notice that the R87P substitution does
not hamper the function of the peptide. Since this mutation, like
most p16 mutations so far detected, is located outside the region
that is shown. to be important for the peptide interaction with the
cdk, it suggests that these mutations will induce conformational
changes of the protein that will effect the central ankyrin like
domain. This hypothesis is strengthened by recent NMR studies
showing that P114 and G101W give rise to global conformational
changes of the protein and that the R87P mutation has been shown to
be temperature sensitive. Similar explanations might also give an
answer to the surprising observations that the peptide loses its
effect when two residues are taken off the N-terminus, suggesting
that these deletions causes conformational changes of the peptide.
The minimal binding peptide (peptide 10) basically consists of two
hydrophobic regions surrounded by polar residues that could form an
amphipatic helical wheel. The p1 8 peptide carries 8 substitutions,
compared to the p16 peptide, and the QT at positions 95 and 96
would at first seem to disturb the binding domain of the peptide
since these disrupts the second hydrophobic pocket. However, this
peptide carries two hydrophobic residues at position 97 and 98
instead which are surrounded by polar residues and it also has the
GFLD region intact, as well as leucine 98, which might suggest that
the second hydrophobic pocket can be moved a few residues toward
the C-terminus without effecting cdk binding.
[0094] Thus, these results show that after initial reductions in
the size of the p16 fragments resulted some reduction in cdk
inhibition (see peptide 6), further reduction in the size of the
p16 fragments once again increased the inhibition- (peptides 7 to
10). This demonstrates that small peptides comprising 16 aa
(peptide 7), 14 aa (peptide 8), 12 aa (peptide 9) and 10 aa
(peptide 10) are able to exert a biological effect which is the
same or analogous to that of full length p16. These results also
support the idea that two motifs of p16 are important for kinase
binding, a FLD motif and a LVVL motif.
[0095] Accordingly, these results indicate that the 20 aa fragment
of p16 disclosed above (residues 84 to 103) can be made at least 50
smaller and still be active.
[0096] Alanine Substitution at Positions 95 and 96
[0097] The results above from an alanine scan substitution series
of the p16 peptide suggested that the two valines at position 95
and 96 could be important for the binding of the peptide to cdk and
for its kinase inhibitory function, and that substitution of
aspartic acid 92 to alanine would potentiation both binding and
kinase inhibitory capacity It is also clear from the peptide
deletion series that these residues are within the 10 residues that
mediate binding of the peptide.
[0098] Accordingly, to investigate this further, we introduced the
VV95;96AA and the D92A mutations into the highly purified peptides
that were linked to the third domain of the Anteinapedia
homeodomain for transporting peptides across biological membranes
and the W95;96AA into the full length is protein in order to see if
residues that seem important for peptide binding to cdk4 and cdk6
also influenced the binding off the full length protein. The
VV95;96AA mutations were introduced into a His-tagged wild type p16
and expressed in E. coli and the corresponding peptides were
synthesized at 99.9% purity.
[0099] FIGS. 6A and 6B show the results of these studies. The
results show that peptide 21, which has the two VV95,96AA
substitutions, is significantly less active in vivo. The same
peptide is also less active in inhibiting the cdk-cyclin D kinase
activity in vitro. This confirms the results of the first alanine
scan described above which suggested that these residues are
important for the peptide to bind to cdk4 and cdk6.
[0100] When the same substitutions are put into the wild type
His-tagged p16 protein a similar loss of kinase inhibitory activity
is observed (in both cases the 50% kinase inhibitory concentration
is increased about 5-fold). While we do not wish to be bound by,
any particular theory, these results further support the view that
these residues are involved in direct binding of the peptide
fragments of p16, as well as the full length protein, to cdk4 and
cdk6, i.e. the mechanism of kinase inhibition is the same in the
full length p16 and the peptide fragments described here.
[0101] In vivo, at the same concentration, the peptide 20 induces
an almost complete block of S-phase entry in HaCaT cells, whereas
the pep21 has only marginal effect at a 10 .mu.M peptide
concentration.
[0102] Response to p16 in Different Cell Lines
[0103] Mouse embryonic fibroblast (MEF) from mice that are p16(-/-)
(knockouts) and from normal p16(+/+) were tested for response to
the p16 peptides linked to Penetratin described above. After 12
hours of treatment with the same amount of the p16 peptide linked
to Penetratin there is an increase of 42i of the (-/-) cells
population in G1 compared to 9% of the (+/+). After 24 hours the
figure is 22% compared to 5%. At the same time the decrease in S
phase is 33% for the (+/+) 30%. Taken together, these results
suggest that p16(-/-) MEFs are more sensitive to the p16 peptide
than the (+/+).
