U.S. patent application number 10/193768 was filed with the patent office on 2003-06-12 for inhibitors of phosphoserine and phosphothreonine-proline-specific isomerases.
This patent application is currently assigned to Max-Planck-Gesellschaft zur Forderung der and Beth Israel Deaconess Medical Center. Invention is credited to Cantley, Lewis, Fischer, Gunter, Lu, Kun Ping, Yaffe, Michael.
Application Number | 20030109423 10/193768 |
Document ID | / |
Family ID | 26737312 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109423 |
Kind Code |
A1 |
Lu, Kun Ping ; et
al. |
June 12, 2003 |
Inhibitors of phosphoserine and phosphothreonine-proline-specific
isomerases
Abstract
Inhibitors of phosphoserine- or phosphothreonine-specific
pepidyl prolyl isomerases are described.
Inventors: |
Lu, Kun Ping; (Newton,
MA) ; Cantley, Lewis; (Cambridge, MA) ; Yaffe,
Michael; (Somerville, MA) ; Fischer, Gunter;
(Halle, DE) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Max-Planck-Gesellschaft zur
Forderung der and Beth Israel Deaconess Medical Center
|
Family ID: |
26737312 |
Appl. No.: |
10/193768 |
Filed: |
July 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10193768 |
Jul 10, 2002 |
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08988842 |
Dec 11, 1997 |
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6462173 |
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60058164 |
Sep 8, 1997 |
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Current U.S.
Class: |
514/19.3 ;
435/184; 514/19.2 |
Current CPC
Class: |
C07K 14/001 20130101;
A61K 38/00 20130101; C07K 1/047 20130101; C07K 1/006 20130101; A61P
35/00 20180101 |
Class at
Publication: |
514/7 ;
435/184 |
International
Class: |
C12N 009/99 |
Goverment Interests
[0002] This invention was made with U.S. Government support under
USPHS Grant Nos. RO1 GM56230 and GM56203. The United States
government has certain rights in the invention.
Claims
What is claimed is:
1. An inhibitor of a phosphoserine- or phosphothreonine-proline
specific peptidyl-prolyl isomerase comprising a molecule that
mimics the structure and conformation of the pSer/pThr-Pro peptide
moiety of the isomerase substrate when the substrate is bound into
the active site of the isomerase.
2. The inhibitor of claim 1 wherein the structure surrounding the
mimic moiety is flanked on one side by hydrophobic residues and on
the other side by hydrophobic or positively charged groups wherein
the groups contact the active site of the isomerase.
3. An inhibitor of a phosphoserine- or phosphothreonine-proline
specific peptidyl-prolyl isomerase comprising a protein, peptide or
peptide mimetic comprising xSer/ThrY wherein x is a negatively
charged tetra or pentavalent moiety and Y is a Pro or a Pro
analog.
4. The inhibitor of claim 3 wherein x is selected from the group
consisting of phosphate, sulfonate, boronate, phosphonate and
sulfonyl amide.
5. The inhibitor of claim 3 wherein the proline analog is a
nitrogen-containing ring structure selected from the group
consisting of imidazole, pyrole, tropolone, benzene, camphor and
heterocyclic aromatic and non-aromatic ring structures.
6. The inhibitor of claim 3 wherein the K.sub.l of the inhibitor is
ten micromolar or less.
7. The inhibitor of claim 3 wherein xSer/Thr-Y is flanked on one
side by hydrophobic residues and on the other side by hydrophobic
residues or positively charged residues.
8. The compound of claim 3 wherein the protein, peptide or peptide
mimetic is linear or cyclic.
9. A method of inhibiting cell growth comprising inhibiting a
mitotic peptidyl-prolyl isomerase in the cell comprising contacting
the cell with an effective amount of the inhibitor of claim 3.
10. The method of claim 9 wherein the mitotic peptidyl-prolyl
isomerase is Pin1.
11. The method of claim 9 wherein the cell is in an individual.
12. The method of claim 9 wherein the cell growth results from a
hyperplastic or neoplastic disorder.
13. The method of claim 9 wherein the cells are eukaryotic
cells.
14. The method of claim 9 wherein the cells are selected from the
group consisting of: mammalian cells, yeast cells and fungal cells.
A composition comprising an inhibitor of claim 3 and a
pharmaceutically-acceptable carrier.
16. A compound that inhibits a phosphoserine- or phospho
threonine-proline specific peptidyl-prolyl isomerase comprising a
protein, peptide and/or a peptide mimetic wherein said protein,
peptide or peptide mimetic has a core sequence of XXXpSer-ProXXX,
wherein X is any L-amino acid, or D-amino acid.
17. The compound of claim 16 wherein the ptrotein, peptide or
peptide mimetic is about eight amino acid residues in length and
comprises the sequence
X.sub.1X.sub.2X.sub.3pS-PX.sub.4X.sub.5X.sub.6 wherein each residue
can be independently selected as follows X.sub.1 is W, Y or F;
X.sub.2 is F or I; X.sub.3 is Y, R, F or W; X.sub.4 is R, F, Y or
W; X.sub.5 is L or I and X.sub.6 is any amino acid.
18. The compound of claim 16 wherein the inhibitor has a K.sub.l of
ten micromolar or less.
19. A peptide inhibitor of a phosphoserine- or
phosphothreonine-proline-sp- ecific peptide prolyl isomerase
comprising Trp-Phen-Tyr-pSer-Pro-Arg.
20. A library of peptides that comprises a mixture of substantially
equimolar amounts of peptides comprising the sequence
NH.sub.2-MAXXXpSXXXAKK, wherein for each peptide X is any amino
acid.
21. A library of compounds that comprises a mixture of
substantially equimolar amounts of peptides comprising the sequence
X.sub.1X.sub.2X.sub.3PS-PX.sub.4X.sub.5X.sub.6, wherein for each
peptide X is any amino acid.
22. A method of identifying a phosphorserine- or
phosphothreonine-specific peptidyl prolyl isomerase inhibitor
comprising the steps of: a) providing a library of compounds that
comprises a mixture of substantially equimolar amounts of peptides
comprising the sequence
X.sub.1X.sub.2X.sub.3pS-PX.sub.4X.sub.5X.sub.6, wherein for each
peptide X is any amino acid; b) contacting the library of a) with
the peptidyl prolyl isomerase of interest under binding conditions
for time sufficient for the isomerase to bind to the peptides; c)
determining the amino acid sequences of peptides bound to the
isomerase of interest; d) synthesizing the peptides of c); and e)
determining the K.sub.l of the peptide wherein a peptide with a
K.sub.l of ten micromolar or less indicates that the peptide is
suitable for use as an inhibitor of the isomerase of interest.
23. A method of identifying a phosphorserine- or
phosphothreonine-specific peptidyl prolyl isomerase inhibitor
comprising the steps of: a) providing the peptidyl prolyl isomerase
of interest; b) mixing the isomerase of interest with: i) a
candidate inhibitor molecule and ii) the substrate of the isomerase
of interest to form an admixture of the isomerase of interest,
candidate molecule and substrate; c) maintaining the admixture of
b) under conditions sufficient for the isomerase of interest to
catalyze the cis/trans isomerazation of the substrate; and d)
determining the K.sub.l of the candidate molecule, wherein a
K.sub.l of 10 micromolar or less is indicative of an inhibitor of
the peptidyl prolyl isomerase of interest.
Description
RELATED APPLICATION
[0001] This application claims priority to U.S. provisional
application Serial No. 60/058,164, the entire teachings of which
are incorporated herein by reference.
BACKGROUND
[0003] Events of the eukaryotic cell cycle are regulated by an
evolutionarily conserved set of protein kinases. The
cyclin-dependent kinases (Cdks) are important for driving cells
through different phases of the cell cycle and their sequential
activation and inactivation are tightly regulated. At the G2/M
transition, activation of the mitotic Cdk, Cdc2, requires multiple
events; these include the synthesis and binding of cyclin B,
phosphorylation on Cdc2 at an activating site by Cak, and finally,
Cdc25-dependent dephosphorylation of inactivating sites that have
been phosphorylated by Wee1 and Myt1 (P. Nurse, Cell 79:547 (1994);
R. W. King, P. K. Jackson, M. W. Kirschner, Cell 79:563 (1994); T.
R. Coleman, W. G. Dunphy, Curr. Opin. Cell Biol. 6:877 (1994)).
[0004] How activation of a Cdk elicits the downstream events of
cell cycle progression is less well understood. Activation of
cyclin B/Cdc2 leads to the phosphorylation of a large number of
proteins, mainly on sites containing a Ser/Thr-Pro motif. Protein
phosphorylation is believed to alter the functions of proteins to
trigger the events of mitosis. In a few cases, mitotic
phosphorylation has been shown to regulate mitotic events (R. Heald
and F. McKeon, Cell 61:579 (1990); E. Bailly, et al., Nature
350:715 (1991); A. Blangy, et al., Cell 83:1159 (1995)). However,
it is not understood how the rapid changes in mitotic
phosphorylation are converted to the sequential events of
mitosis.
[0005] An important experimental tool which has uncovered the
general role of phosphorylation in mitotic regulation is the MPM-2
monoclonal antibody (F. M. Davis, et al., Proc. Natl. Acad. Sci.
USA 80:2926 (1983)). MPM-2 recognizes a Phospho.Ser/Thr-Pro epitope
on approximately 50 proteins which are localized to various mitotic
structures (J. M. Westendorf, P. N. Rao, L. Gerace, Proc. Natl.
Acad. Sci. USA 91:714-8 (1994)). Several important mitotic
regulators are recognized by this antibody, including Cdc25, Wee1,
topoisomerase IIa, Cdc27, Map 4, INCENP and NIMA (Stukenberg, P.
T., K. D. Lustig, T. J. McGarry, R. W. King, J. Kuang and M. W.
Kirschner, Curr Biol 7:338-348 (1997)).
[0006] Currently six kinases have been shown to phosphorylate
proteins in vitro to produce the MPM-2 epitope: Cdc2, Polo-like
kinase (Plk1), NIMA, MAP kinase, a MAP kinase (MEK), and an
unidentified activity ME-H (Kuang, J. and C. L. Ashorn., J Cell
Biol 123:859-868 (1993); Taagepera et al., Mol Biol Cell
5:1243-1251 (1994); Kumagai, A. and W. G. Dunphy, Science
273:1377-1380 (1996); Renzi, L., M. S. Gersch, M. S. Campbell, L.
Wu, S. A. Osmani and G. J. Gorbsky, J. Cell Sci 110:2013-2025
(1997)). However, these kinases also phosphorylate substrates that
do not generate the MPM-2 epitope especially in cell cycle stages
other than mitosis. This suggests that there are additional
features that are required for the recognition by MPM-2.
Determination of the optimal MPM-2 binding sequence have confirmed
the importance of amino acid residues flanking the Phospho
Ser/Thr-Pro motif for the MPM-2 recognition (Westendorf, J. M., P.
N. Rao and L. Gerace. Proc Natl Acad Sci U S A 91:714-718 (1994)).
Westendorf, et al., 1994).