[0104] We have also tested some other cell lines and we see effect
in cells derived from fibroblast, epithelial and muscle origin, and
these show a similar suppression of growth in response to p16
peptide. By way of comparison, no growth suppression was observed
in a Saos 2 (pRb negative cell line), confirming the biological
action of p16 is through the inhibition of pRb phosphorylation.
These results are summarised in Table 1.
[0105] We have also carried out experiments to see whether p16
peptide will inhibit the differentiation of mouse myoblast cells
(C2C12 cells) into myotube cells. These results show that when
C2C12 cells are put in 0.5% FCS medium they will stop growing and
from multinucleated myotubes. However, if they are treated with the
p16 peptide they will, in addition to stopping growing, also
inhibit the formation of multinucleated myotubes in the presence of
0.5% FCS.
1TABLE 1 Tested Growth suppressor cell lines effect by p16 peptide
Source p16(-/-) ++ C2C12 ++ HaCaT +++ human keratinocyte line human
primary +++ keratinocytes MRC5 + human fibroblast MCF7 +++ human
breast cancer derived line MEF+/+ + mouse embiyo fibroblast p16
positive 3T3 ++ mouse fibroblast line Saos2 - pRb negative tumour
cell line
[0106] We have also observed that epithelial cells treated with the
p16 peptide will alter the morphology of the cell-colonies to a
more dense and rounded up phenotype. This is associated with an
increase in the cells adhesion to the tissue culture dish resulting
in 6.times.increase in time before the cells will come off after
trypsin treatment, demonstrating that the peptides will change cell
adhesion properties. The modification of cellular adhesion
properties has applications in preventing the spread of a tumour to
form secondary tumours, providing a method for modulating the
invasive capacity of tumour cells.
[0107] Finally, preliminary results suggest that the peptide are
associated with senescence in human keratinocyte derived cells
(HaCaT cells) as determined by a Beta-Gal assays. This is also
important since it has been suggested that one of the physiological
functions of the p16 protein involves senescence mechanisms which
might be linked to its tumour suppressor role in vivo. The assay
for identifying senescence in cells is described in Dimri et al,
P.N.A.S., 1995, page 9396 seq.
[0108] FACS Analysis
[0109] FIG. 7 shows the results from FACS analysis of HaCaT cells
treated with three different peptides 36 aa peptide consisting of a
16aa Penetratin sequence coupled to a 20aa peptide (peptide 6, the
V95,96A and the D92A mutant p16 peptide). These graphs show that
the cells that do not get FCS are in the G1 phase and have been
arrested properly by the addition of the peptides.
[0110] Conclusions
[0111] These results demonstrate that a 20 aa synthetic peptide
corresponding to residues 84 to 103 of the p16 protein can mimic
essential biochemical and biological properties described for the
full length wild-type p16 protein. Most important is the discovery
that the peptide coupled to a small carrier molecule has the
capacity to inhibit cell proliferation in vivo after direct
addition to the tissue culture medium. This method generically
broadens the application of small peptides in studying biological
events in vivo and, in this case, may allow them to be used to
replace specific suppressor gene function for therapeutic
applications and to serve as models for identifying targets for
novel anti-proliferative drugs.
[0112] As a number of different tumours show defects in the pRb
phosphorylation regulatory pathway, including over expression of
cdk4 and cyclin D1, as well as showing loss of p16 function. All
these tumours are potential candidates for a drug that would
inhibit cdk-cyclin D activity in vivo.
REFERENCES
[0113] The references mentioned in this application are all herein
incorporated by reference.