SUMMARY OF THE INVENTION
[0007] The present invention is based on the discovery that an
essential mitotic peptidyl prolyl isomerase specifically recognizes
phosphorylated serine/threonine-proline bonds present in mitotic
phosphoproteins. As a result of this discovery, a novel class of
moleculular compounds are available with activity to act as
inhibitors of phosphoserine/phosphothre- onine-proline specific
peptidyl prolyl isomerases, in particular the peptidyl prolyl
isomerase, Pin1, and other Pin1-like isomerases. Accordingly, these
molecular inhibitors are useful to treat disorders of cell
proliferation such as hyperplastic or neoplastic disorders, wherein
treatment of the disorder with an inhibitor of the present
invention results in the arrest of mitosis and apoptosis (cell
death) of the target cells.
[0008] The inhibitor compounds of the present invention include any
molecule that binds into the active site of the phosphoserine- or
phosphothreonine-proline specific peptidyl prolyl isomerase and,
upon binding to the isomerase, inhibits the isomerase activity.
Encompassed by the present invention are inhibitor compounds that
mimic the structure and conformation of the substrate moiety when
bound to the catalytic site (also referred to herein as the active
site) of the isomerase. Molecular inhibitors of the the present
invention will typically have an inhibition constant (K.sub.i) in
the nanomolar to micromolar range. Specifically encompassed herein
are organic molecules that mimic the structure and conformation of
pSer/pThr and bind to the isomerase of interest, thereby inhibiting
its activity.
[0009] The inhibitor compounds of the present invention inculde
inhibitors that comprise a core region that mimics the
pSer/pThr-Pro peptide moiety of the phosphoserine- or
phosphothreonine-proline peptidyl prolyl isomerase substrate.
Encompassed by the present invention are inhibitors that comprise
the pSer/pThr mimic moiety with the mimic moiety being flanked on
one side by hydrophobic groups and on the other side by hydrophobic
or positively charged groups, wherein the groups would contact the
active site of the isomerase of interest.
[0010] The inhibitor compounds of the present invention include
compounds that contain a core sequence comprising xSer/xThrY
wherein "x" is a negatively charged tetra-or pentavalent moiety and
"Y" is a Pro (proline) or a Pro analog. More specifically, the
inhibitors of the present invention include compounds that inhibit
a phosphoserine- or phosphothreonine-proline specific
peptidyl-prolyl isomerase comprising a protein, polypeptide,
peptide and/or a peptide mimetic wherein said protein, polypeptide,
peptide or peptide mimetic comprises pSer/pThr. Specifically
encompassed are inhibitors that have the core sequence of
XXXpSer-pProXXX, wherein X is any L-amino acid or D-amino acid.
[0011] Candidate molecules of the present invention are evaluated
for inhibitory activity in competitive inhibition assays. For
example, the assay mixture would include the candidate molecule to
be tested for inhibiting activity, the isomerase of interest and
the intended substrate of the isomerase of interest. This admixture
is maintained for a time sufficient and under conditions sufficient
for the isomerase of interest to bind and catalyze the
isomerization of its intended substrate. The catalytic activity of
the isomerase of interest in the presence of the candidate
inhibitor is then compared with the activity of the isomerase in
the absence of the candidate inhibitor. If the activity of the
isomerase in the presence of the inhibitor is less than the
activity of the isomerase in the absence of the inhibitor, the
candidate inhibitor is suitable for use as an inhibitor of the
isomerase of interest.
[0012] Encompassed by the present invention are inhibitors of
interphase-specific pSer/pThr-Pro specific peptidyl prolyl
isomerases. Specifically encompassed by the present invention are
inhibitors of the essential mitotic peptidyl prolyl isomerase,
Pin1, and other PIN1-like isomerases.
[0013] Also encompassed by the present invention are methods of
inhibiting mitotic peptidyl-prolyl isomerases comprising
administering an effective amount of an inhibitor as described
herein. For example, a composition comprising an effective amount
of the inhibitor and a pharmaceutically acceptable carrier can be
administered to an individual in need thereof. Specifically
encompassed are methods of inhibiting unwanted cell growth
resulting from a hyperplastic or neoplastic disorder. Also
encompassed by the present invention are methods of inhibiting cell
growth in target cells, comprising contacting the cells with an
inhibitor as described herein.
[0014] The present invention also relates to libraries of peptides
that comprises a mixture of substantially equimolar amounts of
peptides comprising the sequence NH.sub.2-MAXXXpSXXXAKK, wherein
for each peptide X is any amino acid.
[0015] The present invention also relates to methods of identifying
a phosphorserine-or phosphothreonine-specific peptidyl prolyl
isomerase inhibitor comprising the steps of:
[0016] a) providing a library of compounds that comprises a mixture
of substantially equimolar amounts of peptides comprising the
sequence X.sub.1X.sub.2X.sub.3pS-PX.sub.4X.sub.5X.sub.6, wherein
for each peptide X is any amino acid;
[0017] b) contacting the library of a) with the peptidyl prolyl
isomerase of interest under binding conditions for time sufficient
for the isomerase to bind to the peptides;
[0018] c) determining the amino acid sequences of peptides bound to
the isomerase of interest;
[0019] d) synthesizing the peptides of c); and
[0020] e) assaying peptides of d) for cis/trans isomerization by
the peptidyl prolyl isomerase of interest to determine which
peptides undergo isomerization by the isomerase of interest, thus
identifying peptides that bind to the isomerase and are suitable
for use as inhibitors of the isomerase of interest.
[0021] The present invention further relates to methods of
identifying a phosphorserine or phosphothreonine-specific peptidyl
prolyl isomerase inhibitor comprising the steps of:
[0022] a) providing the peptidyl prolyl isomerase of interest;
[0023] b) mixing the isomerase of interest with:
[0024] i) a candidate molecule and
[0025] ii) the substrate of the isomerase of interest to form an
admixture of the isomerase of interest, candidate molecule and
substrate;
[0026] c) maintaining the admixture of b) under conditions
sufficient for the isomerase of interest to catalyze the cis/trans
isomerization of the substrate; and
[0027] d) determining the K.sub.l of the candidate molecule,
wherein a K.sub.i of 10 micromolar or less is indicative of an
inhibitor of the peptidyl prolyl isomerase of interest.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 depicts a model for the Pin1-dependent regulation of
mitosis-specific phosphoproteins that are phosphorylated by Cdc2
and other mitotic kinases (M kinase).
[0029] FIG. 2 is a graphic representation of the results of an
experiment showing that Pin1 inhibits mitotic division in Xenopus
embryos.
[0030] FIGS. 3A-C are graphic representations showing that Pin1,
but not the mutant, directly inhibits the ability of Cdc25 to
activate cyclin B/Cdc2.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention is related to the discovery that an
essential mitotic pepidyl-prolyl isomerase (PPIase) recognizes
phosphorylated serine/threonine (pSer/pThr) bonds present in
mitotic phosphoproteins. Pin1 is an essential peptidyl-prolyl
cis-trans isomerase (PPIase). It is distinct from two other
well-characterized PPIase families: the cyclophilins and the
FK-506-binding proteins (FKBPs), which are targets for the
immunosuppressive drugs cyclosporin A and FK506, respectively
(reviewed in Schreiber, S. L., Science 251:283-287 (1991)).
[0032] PPIases are ubiquitous enzymes that catalyze rotation about
the peptide bond preceding a Pro residue, and may accelerate the
folding and trafficking of some proteins (reviewed in Schmid, F.
X., Curr. Biol. 5:993-994 (1995)). Interestingly, inhibition of
PPIase activity is not required for the immunosuppressive property
of cyclosporin A and FK506. Furthermore, neither the cyclophilins
nor the FKBPs are essential for normal cell growth. Thus, evidence
for the biological importance of PPIase enzymatic activity has been
limited.
[0033] In contrast, Pin1 contains a PPIase domain that is essential
for cell cycle progression and its subcellular localization is
tightly regulated at the G2/M transition (Lu, K. P., S. D. Hanes
and T. Hunter, Nature 380:544-547 (1996)). Pin1 is localized in a
defined nuclear substructure in interphase, but is concentrated to
the condensed chromatin, with some staining in other structures,
during mitosis. Furthermore, depletion of Pin1 protein in HeLa
cells or Pin1/Ess1p in yeast results in mitotic arrest, whereas
overexpression of Pin1 induces a G2 arrest. These results suggest
that Pin1 is an essential mitotic regulator that both negatively
regulates entry into mitosis and is required for progression
through mitosis.
[0034] As described herein, Pin1-binding proteins have been
identified in human cells and Xenopus extracts. Pin1 has been
identified in all eukaryotic organisms where examined, including
plants, yeast, Aspergillus, and mammals (sequences deposited in
GenBank). Results indicate that although Pin1 levels are constant
throughout the cell cycle, the interaction of Pin1 and its targets
is cell cycle-regulated and depends upon mitotic phosphorylation of
target proteins.
[0035] Pin1 directly interacts with a large subset of
mitosis-specific phosphoproteins, which includes Cdc25, Wee1, Myt1,
Plk1, Cdc27 and E-MAP115 as well as some others recently identified
by a screen for mitotic phosphoproteins (Stukenberg, P. T., K. D.
Lustig, T. J. McGarry, R. W. King, J. Kuang and M. W. Kirschne,
Curr Biol 7:338-348 (1997)). Many of these Pin1-interacting
proteins are also recognized by the MPM-2 antibody. In functional
assays, microinjection of Pin1 inhibits mitotic division in Xenopus
embryos and entry into mitosis in Xenopus extracts, as is the case
in HeLa and yeast cells. Furthermore, Pin1 binds the mitotically
phosphorylated form of Cdc25 in vitro and in vivo, and it binds
Cdc25 on the important phosphorylation sites and inhibits its
activity. This characterization of the Pin1-Cdc25 interaction can
at least partially explain the ability of Pin1 to inhibit the G2/M
transition. All these activities of Pin1 are dependent upon the
ability of Pin1 to mitotic phosphoproteins since the activities are
disrupted by point mutations which inhibit the ability of Pin1 to
recognize this unique class of phosphoproteins.
[0036] Also as described herein, Pin1 is a sequence-specific and
phosphorylation-dependent PPIase that can specifically recognize
the phosphorylated Ser/Thr-Pro bonds present in mitotic
phosphoproteins. These results suggest that Pin1 acts as a general
modulator of mitotic phosphoprotein activity, presumably by
catalyzing phosphorylation-depende- nt Pro isomerization.
[0037] The crystal structure of human Pin1 complexed with an
Ala-Pro dipeptide suggests that the isomerization mechanism of Pin1
includes general acid-base and covalent catalysis during peptide
bond isomerization (Ranganathan et al., Cell 89:875-886 (1997)).
More interestingly, Pin1 displays a unique substrate specificity.
It prefers an acidic residue N-terminal to the isomerized Pro bond
due to interaction of the acidic side chain with a basic cluster in
Pin1. This basic cluster consists of the highly conserved residues
Lys63, Arg68, and Arg69 at the entrance to the active site. In the
crystal structure, this conserved triad sequestered a sulfate ion
in close proximity to the .beta. methyl group of the Ala residue in
the bound Ala-Pro dipeptide. One candidate for this negatively
charged residue is Phospho-Ser/Thr.
[0038] To investigate how Pin1 interacts with essential mitotic
proteins, a glutathione S-transferase (GST) Pin1 fusion protein was
used to screen oriented degenerate peptide libraries. The oriented
peptide library approach (Z. Songyang et al. Cell 72:767 (1993) was
used to screen for optimal peptides. All amino acids except Cys
were incorporated at equimolar amounts in each degenerate position,
yielding a total theoretical degeneracy for both libraries of
19.sup.6=4.7.times.10.sup.7 distinct peptide sequences. Pin1-GST
beads and MPM2 antibody bound to protein-G beads were incubated
with the peptide library mixtures and washed extensively. Bound
peptides were eluted with 30% acetic acid and sequenced. The
crystal structure of Pin1 containing an Ala-Pro dipeptide substrate
revealed a sulfate ion located 5 .ANG. from the C.sub..beta. carbon
of Ala (A), suggesting that phosphorylated Ser (pSer) might be
preferred at this site (R. Ranganathan, K. P. Lu, T. Hunter, J. P.
Noel, Cell 89:875 (1997)).
[0039] Next a pS-containing degenerate peptide library of general
sequence NH.sub.2-MAXXXpSXXXAKK, where X includes every amino acid
except Cys, was prepared. GST-Pin1 protein preferentially bound a
subset of peptides with Pro (P) immediately COOH-terminal to
pSer.
[0040] To investigate whether peptides containing pS-P were
preferred substrates for the isomerase activity of Pin1,
oligopeptide substrates were synthesized and assayed for cis/trans
isomerization by Pin1 and by members of the cyclophilin (Cyp18) and
FKBP (FHBP12) families of PPIases. The chromogenic oligopeptides
were synthesized (A. Bernhardt, M. Drewello, & M. Schutkowski,
Int. J. Peptide Protein Res. 50:143 (1997) and confirmed by NMR.
Standard peptides were purchased from Bachem. PPIase activity was
assayed and the bimolecular rate constants k.sub.cat/K.sub.m were
calculated according to the equation
k.sub.cat/K.sub.m=(k.sub.obs-k.sub.u)/[PPIase], where k.sub.u is
the first-order rate constant for spontaneous cis/trans
isomerization and k.sub.obs is the pseudo-first-order rate constant
for cis/trans isomerization in the presence of PPIase, as described
in G. Fischer, H. Bang, C. Mech, Biomed. Biochim. Acta 43:1101
(1984) and in J. L. Kofron et al. Biochemistry 30:6127 (1991).
Affinity of Pin1 for peptides was measured as described in
Schutkowski, M., Wollner, S., & Fischer, G. Biochemistry
34:13016, (1995)). Neither Cyp18 nor FKBP12 effectively catalyzed
isomerization of peptides with pS/pT-P moieties (Table 1). In
contrast, either Y--P or pY--P bonds were good substrates for both
enzymes. Thus phosphorylation on S/T-P, but not Y--P, renders the
prolyl-peptidyl bond resistant to the catalytic action of
conventional PPIases, and suggests the need for a different enzyme
to catalyze this reaction.
1TABLE 1 Interaction between Pin1 and Selected Mitotic
Phosphoproteins Phosphoproteins Interphase Mitotic Cdc25* - +++
Plk1* - +++ Plx1 - +++ Wee1 + ++ Mos + ++ Cdc27* - +++ NIMA - +++
Sox3 - +++ Xbr-1b - +++ MP75 (E-MAP-115) - +++ MP110 (Cdc5) - +++
MP68 - +++ MP30 - ++ MP105 + + MP48 - - Cyclin B* - -
[0041] The binding between Pin1 and all selected mitotic
phosphoproteins was assayed by incubating synthesized proteins with
interphase and mitotic Xenopus extracts, followed by precipitation
with GST-Pin1 beads. The Pin1 interactions with those proteins
indicated with * were also confirmed by GST-Pin1pull-down assay
from endogenous interphase and mitotic HeLa cell extracts. +, a
week but above background interaction; ++, readily detectable
interaction; +++, strong interaction.
[0042] In contrast to cyclophilins and FKBPs, Pin1 isomerase
activity was highly specific for peptides with pS/pT-P bonds (Table
2). Pin1 displayed little isomerase activity for substrates
containing S/T-P bonds. However, phosphorylation of these peptides
on S or T residues increased the k.sub.cat/K.sub.m values up to
300-fold.
2TABLE 2 Sequence-specific and phosphorylation-dependent PPlase
activity of Pin1 Pplase Activity kcat/KM Substrate
(mM.sup.-1s.sup.-1) AAPL-pNA 55 AAAPR-pNA 121 AAPM-pNA 134 ADPY-pNA
220 AEPF-pNA 3410 AYPY-pNA 5 ApYPY-pNA 3 ASPY-pNA 269 ApSPY-pNA
3370 ATPY-pNA 635 ApTPY-pNA 2480 AAEPF-pNA 220 AASPF-pNA 9
AapSPF-pNA 3760 AATPF-pNA 4 AapTPF-pNA 1370 AASPR-pNA 7 AApSPR-pNA
9300 WypSPRT-pNA 14100 AapTPR-pNA 3700 WFYSPR-pNA 170 WFYpSPR-pNA
20160 Assays were done as described in Table 1 except that trypsin
was used instead of chymotrypsin as an isomer-specific protease
when peptides with Pro-Arg-pNA were used as substrates.
[0043] Pin1 had low isomerization activity for peptides containing
an Ala-Pro peptide bond, whereas incorporation of Glu (E) or Asp
(D) immediately preceding P to mimic the pS, increased
isomerization activity. Peptides containing a Y or pY preceding Pro
were poor substrates for Pin1. This substrate specificity
distinguishes Pin1 from the conventional PPIases in the cyclophilin
and FKBP families.
[0044] To further define the sequence specificity of Pin1, a
degenerate peptide library containing a fixed pS-P sequence flanked
by 3 degenerate positions on each side was used. Pin1 selected Arg
or aromatic residues at the -1 and +1 positions of the pS-P motif
(Table 3). Aromatic amino acids were also selected at the -3,
Phe/Ile at the -2, and Leu/Ile at the +2 position of pS-P. On this
basis, several additional peptides were synthesized as Pin1
substrates, as described above. Peptides with Arg introduced at the
P+1 position proved better substrates with specificity constants
increased up to 1300 fold compared to their non-phosphorylated
counterparts (Table 2). Placing aromatic residues NH.sub.2-terminal
to the pS-P position made these peptides even better substrates
(Table 2). The best substrate identified thus far
(Trp-Phe-Tyr-pSer-Pro-Arg-pNA) is the optimal sequence selected
from the peptide library (Table 2 and 3). The apparent Km of Pin1
towards this peptide was 10 .mu.M.
3TABLE 3 Binding specificity of Pin1 and MPM-2 -3 -2 -1 0 +1 +2 +3
Pin1 W F Y pS P R L X Y I R F I F F Y W W MPM-2 Y W F pS P L X X F
F L I W I V F M GST-Pin1 and MPM-2 were incubated with the pS-P
oriented degenerate peptide library NH.sub.2-MAXXXpSXXXAKK, where X
contains every amino acid except Cys. After an extensive wash,
peptides bound with GST-Pin1 were eluted and sequenced. Amino acids
with a significant selection at each degenerate position are
shown.
[0045] As described herein, Pin1 binds a large subset of mitotic
phosphoproteins also recognized by the monoclonal antibody MPM-2.
Therefore, the sequence specificity for MPM-2 recognition was
evaluated. When immobilized MPM-2 antibody was probed with a
peptide library containing only a pS as the orienting residue,
there was a strong selection for peptides with P at the pS+1
position. Using the pS-P degenerate peptide library, MPM-2 strongly
selected peptides with aromatic and aliphatic amino acids at the
-3, -1 and +1 positions relative to pS-P (Table 3). This MPM-2
binding motif is similar to the sequence motif selected by Pin1
(Table 3) and explains the observation that Pin1 specifically
interacts with MPM-2 antigens.
[0046] To determine the structural basis for the Pin1 substrate
specificity, molecular model-building was performed and tested by
site-directed mutagenesis. A peptide (Trp-Phe-Tyr-pSer-Pro-Arg) was
modeled into the Pin1 structure (R. Ranganathan, K. P. Lu, T.
Hunter, J. P. Noel, Cell 89:875 (1997) assuming that the phosphate
group of pS occupies the position of of sulfate in the structure.
The phosphate of pS in the modeled peptide was superimposed on the
co-crystallizing SO.sub.4 ion in the original Pin1 structure, and P
residue displacements minimized with respect to the Ala-Pro ligand
in the original Pin1 structure using molecular modeling programs
GRASP (A. Nicholls, K. Sharp, B. Honig. Proteins 11:281 (1991),
Molscript and Raster3d. In this model, R68 and R69 of Pin1
coordinate the pS phosphate, a hydrophobic groove accepts the
preceding aromatic tripeptide, and the side chain of C113 and H59
coordinate the isomerizing pS-P peptide bond with
.omega.=90.degree., stabilizing the transition state between cis
and trans configuration. To test these predictions from the model,
site-directed Pin1 mutants were generated and their PPIase activity
assayed as described herein and in K. P. Lu, S. D. Hanes, T.
Hunter, Nature 380:544 (1996); K. P. Lu and T. Hunter, Cell 81:413
(1995).
[0047] Substitution of both R68 and R69 by Ala reduced the
k.sub.cat/K.sub.m to 1/500 that of wild type Pin1 for the
phosphorylated substrate. The catalytic activity of Pin1.sup.R68,
69A was the same as wild-type Pin1 for the unphosphorylated peptide
substrate. Thus, this cluster of basic residues appears to
participate in coordinating the phosphate of pS/pT. Parvulin (J. U.
Rahfeld, et al., FEBS Lett. 352:180 (1994);idid 343:65 (1994); K.
E. Rudd, et al., TIBS 20:12 (1995), the prototype of the Pin1
family of PPIases has R68 and R69 replaced by Glu and failed to
catalyze the isomerization of pS-P peptidyl bonds, though it was
very effective in catalyzing the P isomerization of the
unphosphorylated peptide. Replacement of the catalytic H59 residue
of Pin1 with Ala dramatically decreased the PPIase activity for
both phosphorylated and unphosphorylated peptides; however, the
specificity for phosphorylated over unphosphorylated substrates was
unchanged. For Pin1 the K.sub.cat/K.sub.m for the phosphorylated
versus unphosphorylated substrate is 19,400/7, which is
approximately equal to the similar ratio of 1120/<1 for
Pin1.sup.H59A. Thus H59 appears to play an important role in
catalyzing P isomerization and/or binding the substrate P
residue.
[0048] On the basis of amino acid preferences deduced in the 6
positions surrounding the pS-P motif for optimal Pin1 binding, a
weighted screening of the SWISS-PROT sequence database. Protein
sequence database screening was performed with the program
INDOVINATOR, using an entropy-based weighing technique to score for
relative information content at each amino acid position flanking
the pS/pT-P motif with the quantitative peptide library results,
which are shown qualitatively in Table 3. This scan revealed within
the top 5% of highest scores several potentially important mitotic
phosphoprotein targets for Pin1. Many of these proteins are
involved in regulation of the cell cycle, cytoskeletal/spindle
structure, DNA replication, transcription or RNA processing (Table
4).