[0114] 1. Serrano, M.et al. Nature, 366, 704-707 (1993)
[0115] 2. Serrano, M. et al. Science, 267, 249-252 (1995)
[0116] 3. Lukas, J. et al. Nature, 375, 503-506 (1995)
[0117] 4. Koh, J. et al. Nature, 375, 506-510 (1995)
[0118] 5. Parry, D. et al. EMBO J., 14, 503-511 (1995)
[0119] 6. Weinberg, R., Cell 81, 323-330 (1995)
[0120] 7. Sherr, C. & Roberts, J., Genes & Development 9,
1149-1163 (1995)
[0121] 8. Hunter, T. & Pines, J., Cell 79, 573-582 (1994)
[0122] 9. Kamb, A. et al., Nature Genetics 8, 22-26 (1994)
[0123] 10. Caldas, C. et al, Nature Genetics 8, 27-31 (1994)
[0124] 11. Schmidt, E. et al,. Cancer Research 54, 6321-6324
(1994)
[0125] 12. Ranade, K. et al., Nature genetics 10, 114-116
(1995)
[0126] 13. Okamoto, A. et al., P.N.A.S. 91, 11045-11049 (1994)
[0127] 14. Mori, T. et al., Canc. Res. 54, 3396-3397 (1994)
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[0129] 16. Warbrick, E. et al., Current Biology 5, 275-282
(1995)
[0130] 17. Picksley, S. M.et al., Oncogene 9, 2523-2529
[0131] 18. Moodie, S. A & Wolfman, A, Trends Genet. 10, 44-48
(1994)
[0132] 19. Pawson, T. & Schlessinger, J., Current Biology 3,
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[0135] 22. Hirai, H. et al., Mol. & Cell. Biol. 15, 2672-2681
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[0137] 24. Derossi, D. et al., J. Biol. Chem. 269, 10444-10450
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[0139] (26. Mittnacht, S. & Weinberg, R., Cell 65, 381-393
(1991)
Sequence CWU 1
1
16 1 20 PRT Artificial Sequence Description of Artificial Sequence
synthetic peptide 1 Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val
Val Leu His Arg 1 5 10 15 Ala Gly Ala Arg 20 2 4 PRT Artificial
Sequence Description of Artificial Sequence synthetic peptide 2 Leu
Val Val Leu 1 3 16 PRT Artificial Sequence Description of
Artificial Sequence synthetic peptide 3 Arg Gln Ile Lys Ile Trp Phe
Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 4 4 PRT Artificial
Sequence Description of Artificial Sequence synthetic peptide 4 Ser
Gly Ser Gly 1 5 4 PRT Artificial Sequence Description of Artificial
Sequence synthetic peptide 5 Gly Phe Leu Asp 1 6 8 PRT Artificial
Sequence Description of Artificial Sequence synthetic peptide 6 Phe
Leu Asp Xaa Leu Val Val Leu 1 5 7 20 PRT Artificial Sequence
Description of Artificial Sequence synthetic peptide 7 Asp Ala Ala
Arg Ala Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu 1 5 10 15 Phe
Gln Ala Asp 20 8 20 PRT Artificial Sequence Description of
Artificial Sequence synthetic peptide 8 Asp Ala Ala Arg Glu Gly Phe
Leu Asp Thr Leu Val Val Leu His Gly 1 5 10 15 Ser Gly Ala Arg 20 9
20 PRT Artificial Sequence Description of Artificial Sequence
synthetic peptide 9 Asp Ala Ala Pro Glu Gly Phe Leu Asp Thr Leu Val
Val Leu His Arg 1 5 10 15 Ala Gly Ala Arg 20 10 20 PRT Artificial
Sequence Description of Artificial Sequence synthetic peptide 10
Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg 1 5
10 15 Ala Gly Ala Arg 20 11 16 PRT Artificial Sequence Description
of Artificial Sequence synthetic peptide 11 Asp Ala Ala Arg Glu Gly
Phe Leu Asp Thr Leu Val Val Leu His Arg 1 5 10 15 12 18 PRT
Artificial Sequence Description of Artificial Sequence synthetic
peptide 12 Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg
Ala Gly 1 5 10 15 Ala Arg 13 16 PRT Artificial Sequence Description
of Artificial Sequence synthetic peptide 13 Glu Gly Phe Leu Asp Thr
Leu Val Val Leu His Arg Ala Gly Ala Arg 1 5 10 15 14 14 PRT
Artificial Sequence Description of Artificial Sequence synthetic
peptide 14 Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg
1 5 10 15 12 PRT Artificial Sequence Description of Artificial
Sequence synthetic peptide 15 Phe Leu Asp Thr Leu Val Val Leu His
Arg Ala Gly 1 5 10 16 10 PRT Artificial Sequence Description of
Artificial Sequence synthetic peptide 16 Phe Leu Asp Thr Leu Val
Val Leu His Arg 1 5 10
* * * * *