4 TABLE 4 Predicted Cell Cycle Regulatory Binding Binding Proteins
Site(s) Confirmed NIMA YVGTPFYM Yes FYMSPEIC ILNTPVIR ESRTPFTR
KSRSPHRR EMPSPFLA Cdc25C YLGSPITT Yes Plk1 ANITPREG Yes Wee1
GRRSPRPD Yes Cdc27 FLWSPFES Yes Cytoskeletal/Spindle Proteins
E-MAP-115 ASCSPIIM Yes Centromere LRKSPFCR protein A Nedd 5
YFISPFGH Nuclear/Splicing/Transcriptional Proteins Splicing factor
RSRSPRRR Yes SC35 DNA topoisomerase DSASPRYI II - alpha Lim 1
homeobox FFRSPRRM Protein Laminin beta-1 DPYSPRIQ Nuclear pore
FGFSPSGT complex Unp214 Guanine-Nucleotide-related Proteins Rab 4
QLRSPRRT Yes Rab GDP YGKSPYLY dissociation inhibitor Protein
Kinases/Phosphatases S6 kinase KIRSPRRF Yes Mkk2 PCYTPYYV Ab12
GFFTPRLI Erk3 WYRSPRLL Jnk1, 2 FMMTPYVV PP2A WGISPRGA Tyrosine
NWRSPRLR phosphatase PTP-H1
[0049] Predicted and/or Confirmed Pin1 Substrates
[0050] Based on the amino acid preference values in each of the 6
positions surrounding the pS/pT-P motif for optimal Pin1 binding
(Table 3), a weighted screening of the SWISS-PROT sequence database
was undertaken. This is a partial list of selected proteins with
the top scores; human sequences are used whenever possible.
Interactions between Pin1 and some of the identified proteins have
been confirmed in vitro. Pin1 not only binds these two proteins,
but also suppresses their functions .paragraph., the interaction
between Pin1 and SC35 is inferred from their colocalization.
[0051] Several of these predicted proteins, such as Rab4, Cdc25 and
NIMA, undergo mitosis-specific phosphorylation (R. Heald and F.
McKeon, Cell 61:579 (1990); E. Bailly, et al., Nature 350:715
(1991); A. Blangy, et al., Cell 83:1159 (1995): J. Kuang, et al.,
Mol. Biol. Cell 5:135 (1994); X. S. Ye, et al., EMBO J. 14:986
(1995)). Cdc25 and NIMA also binds to Pin1 in a
phosphorylation-dependent manner, as described herein. Other
proteins identified in this search, however, had not been
previously suspected of interacting with Pin1; therefore, a few
were further investigated as example cases. Rab4 and ribosomal S6
kinases were found to interact with Pin1 specifically in mitotic,
but not in interphase extracts. Thus, Pin1 binds a wide functional
range of mitotic phosphoproteins.
[0052] Differences in isomerase activity of Pin1 and other PPIases
result from different organization of the X-P binding pocket. In
all PPIases, a hydrophobic pocket sequesters the aliphatic P side
chain (S. L. Schreiber, Science 251:2881 (1991); G. Fischer, Angew.
Chem. Intl. Ed. Engl. 33:1415 (1994); F. X. Schmid, Curr. Biol.
5:993 (1995)), hence the residues responsible for determining
substrate preference must reside at the entrance to the P-binding
pocket. In Pin1 and its homologues (K. P. Lu, S. D. Hanes, T.
Hunter, Nature 380:544 (1996); K. P. Lu and T. Hunter, Cell 81:413
(1995), S. D. Hanes, et al., Yeast 5:55 (1989); R. Maleszka, et
al., Proc. Natl. Acad. Sci. USA 93:447 (1996)), a cluster of basic
residues coordinate the pS phosphate, and determine the specificity
of this isomerase. Absence of a basic pocket in the cyclophilins,
FKBPs, and other members of the parvulin families of PPIases may
explain their failure to isomerize the pS/pT-P bonds.
[0053] The specificity of Pin1 rationalizes Pin1-binding proteins
and also predicts a number of novel potential Pin1 substrates, some
of which have been confirmed as in vitro Pin1 targets. Furthermore,
Pin1 and MPM-2 bind similar sequences and proteins, and have
similar phenotypes, indicating that the wide conservation of MPM-2
epitopes across various species (J. Kuang, et al., Mol. Biol. Cell
5:135 (1994); X. S. Ye, et al., EMBO J. 14:986 (1995), F. M. Davis,
et al., Proc. Natl. Acad. Sci. USA 80:2926 (1983); J. M.
Westendorf, P. N. Rao, L. Gerace, idid 91:714-8 (1994); S.
Taagepera, et al., Mol. Biol. Cell 5:1243 (1994); A. Kumagai, W. G.
Dunphy, Science 273:1377-80 (1996) can be explained by recognition
of this epitope by a highly conserved mitotic regulator, Pin1.
[0054] Based on the determination of specific substrates for Pin1,
as described herein, inhibitors of Pin1, Pin1-like isomerases and
other phospho-Ser/Thr-specific PPIases can be produced. Thus, the
present invention provides compounds that inhibit phosphoserine-
and phosphothreonine-specific peptidyl-prolyl isomerases.
Specifically encompassed by the present invention are peptidyl
prolyl isomerases that recognize phosphorylated
serine/threonine-proline bonds present in mitotic
phosphoproteins.
[0055] The inhibitor compounds of the present invention include any
molecule that binds into the active site of the phosphoserine- or
phosphothreonine-proline specific peptidyl prolyl isomerase and,
upon binding to the isomerase, inhibits the isomerase activity.
Encompassed by the present invention are inhibitor compounds that
mimic the structure and conformation of the substrate moiety when
bound to the catalytic site (also referred to herein as the active
site) of the isomerase. Molecular inhibitors of the the present
invention will typically have an inhibition constant (K.sub.l) of
ten micromolar, or less. Specifically encompassed are organic
molecules that mimic the structure and conformation of pSer/pThr
and bind to the isomerase of interest, thereby inhibiting its
activity. Such inhibitors are of a size and conformation so that it
will bind into the active site of the isomerase of interest.
Typically the inhibitors will have a three-dimensional conformation
of about 12 angstroms wide, about 15 angstroms long and about 15
angstroms deep. However the inhibitor can be as large as about 19
angstroms wide, about 20 angstroms long and 15 angstroms deep.
[0056] The inhibitor compounds of the present invention inculde
inhibitors that comprise a core region (or moiety) that mimics the
pSer/pThr moiety of the phosphoserine- or phosphothreonine-proline
peptidyl prolyl isomerase substrate. Encompassed by the present
invention are inhibitors that comprise the pSer/pThr mimic moiety
with the mimic moiety being flanked on one side by hydrophobic
groups and the other side of the mimic moiety being flanked by
hydrophobic or positively charged groups, wherein the groups would
contact the active site of the isomerase of interest.
[0057] Specifically encompassed by the present invention are
inhibitor compounds comprising proteins, polypeptides and peptides.
The proteins, polypeptides and peptides of the present invention
comprise naturally-occurring amino acids (e.g., L-amino acids),
non-naturally amino acids (e.g., D-amino acids), and small
molecules that biologically and biochemically mimic the inhibitor
peptides, referred to herein as peptide analogs, derivatives or
mimetics. (Saragovi, H. U., et al., Bio/Technology, 10:773-778
(1992)). The protein, polypeptide or peptide inhibitors of the
present invention can be in linear or cyclic conformation.
[0058] Compounds that have PPIase inhibiting activity can be
identified using oriented degenerate peptide libraries as described
herein. For example, a library of xSer/Thr-X-containing peptides of
a defined length can be screened for specific binding to the PPIase
of interest. Peptides that specifically bind to the PPIase of
interest can be further evaluated for PPIase inhibiting activity as
described herein.
[0059] The phosphoserine and phosphothreonine-specific
peptidyl-prolyl isomerase inhibitors, or PPIase inhibitors, of the
present invention can comprise a core sequence of xSer/Thr-Y
wherein x can be any negatively charged tetra- or penta-valent
moiety and Y can be Pro or any Pro analog. Preferred moieties for x
can be phosphate, sulfonate, boronate, phosphonate or a sulfonly
amide. The Pro analog can be any nitrogen-containing ring
structure, including imidazole, pyrole, tropolone, benzene,
camphor, and hetrerocyclic aromatic and non-aromatic ring
structures. Typically, the xSer/Thr-Y core sequence is flanked by
hydrophobic residues or Arg, where the hydrophobic residues (e.g.,
Phe, Tyr, Trp and Ile) typically precede the xSer/Thr residue and
Arg follows the Y residue. Specifically encompassed by the present
invention are inhibitors comprising the core sequence
phosphoserine/phosphothreonine-pr- oline.
[0060] The inhibitors of the present invention can be anywhere from
2 to 200 amino acid residues in length. The inhibitors are
typically 2-20 residues in length, and more typically 2-10 residues
in length. Most typically the PPIase inhibitors are about eight
residues in length, as represented by the consensus sequence,
XXXpSer/pThrXXX, wherein X can be any amino acid residue.
[0061] Encompassed by the present invention are compounds that are
about eight amino acid residues in length and comprise the core
sequence X.sub.1X.sub.2X.sub.3pS-PX.sub.4X.sub.5X.sub.6 wherein
each residue can be independently selected as follows X.sub.1 is W,
Y or F; X.sub.2 is F or I; X.sub.3 is Y, R, F or W; X.sub.4 is R,
F, Y or W; X.sub.5 is L or I and X.sub.6 is any amino acid.
[0062] Specifically encompassed by the present invention is the
inhibitor of a phosphoserine- or phosphothreonine-proline-specific
peptide prolyl isomerase comprising Trp-Phen-Tyr-pSer-Pro-Arg.
[0063] The inhibitors of the present invention can be synthesized
using standard laboratory methods that are well-known to those of
skill in the art, including standard solid phase techniques.
Inhibitors comprising naturally occurring amino acids can also be
produced by recombinant DNA techniques known to those of skill, and
subsequently phosphorylated.
[0064] The inhibitors of the present invention can comprise either
the 20 naturally occurring amino acids or other synthetic amino
acids. Synthetic amino acids encompassed by the present invention
include, for example, naphthylalanine, L-hydroxypropylglycine,
L-3,4-dihydroxyphenylalanyl, .alpha.-amino acids such as
L-.alpha.-hydroxylysyl and D-.alpha.-methylalanyl,
L-.alpha.-methyl-alanyl, .beta. amino-acids such as .beta.-analine,
and isoquinolyl.
[0065] D-amino acids and other non-naturally occurring synthetic
amino acids can also be incorporated into the inhibitors of the
present invention. Such other non-naturally occurring synthetic
amino acids include those where the naturally occurring side chains
of the 20 genetically encoded amino acids (or any L or D amino
acid) are replaced with other side chains, for instance with groups
such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl,
amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,
hydroxy, carboxy and the lower ester derivatives thereof, and with
4-, 5-, 6-, to 7-membered heterocyclic. In particular, proline
analogs in which the ring size of the proline residue is changed
from 5 members to 4,6, or 7 members can be employed.
[0066] As used herein, "lower alkyl" refers to straight and
branched chain alkyl groups having from 1 to 6 carbon atoms, such
as methyl, ethyl propyl, butyl and so on. "Lower alkoxy"
encompasses straight and branched chain alkoxy groups having from 1
to 6 carbon atoms, such as methoxy, ethoxy and so on.
[0067] Cyclic groups can be saturated or unsaturated, and if
unsaturated, can be aromatic or non-aromatic. Heterocyclic groups
typically contain one or more nitrogen, oxygen, and/or sulphur
heteroatoms, e.g., furazanyl, furyl, imidazolidinyl, imidazolyl,
imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.
morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl
(e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,
pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,
thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.
thiomorpholino), and triazolyl. The heterocyclic groups can be
substituted or unsubstituted. Where a group is substituted, the
substituent can be alkyl, alkoxy, halogen, oxygen, or substituted
or unsubstituted phenyl. (See U.S. Pat. No. 5,654, 276 and U.S.
Pat. No. 5,643,873, the teachings of which are herein incorporated
by reference). Biologically active derivatives or analogs of the
above-described inhibitors, referred to herein as peptide mimetics,
can be designed and produced by techniques known to those of skill
in the art. (See e.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and
5,654,276, the teachings of which are herein incorporated by
reference). These mimetics are based on a specific peptide PPIase
inhibitor sequence and maintain the relative positions in space of
the corresponding peptide inhibitor. These peptide mimetics possess
biologically activity (i.e., PPIase inhibiting activity) similar to
the biological activity of the corresponding peptide compound, but
possess a "biological advantage" over the corresponding peptide
inhibitor with respect to one, or more, of the following
properties: solubility, stability, and susceptibility to hydrolysis
and proteolysis.
[0068] Methods for preparing peptide mimetics include modifying the
N-terminal amino group, the C-terminal carboxyl group, and/or
changing one or more of the amino linkages in the peptide to a
non-amino linkage. Two or more such modifications can be coupled in
one peptide mimetic inhibitor. The following are examples of
modifications of peptides to produce peptide mimetics as described
in U.S. Pat. Nos. 5,643,873 and 5,654,276, the teachings of which
are incorporated herein by reference.
[0069] Modification of the N-Amino Terminus
[0070] After solid phase synthesis of the peptide inhibitor, the
blocking group on the N-terminus amino group can be selectively
removed so as to provide for a peptide sequence blocked at all
positions other than the N-terminal amino group and attached to a
solid resin through the C-terminus. One can then modify the amino
terminus of the peptides of the invention to produce peptide
mimetics of the invention.
[0071] Amino terminus modifications include alkylating,
acetylating, adding a carbobenzoyl group, forming a succinimide
group, etc. Specifically, the N-terminal amino group can then be
reacted as follows:
[0072] (1) to form an amide group of the formula RC(O)NH-- where R
is as defined above by reaction with an acid halide (e.g., RC(O)Cl)
or acid anhydride. Typically, the reaction can be conducted by
contacting about equimolar or excess amounts (e.g., about 5
equivalents) of an acid halide to the peptide in an inert diluent
(e.g., dichloromethane) preferably containing an excess (e.g.,
about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine, to a scavenge the acid generated during
reaction. Reaction conditions are otherwise conventional (e.g.,
room temperature for 30 minutes). Alkylation of the terminal amino
to provide for a lower alkyl N-substitution followed by reaction
with an acid halide as described above will provide for
N-alkylamide group of the formula RC(O)NR--;
[0073] (b) to form a succinimide group by reaction with succinic
anhydride. As before, an approximately equimolar amount or an
excess of succinic anhydride (e.g., about 5 equivalents) can be
employed and the amino group is converted to the succinimide by
methods well known in the art including the use of an excess (e.g.,
ten equivalents) of a tertiary amine such as diisopropylethylamine
in a suitable inert solvent (e.g., dichloromethane). See, for
example, Wollenberg, et al., U.S. Pat. No. 4,612,132 which is
incorporated herein by reference in its entirety. It is understood
that the succinic group can be substituted with, for example,
C.sub.2-C.sub.6 alkyl or --SR substituents which are prepared in a
conventional manner to provide for substituted succinimide at the
N-terminus of the peptide. Such alkyl substituents are prepared by
reaction of a lower olefin (C.sub.2-C.sub.6) with maleic anhydride
in the manner described by Wollenberg, et al., supra. and --SR
substituents are prepared by reaction of RSH with maleic anhydride
where R is as defined above;
[0074] (c) to form a benzyloxycarbonyl-NH-or a substituted
benzyloxycarbonyl-NH-group by reaction with approximately an
equivalent amount or an excess of CBZ-Cl (i.e., benzyloxycarbonyl
chloride) or a substituted CBZ-Cl in a suitable inert diluent
(e.g., dichloromethane) preferably containing a tertiary amine to
scavenge the acid generated during the reaction;
[0075] (d) to form a sulfonamide group by reaction with an
equivalent amount or an excess (e.g., 5 equivalents) of
R--S(O).sub.2Cl in a suitable inert diluent (dichloromethane) to
convert the terminal amine into a sulfonamide where R is as defined
above. Preferably, the inert diluent contains excess tertiary amine
(e.g., ten equivalents) such as diisopropylethylamine, to scavenge
the acid generated during reaction. Reaction conditions are
otherwise conventional (e.g., room temperature for 30 minutes);
[0076] (e) to form a carbamate group by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R--OC(O)Cl or
R--OC(O)OC.sub.6H.sub.4-p-NO.sub.2 in a suitable inert diluent
(e.g., dichloromethane) to convert the terminal amine into a
carbamate where R is as defined above. Preferably, the inert
diluent contains an excess (e.g., about 10 equivalents) of a
tertiary amine, such as diisopropylethylamine, to scavenge any acid
generated during reaction. Reaction conditions are otherwise
conventional (e.g., room temperature for 30 minutes); and
[0077] (f) to form a urea group by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R--N.dbd.C.dbd.O in a
suitable inert diluent (e.g., dichloromethane) to convert the
terminal amine into a urea (i.e., RNHC(O)NH--) group where R is as
defined above.
[0078] Preferably, the inert diluent contains an excess (e.g.,
about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine. Reaction conditions are otherwise
conventional (e.g., room temperature for about 30 minutes).
[0079] Modification of the C-Terminus
[0080] In preparing peptide mimetics wherein the C-terminal
carboxyl group is replaced by an ester (i.e., --C(O)OR where R is
as defined above), the resins used to prepare the peptide acids are
employed, and the side chain protected peptide is cleaved with base
and the appropriate alcohol, e.g., methanol. Side chain protecting
groups are then removed in the usual fashion by treatment with
hydrogen fluoride to obtain the desired ester.
[0081] In preparing peptide mimetics wherein the C-terminal
carboxyl group is replaced by the amide --C(O)NR.sup.3R.sup.4, a
benzhydrylamine resin is used as the solid support for peptide
synthesis. Upon completion of the synthesis, hydrogen fluoride
treatment to release the peptide from the support results directly
in the free peptide amide (i.e., the C-terminus is --C(O)NH.sub.2).
Alternatively, use of the chloromethylated resin during peptide
synthesis coupled with reaction with ammonia to cleave the side
chain protected peptide from the support yields the free peptide
amide and reaction with an alkylamine or a dialkylamine yields a
side chain protected alkylamide or dialkylamide (i.e., the
C-terminus is --C(O)NRR.sup.1 where R and R.sup.1 are as defined
above). Side chain protection is then removed in the usual fashion
by treatment with hydrogen fluoride to give the free amides,
alkylamides, or dialkylamides.
[0082] Alternatively, the C-terminal carboxyl group or a C-terminal
ester can be induced to cyclize by internal displacement of the
--OH or the ester (--OR) of the carboxyl group or ester
respectively with the N-terminal amino group to form a cyclic
peptide. For example, after synthesis and cleavage to give the
peptide acid, the free acid is converted to an activated ester by
an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC) in solution, for example, in
methylene chloride (CH.sub.2Cl.sub.2), dimethyl formamide (DMF)
mixtures. The cyclic peptide is then formed by internal
displacement of the activated ester with the N-terminal amine.
Internal cyclization as opposed to polymerization can be enhanced
by use of very dilute solutions. Such methods are well known in the
art.
[0083] Modification to Incorporate a Non-Peptidyl Linkage
[0084] Peptide mimetics wherein one or more of the peptidyl
linkages [--C(O)NH--] have been replaced by such linkages as a
--CH.sub.2-carbamate linkage, a phosphonate linkage, a
--CH.sub.2-sulfonamide linkage, a urea linkage, a secondary amine
(--C.sub.2NH--) linkage, and an alkylated peptidyl linkage
[--C(O)NR.sup.6-- where R.sup.6 is lower alkyl] are prepared during
conventional peptide synthesis by merely substituting a suitably
protected amino acid analogue for the amino acid reagent at the
appropriate point during synthesis.
[0085] Suitable reagents include, for example, amino acid analogs
wherein the carboxyl group of the amino acid has been replaced with
a moiety suitable for forming one of the above linkages. For
example, if one desires to replace a --C(O)NR-- linkage in the
peptide with a --CH.sub.2-- carbamate linkage
(--CH.sub.2OC(O)NR--), then the carboxyl (--COOH) group of a
suitably protected amino acid is first reduced to the --CH.sub.2OH
group which then converted by conventional methods to a --OC(O)Cl
functionality or a para-nitrocarbonate --OC(O)O--C.sub.6H.sub.4-
-p-NO.sub.2 functionality. Reaction of either of such functional
groups with the free amine or an alkylated amine on the N-terminus
of the partially fabricated peptide found on the solid support
leads to the formation of a --CH.sub.2OC(O)NR-linkage. For a more
detailed description of the formation of such --CH.sub.2-carbamate
linkages.
[0086] Similarly, replacement of an amino linkage in the peptide
with a phosphonate linkage can be achieved using techniques known
to those of skill in the art.
[0087] Replacement of an amino linkage in the peptide with a
--CH.sub.2-sulfonamide linkage can be achieved by reducing the
carboxyl (--COOH) group of a suitably protected amino acid to the
--CH.sub.2OH group and the hydroxyl group is then converted to a
suitable leaving group such as a tosyl group by conventional
methods. Reaction of the tosylated derivative with, for example,
thioacetic acid followed by hydrolysis and oxidative chlorination
will provide for the --CH.sub.2--S(O).sub.2Cl functional group
which replaces the carboxyl group of the otherwise suitably
protected amino acid. Use of this suitably protected amino acid
analogue in peptide synthesis provides for inclusion of an
--CH.sub.2S(O).sub.2NR-- linkage which replaces the amino linkage
in the peptide thereby providing a peptide mimetic.
[0088] Replacement of an amino linkage in the peptide with a urea
linkage can be achieved using techniques known to those of skill in
the art.
[0089] Secondary amine linkages wherein a --CH.sub.2NH-- linkage
replaces the amino linkage in the peptide can be prepared by
employing, for example, a suitably protected dipeptide analogue
wherein the carbonyl bond of the amino linkage has been reduced to
a CH.sub.2 group by conventional methods. For example, in the case
of diglycine, reduction of the amide to the amine will yield after
deportection H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2COOH which is then
used in N-protected form in the next coupling reaction. The
preparation of such analogues by reduction of the carbonyl group of
the amino linkage in the dipeptide is well known in the art.
[0090] The suitably protected amino acid analog is employed in the
conventional peptide synthesis in the same manner as would the
corresponding amino acid. For example, typically about 3
equivalents of the protected amino acid analogue are employed in
this reaction. An inert organic diluent such as methylene chloride
or DMF is employed and, when an acid is generated as a reaction
by-product, the reaction solvent will typically contain an excess
amount of a tertiary amine to scavenge the acid generated during
the reaction. One particularly preferred tertiary amine is
disopropylethylamine which is typically employed in about 10 fold
excess. The reaction results in incorporation into the peptide
mimetic of an amino acid analogue having a non-peptidyl linkage.
Such substitution can be repeated as desired such that from zero to
all of the amino bonds in the peptide have been replaced by
non-amino bonds.
[0091] The inhibitors of the present invention can also be cyclic
protein, peptides and cyclic peptide mimetics. Such cyclic
inhibitors can be produced using known laboratory techniques, e.g.,
as described in U.S. Ser. No. 08/864,392, filed on May 28, 1997,
the teachings of which are herein incorporated in their entirety by
reference, and U.S. Pat. No. 5,654,276, the teachings of which are
herein incorporated in their entirety by reference).
[0092] Inhibitors of the present invention are evaluated for
biological activity as described herein. For example, the candidate
compounds can be screened in an assay that determines the
displacement of a labeled high affinity molecule (e.g., a
competitive inhibition asay) in an assay utilizing immobilized
molecules on a grid, as well as screening libraries of candidate
molecules. These techniques are known to those of skill in the
art.
[0093] As defined herein, biological activity of the PPIase
inhibitors include specific binding to the PPIase of interest
(e.g., specific binding to Pin1) and/or specific inhibition of the
peptidyl prolyl isomerase activity as measured as described in
Schutkowski, M. et al., Biochemistry, 34:13016 (1995). Specific
binding to the PPIase of interest can be determined as described
herein. Further evaluation of candidate inhibitors (e.g.,
inhibitors that specifically bind to the PPIase of interest, for
inhibiting activity can be determined by competitive inhibition
assay. Alternatively, candidate moleucules of the present invention
can be directly evaluated for their inhibitory activity withour
prior determination of their specific binding to the isomerase of
interest. Inhibitor compounds of the present invention typically
have a K.sub.i in the nanomolar or micromolar range. Methods to
determine K.sub.i are known to those of skill in the art.
[0094] The inhibitors of the present invention can be used in vitro
to study cell cycle regulation and mitotic events. For example, the
inhibitors of the present invention can be used to evaluate mitotic
events in mammalian cells by inhibiting a specific isomerase and
evaluating the effects on the cell cycle.
[0095] The inhibitors of the present invention can also be used to
interfere with eucaryotic cell growth. The inhibitors can be used
to inhibit cell growth, and to kill targeted cells. For example,
the inhibitors of the present invention can be used to treat fungal
and yeast, including Aspergillus, and parasitic infections (e.g.,
malaria) in mammals. As defined herein, mammals include
domesticated animals and humans. Specifically, the inhibitors of
the present invention can be used to treat hyperplastic and
neoplastic disorders in mammals, including humans.
[0096] For example, Pin1 is an important molecule in controlling
the sequential events of mitosis (FIG. 1). Entry and exit from
mitosis are accompanied by abrupt changes in kinase activities,
which lead to changes in the phosphorylation state of numerous
proteins that trigger specific events in mitosis. Pin1 binding and
consequent inhibition of target protein activity may provide a
means for temporally synchronizing and/or amplifying the activity
of mitotic proteins. Inhibition of Pin1 induces mitotic arrest and
apoptosis. Thus, the Pin1 mediated mechanism of regulating mitotic
events is a therapeutic target for cancer.
[0097] Neoplastic and hyperplastic disorders include all forms of
malignancies, psoriasis, retinosis, athrosclerosis resulting from
plaque formation, leukemias and benign tumor growth. For example,
such disorders include lymphomas, papilomas, pulmonary fibrosis,
rheumatoid arthritis and multiple sclerosis.
[0098] The inhibitors of the present invention can be formulated
into compositions with an effective amount of the inhibitor as the
active ingredient. Such compositions can also comprise a
pharmaceutically acceptable carrier, and are referred to herein as
pharmaceutical compositions. The inhibitor compositions of the
present invention can be administered intraveneously, parenterally,
orally, by inhalation or by suppository. The inhibitor composition
may be administered in a single dose or in more than one dose over
a period of time to achieve a level of inhibitor which is
sufficient to confer the desired effect.
[0099] Suitable pharmaceutical carriers include, but are not
limited to water, salt solutions, alcohols, polyethylene glycols,
gelatin, carbohydrates such as lactose, amylose or starch,
magnesium stearate, talc, silicic acid, viscous paraffin, fatty
acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. The
pharmaceutical preparations can be sterilized and desired, mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsiers, salts for influencing
osmotic pressure, buffers, coloring, and/or aromatic substances and
the like which do not deleteriously react with the active
compounds. They can also be combined where desired with other
active agents, e.g., enzyme inhibitors, to reduce metabolic
degradation.
[0100] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Ampoules are convenient unit dosages.
[0101] It will be appreciated that the actual effective amounts of
the inhibitor in a specific case will vary according to the
specific compound being utilized, the particular composition
formulated, the mode of administration and the age, weight and
condition of the patient, for example. As used herein, an effective
amount of inhibitor is an amount of inhibitor which is capable of
inhibiting the isomerase activity of the isomerase of interest,
thereby inhibiting target cell growth and resulting in target cell
death. Dosages for a particular patient can be determined by one of
ordinary skill in the art using conventional considerations, (e.g.
by means of an appropriate, conventional pharmacological
protocol).
[0102] The present invention is illustrated by the following
examples, which are not intended to be limited in any way.
EXAMPLE 1
Expression, Purification and Kinetic Analysis of Recombinant Pin1
Proteins
[0103] Pin1 was expressed and purified by Ni.sup.2+-NTA agarose
column as an N-terminally His.sub.6-tagged fusion protein, followed
by removing the tag using thrombin, as described in Lu et al.,
1996; and Ranganathan et al., 1997). To generate an N-terminally
GST-Pin1 fusion protein, Pin1 cDNA was subcloned into a pGEX vector
and the resulting fusion protein was expressed and purified by
glutathione agarose column, as described in Lu et al., 1993; Lu, et
al., 1996. GST-Pin1 was stored in the agarose bead at 4.degree. C.
for 2 weeks or eluted from the beads and concentrated to 20 mg/ml
with a Centricon-10 (Amicon), followed by storing at -80.degree. C.
Both preparations were stored in a buffer containing 20 mM HEPES,
pH 7.5, 50 mM NaCl and 1 mM DTT, as described in Ranganathan et
al., 1997. All proteins were quantified by the method of Bradford
(Biorad) using BSA as a standard.
[0104] Site-directed mutations of Pin1 were introduced using
PCR-based techniques and verified by DNA sequencing. The
corresponding mutant proteins were expressed and purified using the
same procedures as those described for wild-type Pin1. PPIase
activity was measured, as described previously (Lu et al., 1996),
with the exception that the absorbance of p-nitroaniline (at 395
nM) was followed every second for 2-10 min. and data were analyzed
offline using a kinetic computer program written by G.
Tucker-Kellogg in the C. Walsh lab at Harvard Medical School.
EXAMPLE 2
Analysis of Pin1 and its Binding Proteins During Cell Cycle
[0105] HeLa cells were arrested at the G1/S boundary using double
thymidine and aphidicolin block, and released to enter the cell
cycle, as described in Heintz, N., H. L. Sive and R. G. Roeder, Mol
Cell Biol 3:539-550 (1983) and Lu, K. P. and T. Hunter, Cell
81:413-424 (1995)). To accumulate cells at mitosis, nocodazole (50
ng/ml) was added to cells at 8 h after the release for the
specified period of time. To obtain a large quantity of interphase
and mitotic cells, HeLa cells were incubated with double thymidine
and aphidicolin or nocodazole for 16 h, which resulted in over 90%
of cells being arrested at the G1/S boundary or mitosis,
respectively. Cells were harvested and a aliquot of cells was
subjected to flow cytometry analysis, as described in Lu and
Hunter, 1995). The remaining cells were lysed in RIPA buffer (10 mM
sodium phosphate pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 10
.mu.g/ml aprotinin, 50 .mu.g/ml phenylmethylsulfonyl fluoride and 1
mM DTT) and same amount of total proteins were subjected to
immunoblotting analysis using various antibodies or Farwestern
analysis using GST-Pin1 as a probe. For Farwestern analysis, after
blocking with 5% BSA, membranes were incubated with 2 .mu.g/ml
GST-Pin1 in TBST for 2 hr, followed by incubation with anti-GST
monoclonal antibodies (UBI) and the ECL detection procedures.
EXAMPLE 3
Microinjection of Xenopus Embryos
[0106] Unfertilized eggs were incubated with sperm, dejellied, and
4 .mu.M of the indicated protein (about 10 fold above the estimated
endogenous levels) was injected in one cell of two cell stage
embryos (30 embryos each protein). The injected embryos were
allowed to develop at 18.degree. C. to stage 8 and pictures were
taken of typical embryos. The titration of Pin1 and the mutants was
essentially as described above except that the indicated protein
was injected into one cell of 4 cell stage (18 embryos each Pin1
concentration) to the indicated final concentration and allowed to
develop for 3 h. The cell cycle blocks by GST-Pin1 were not
homogeneous as cells that were injected with greater concentrations
of GST-Pin1 were cleaved fewer times indicating a tighter cell
cycle block. To be consistent, cell cycle blocked embryo's were
scored as those that contained at least one cell on the injected
side that was greater than 5 times larger than uninjected cells
(FIG. 2).
EXAMPLE 4
Preparation of Xenopus CSF Extracts
[0107] Xenopus CSF extracts were prepared from unfertilized eggs,
as previously described (Murray, 1991) and used immediately. To
examine the effect of Pin1 on mitotic entry, a fresh CSF extract
containing demembranated sperm (150 .mu.l) and rhodamine tubulin (2
.mu.g/ml) was activated by addition of 0.4 mM calcium chloride for
15 min, before the indicated concentrations of various Pin1
proteins were added and mitotic entry was followed for 2 h by
nuclear morphology, nuclear envelope breakdown, spindle formation
and Cdc2 activity, as described previously (Murray, A. W., Methods
Cell Biol 36:581-605 (1991)). The cell cycle state of nuclei within
the extracts were over 90% synchronous and typical nuclei were
photographed.
EXAMPLE 5
Synthesis of Mitotic Phosphoproteins
[0108] The mitotic phosphoproteins were translated in vitro using
the TNT coupled transcription/translation kit (Promega) in a total
volume of 10 .mu.l in the presence of 8 .mu.Ci [.sup.35S]methionine
(1000 Ci/mmol) for 2 h at 30.degree. C. They were then incubated in
Xenopus interphase and mitotic extracts as described (Stukenberg et
al., 1997). These incubated clones were precipitated by Pin1 beads
as described below. The Xenopus Mos and Wee1 clones were a kind
gift of M. Murakami, G, F. Woude and J. Cooper; the Xenopus Cdc25
clone was a generous gift of W. Dunphy, T3 and T3S2 Cdc25 mutants
were kindly provided by J. Maller (Izumi and Maller, 1993).
EXAMPLE 6
Production of Pin1 and Cdc25 Antibodies
[0109] Since antibodies that we previously raised against
C-terminal peptide of Pin1 (Lu, et al., 1996) did not have a high
sensitivity for detecting Pin1, especially for Xenopus Pin1,
rabbits were immunized with His-Pin1 as an antigen. After 2 months,
antisera specifically recognize a single 18 kDa Pin1 protein in
human cells and Xenopus extracts.
[0110] To raise antibodies against Xenopus Cdc25, recombinant
GST-Cdc25 (the clone was a kind gift of A. Nebrada and T. Hunt) was
affinity purified as described by the manufacture (Pharmacia). The
protein was further purified by SDS-PAGE and a gel slice containing
Cdc25 was used to immunize rabbits.
EXAMPLE 7
GST Pull-down, Imunoprecipitation and Immunoblotting Analysis
[0111] To detect Pin1-binding proteins, either HeLa cells were
lysed in or Xenopus extracts were diluted in a buffer (buffer A)
containing 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 100 mM NaF, 1 mM
sodium orthovanadate, 10% glycerol, 1% Triton X100, 10 .mu.g/ml
aprotinin, 50 .mu.g/ml phenylmethylsulfonyl fluoride and 1 mM DTT.
The lysates were preclarified with boiled S. aureus bacteria
(CalBiochem) and then incubated with 10 .mu.l of agarose beads
containing various GST-Pin1 proteins or control GST for 2 h at
4.degree. C. The precipitated proteins were washed 5 times in the
same buffer and subjected to immunoblotting analysis.
Immunoprecipitation and immunoblotting analysis using MPM-2
antibody (Davis et al. 1983), which was kindly provided by J.
Kuang, Pin1 antibodies (Lu et al., 1996, kindly provided by M.
White or newly generated), anti-phospho.Tyr antibody (UBI),
anti-Cdc25C (Ogg, et al., 1994) (from H. Piwnica-Worms and Santa
Cruz Biotechnology), anti-Cdc27, anti-Plk1 (Zymed), anti-Cdc2
(Solomon, M. J., M. Glotzer, T. H. Lee, M. Philippe and M. W.
Kirschner. Cell 63:1013-1024 (1991), anti-human Myt1, anti-human
cyclin B1 and anti-Xenopus cyclin B were performed, as described
previously in Lu and Hunter, 1995; Lu et al., 1996).
EXAMPLE 8
Coimmunoprecipitation of Pin1 and Cdc25
[0112] To detect Pin1 and Cdc25 interaction during the Xenopus cell
cycle, about 500 eggs were fertilized in a minimal volume of MMR
(100 m NaCl, 2 mM KCl, 1 mM MgCl2, 2m CaCl2, 0.1 mM EDTA, 5 mM
HEPES, pH 7.8), diluted in 0.1.times. MMR for 10 minutes, dejellied
as described in Murray, (1991) and incubated in CSF-XB (100 mM KCl,
0.1 mM CaCl2, 2 mM MgCl2, 10 mM K-HEPES, pH 7.7, 50 mM Sucrose 5 mM
EGTA, pH 7.7). At the indicated time after fertilization 15 eggs
were crushed into 150 .mu.l of ice cold CSF-XB with 1 .mu.M okadeic
Acid, microcenfuged for 20 seconds, the layer between the yolk and
the pellet was removed to a fresh chilled tube. This solution was
mixed well and 5 .mu.l was frozen in liquid nitrogen for future H1
kinase assays, and 30 .mu.l was diluted in 10 .mu.l of either
.alpha.-cdc25 or control rabbit sera beads in 100 .mu.l of buffer A
(containing 5 mM EDTA and 1 .mu.M microcystein but not vanadate).
The immunoprecipitation reactions were rotated for approximately 40
minutes at 4.degree. C., washed 4 times in and subjected to
immunoblotting with anti-Pin1 antibodies. The associated Pin1 was
quantified as described (Stukenberg et al., 1997).
EXAMPLE 9
Cdc2 and Cdc25 Assays
[0113] Cdc2 was assayed using histone H1 as a substrate, as
previously described in Murray, 1991; Lu and Hunter, (1995). Cdc25
activity was assayed by using the activation of its endogenous
substrate, Cdc2/cyclin B complex phosphorylated on Thr161, Tyr15,
Thr14 as an indicator using a variation of an established protocol
(Kumagai, A. and W. G. Dunphy. 1996. Science 273:1377-1380 (1996).
When cyclin B is added to a Xenopus interphase extract at levels
insufficient to activate mitosis (referred to as a "subthreshold
cyclin concentration"), the added cyclin B binds Cdc2 and the Cdc2
in the complex is phosphorylated by CAK, Wee1 and Myt 1 to
accumulate in an inactive form (Solomon et al. 1991).
[0114] A subthreshold concentration of GST cyclin B (10 .mu.g) was
added to 1 ml of Xenopus interphase extract for thirty min at room
temperature (Solomon, M. J., M. Glotzer, T. H. Lee, M. Philippe and
M. W. Kirschner, Cell 63:1013-1024 (1990). This was diluted 8 fold
in XB+ 3 mM DTT, rotated for 1 h with 3 ml of GST agarose beads,
washed 3 times in XBIP (XB+500 mM NaCl and 1% NP40+2 mM DTT),
washed 2 times (once overnight) in EB (80 mM .beta. glycerol
phosphate, 15 mM EGTA, 15 mM MgCl2)+2 mM DTT, 500 mM NaCl and 1%
NP40, and finally twice with EB +10 mM DTT. These Cdc25 assay beads
were stored at 4.degree. C. for up to 1 month.
[0115] Mitotic GST-Cdc25 was purified by incubating 22 .mu.g of
GST-Cdc25, in a Xenopus mitotic extract for 30 min at 23.degree.
C., this was diluted 8 fold in XB and rotated with 50 .mu.l of
glutathione-Agarose beads (Sigma) for 1 hr at 4.degree. C. The
beads were washed 5 times in XB-IP, twice in XB+2 mM DTT and eluted
in 25 .mu.l XB+2 mM reduced GSH. The final concentration of Mitotic
GST-Cdc25 was 0.36 mg/ml. A 27 fold dilution of this mitotic
GST-Cdc25 could fully activate Cdc2 in the assay below, while
GST-cdc25 isolated from Interphase extracts in parallel lost
activity after a 3 fold dilution. Thus the mitotic extract
stimulated the Cdc25 at least 9 times over interphase extracts. To
assay Cdc25 activity 1 .mu.M mitotic GST-cdc25, and the indicated
concentration of either Pin1, Pin1.sup.R68, 69A or BSA were
incubated in a 20 .mu.l reaction in XB+1 mM ATP for 10 minutes at
room temperature. These reactions were sequentially diluted (1/1,
1/3, 1/9, 1/27) into XB+1 mM ATP and 10 .mu.l of each was mixed
with 10 .mu.l of cdc25 assay beads for 10 minutes at room
temperature with constant shaking. The Cdc25 assay beads were
washed 3 times in XB-IP, 2 times in EB+1 mM DTT and assayed for H1
kinase activity. Phosphoimager analysis of the H1 kinase assays
were quantified by the Molecular Dynamics ImageQuant 3.3 software.
As described herein an assay with 1 .mu.M mitotic GST-cdc25, 0.67
.mu.M of either Pin1, Pin1.sup.R68, 69A or 16 .mu.M BSA then
diluted 27 fold before being mixed with the Cdc25 assay beads and
the amount of H1kinase activity is relative to the amount of
activity of the beads without cdc25 being zero and the BSA reaction
being 100%. The most reproducible way to quantify the Cdc25
activity in this assay was by determining the endpoint dilution of
Cdc25 which could activate Cdc2. Therefore the Cdc25 activity is
quantified by the endpoint dilution of the mitotic GST-Cdc25 at
which Cdc2 on the beads could still be significantly activated.
EXAMPLE 10
Pin1 Levels are Constant through the Cell Cycle
[0116] Whereas overexpression of Pin1 results in G2 arrest,
depletion of Pin1 induces mitotic arrest without affecting DNA
synthesis. To determine the basis for this cell cycle-specificity,
it was determined whether Pin1 protein level fluctuated during the
cell cycle. To address this question, HeLa cells were synchronized
at the G1/S boundary. At different times following the release from
the block, cells were harvested and analyzed by flow cytometry or
lysed and analyzed for protein expression by immunoblotting.
Analysis of DNA content and cyclin BY levels indicated that the
HeLa cells synchronously progressed through different phases of the
cell cycle. However, total Pin1 levels did not change significantly
during the cell cycle.
EXAMPLE 12
Pin1 Directly Binds a Subset of Conserved Mitotic
Phosphoproteins
[0117] Since the levels of Pin1 do not fluctuate during the cell
cycle, its mitosis-specific function is likely conferred by some
other mechanisms. There are many such possibilities. Pin1 could be
subjected to post-translational modifications, such as
phosphorylation, or allosteric interactions with a transiently
appearing subunit, like a cyclin which regulates its activity.
Alternatively, the interaction of Pin1 and its targets may be cell
cycle-regulated. Initial experiments suggested no evidence for Pin1
phosphorylation or for interaction of Pin1 with a regulatory
subunit. A cell cycle-dependent interaction of Pin1 with its
binding proteins was tested for.
[0118] A glutathione-S-transferase (GST) fusion protein containing
full length Pin1 was bacterially expressed, purified, and then used
to probe for interacting proteins in S-phase, mitosis or G1-phase
by Farwestern analysis. The ability of Pin1 to interact with
cellular proteins remained relatively low during S, increased when
cells progressed though G2/M (10 h point), and was almost
completely lost when cells moved to the next G1 (14 h point).
However, if cells were not allowed to progress into the next cell
cycle, but rather were blocked at mitosis by adding nocodazole
(14+Noc), Pin1-binding activity increased even further. Since the
binding activity was detected using denatured proteins, the
protein-protein interaction between Pin1 and these proteins must be
direct.
[0119] To examine whether this Pin1 interaction with its target
proteins occur under nondenaturing conditions and to estimate the
number of Pin1-interacting proteins, glutathione beads containing
GST and GST-Pin1 were incubated with interphase and mitotic
extracts, and beads were extensively washed and proteins bound to
beads were separated on SDS-containing gels and stained with
Coomassie blue. Whereas GST did not precipitated any detectable
proteins from either interphase or mitotic extracts, GST-Pin1
specifically precipitated about 30 clearly Coomassie-stainable
bands from mitotic extracts, but only 4-7 minor bands from
interphase extracts. Together, these two results indicate that Pin1
mainly interacts with a subset of proteins in a mitosis-specific
manner.
[0120] The crystal structure of Pin1 suggests that Pin1 could
strongly interact with a Phospho.Ser/Thr-Pro motif (Ranganathan, et
al., Cell 89:875-886 (1997)). A large number of proteins have been
shown to be phosphorylated at such a motif specifically during
mitosis and many of these phosphoproteins are recognized by the
MPM-2 antibody. Therefore, interactions between Pin1 and MPM-2
antigens were examined. After incubation with soluble proteins
prepared from interphase and mitotic HeLa cells, GST-Pin1 and
control GST glutathione beads were washed extensively and the
interacting proteins are detected by immunoblotting with the MPM-2
antibody. Many of the GST-Pin1-binding proteins reacted with MPM-2
only in the mitotic extracts, including a strong band of 55 kDa
(p55). p55 has been previously shown to be the most prominent MPM-2
antigen in HeLa cells (Zhao et al., FEBS Lett 249:389-395 (1989),
although its identity remains to be determined. In contrast,
control GST glutathione beads precipitated just two proteins
(p58/60) from either lysate. In addition, when MPM-2
immunoprecipitates were subjected to Farwestern analysis using
GST-Pin1 as a probe, Pin1 directly bound MPM-2 antigens on
membranes.
[0121] To determine whether GST-Pin1 can deplete MPM-2 antigens and
to estimate what concentrations of Pin1 are required to completely
deplete MPM-2 antigens, mitotic extracts were incubated with
different amounts of GST-Pin1, followed by analyzing MPM-2 antigens
remaining in the depleted supernatants. The total cellular Pin1
concentration in HeLa cells was estimated to be about 0.5 .mu.M,
based on immunoblotting analysis using anti-Pin1 antibodies with
recombinant Pin1 protein as a standard. At a concentration (8
.mu.M) that was about 15 fold higher than the endogenous level,
Pin1 depleted the majority of MPM-2 antigens, indicating that Pin1
strongly interacts with most MPM-2 antigens. The above results
demonstrate that Pin1 interacts with MPM-2 antigens in vitro.
[0122] To determine if endogenous Pin1 interacts with MPM-2
antigens in the cell, Pin1 was immunoprecipitated from either
interphase or mitotic HeLa extracts using anti-Pin1 antibodies in
the presence of various phosphatase inhibitors. The resulting Pin1
immunoprecipitates were probed with MPM-2. As described above,
several MPM-2 antigens were co-immunoprecipitated with anti-Pin1
antibodies. These results indicate that stable complexes between
Pin1 and MPM-2 antigens exist in the cell and that Pin1 does not
form complexes with all Pin1-binding proteins at the same time in
vivo.
[0123] Since Pin1 and MPM-2 antigens are highly conserved, it is
possible that Pin1-binding proteins are also conserved. To examine
this possibility, the interaction between human Pin1 and mitotic
phosphoproteins in Xenopus extracts was observed. When GST-Pin1 was
incubated with interphase or mitotic egg extracts, Pin1
specifically precipitated a subset of MPM-2 antigens from mitotic
extracts, with molecular weights similar, although not identical,
to those present in human cells. Again, this interaction between
Pin1 and Xenopus MPM-2 antigens was specific as it was not detected
if the precipitation was performed with control GST glutathione
beads. These results demonstrate that Pin1 also interacts with a
subset of conserved mitosis-specific phosphoproteins in
Xenopus.
EXAMPLE 13
Mutations in the Binding Pocket Abolish the Ability of Pin1 to
Interact with Most Mitotic Phosphoproteins
[0124] The above results demonstrate that Pin1 directly binds
numerous conserved mitotic phosphoproteins in a mitosis-dependent
manner. To insure that this interaction is highly specific for
Pin1, site-specific mutations were introduced into Pin1. A high
resolution X-ray structural and preliminary functional analysis of
Pin1 suggest that a basic cluster consisting of Lys63, Arg68, and
Arg69 is likely to be coordinate the putative phosphate group in
the substrate. Ala substitutions at these residues (Pin1.sup.R68,
69A) should cause a reduction in the ability to bind phosphorylated
residues N-terminal to the target Pro residue in the substrate. In
addition, His59 has been shown to have an intimate contact with the
cyclic side chain of the catalyzing Pro residue. An Ala
substitution at His-59 of Pin1 (Pin1.sup.H59A) should therefore
disrupt the interaction between Pin1 and the substrate Pro
residue.
[0125] The mutant proteins were expressed and purified as GST
fusion proteins, and both their PPIase activity and their ability
to bind mitotic phosphoproteins were determined. PPIase activity
was assayed with two peptide substrates: AEPF, which has an acidic
residue at the position N-terminal to the catalytic Pro residue,
and AAPF, which does not. Pin1 had a strong preference for the AEPF
substrate. The PPIase activity of Pin1.sup.R68, 69A was reduced
more than 90% against AEPF, whereas the reduction was very small
against AAPF. Moreover, Pin1.sup.R68, 69A had little preference for
either substrate. These results confirm that residues Arg68 and
Arg69 are critical for promoting strong selection for a negatively
charged residue at the position N-terminal to the substrate Pro
residue. The PPIase activity of Pin1.sup.H59A was barely detectable
against either peptide substrate, confirming the importance of
His59 in Pin1 substrate binding and/or catalysis.
[0126] To determine if the Pin1 mutants interact with mitotic
phosphoproteins, GST-Pin1, -Pin1.sup.R68, 69A and -Pin1.sup.H59A
fusion proteins were incubated with interphase or mitotic HeLa cell
extracts and associated proteins subjected to MPM-2 immunoblotting
analysis. Pin1 specifically interacted with MPM-2 antigens in two
independently prepared mitotic extracts, but the binding activity
of both Pin1.sup.R68, 69A and Pin1.sup.H59A was significantly
reduced compared to the wild-type protein. A few proteins including
the most strongly reacting p55 band could still be recognized. The
two Pin1 mutants also failed to bind most mitotic phosphoproteins
from Xenopus extracts. Thus, mutating the residues that are
implicated in binding either the substrate's putative phosphate
group or the substrate's Pro residue abolish the ability of Pin1 to
bind MPM-2 antigens. This suggests that Pin1 must recognize both
the Phospho Ser/Thr and the Pro residues to bind MPM-2 antigens.
These results also demonstrate that mitotic phosphoproteins
specifically interact with active site residues of Pin1
EXAMPLE 14
Identification of Several Mitotic Regulators as Pin1 Targets
[0127] Several known mitotic regulators such as cyclin B, Cdc25,
Myt1, Plk1 and Cdc27 are phosphorylated at mitosis. To identify at
least a few of the many Pin1 binding proteins, Pin1-binding
proteins were precipitated from HeLa cells, or Xenopus extracts and
probed with antibodies specific for known mitotic phosphoproteins.
As shown previously, levels of Plk1 and cyclin B1 increased at
mitosis, whereas similar amounts of Cdc25C were present in
interphase and mitotic HeLa cells. Moreover, a significant fraction
of Cdc25C, Plk1, Myt1, Cdc27 and PTP-1B became hyperphosphorylated
during mitosis and exhibited a shift in electrophoretic mobility by
SDS-PAGE. Although cyclin B1 and PTP-1B were not precipitated by
Pin1 in either interphase or mitotic extracts, Pin1 bound
selectively only to the mitotically hyperphosphorylated form of
Cdc25C, Plk1, Myt1 and Cdc27. Furthermore, neither mutant
Pin1.sup.R68, 69A nor Pin1.sup.H59A interacted with Cdc25 or Cdc27,
indicating that the residues that are implicated in binding either
the substrate's putative phosphate group or the substrate's Pro
residue are necessary for Pin1 to bind Cdc25 and Cdc27. Similarly,
only the mitotic, but not the interphase form of Xenopus Cdc27 was
precipitated by Pin1. Moreover, pretreatment of the mitotic extract
with calf intestine phosphatase (CIP) completely dephosphorylated
Cdc27 and abolished the interaction between Pin1 and Cdc27,
demonstrating a phosphorylation-dependent interaction. These
results indicate that the interaction between Pin1 and Cdc25 or
Cdc27 is likely to be mediated by a Phospho.Ser/Thr-Pro motif.
[0128] To gain a sense of the generality of the interaction between
Pin1 and mitotic phosphoproteins and to confirm the Pin1
interaction with target proteins is indeed mediated by
phosphorylation, the ability of Pin1 to bind other known mitotic
phosphoproteins and a set of mitotic phosphoproteins identified by
a systematic phosphoprotein screen (Stukenberg et al., Curr Biol
7:338-348 (1997). Proteins synthesized in vitro were phosphorylated
in a cell cycle specific manner by incubating them in either
Xenopus interphace or mitotic extracts. These labeled protein were
subsequently incubated with GST-Pin1 beads that were extensively
washed and the bound proteins analyzed by SDS-PAGE. To validate
this method, Cdc25 was first tested. Again, the mitotically
phosphorylated form of in vitro translated Cdc25 could be
precipitated by GST-Pin1 beads. However, Cdc25 was not recognized
by Pin1 if it was incubated in interphase extracts. Moreover, Pin1
did not interact with Cdc25 if the mitotically phosphorylated Cdc25
was treated with phosphatase prior to the GST-Pin1 incubation.
These results demonstrate that this method can be used to detect
mitosis-specific and phosphorylation-dependent interactions between
Pin1 and phosphoproteins. Out of the 13 mitotic phosphoproteins
examined, Pin1 bound 10 in a mitosis and phosphorylation-dependent
manner (summarized in Table 1), including Wee1, MP75 and MP110,
MP75 and MP110 are Xenopus proteins related to
microtubule-associated protein E-MAP-115 and the fission yeast G2
transcription factor Cdc5, respectively. These results indicate
that Pin1 may target many mitosis-specific phosphoproteins.
EXAMPLE 15
Pin1 Blocks Cell Cycle Progression in Xenopus Embryos and Entry
into Mitosis in Xenopus Extracts
[0129] Since Pin1 is conserved from yeast to humans, it is likely
that Pin1 exists in Xenopus. To confirm this, Xenopus egg extracts
were immunoblotted with two separate anti-human Pin1 antisera. Both
antibodies, but not their respective preimmune sera, specifically
recognized a band which comigrated with human Pin1 at 18 kDa,
indicating that Pin1 is present in Xenopus.
[0130] Overexpression of Pin1 has been shown to inhibit cell cycle
progression in both yeast and HeLa cells. To examine whether
increasing the concentration of Pin1 has similar biological effects
in Xenopus, Pin1 or Pin1 mutants were injected into one cell of 2
cell stage embryos and allowed the embryos to develop for 3 h
(about 5 divisions). Wild-type Pin1 injected cells failed to cleave
or cleaved slowly when compared to the cells in the uninjected
side. A similar concentration (4 .mu.M final) of either Pin1 mutant
had little effect on the cell cycle. In a separate experiment Pin1
blocked cleavage of the injected cells in a concentration-dependent
manner, and at a concentration approximately 20 fold above the
estimated endogenous levels (10 .mu.M), completely inhibited the
cell cycle (FIG. 1). In contrast, higher concentrations of the
mutant proteins were needed to block the cell cycle (FIG. 1).
Injection of control BSA had no obvious effect on cell cycle
progression. These results suggest that Pin1 must bind mitotic
phosphoproteins in order to block cell cycle progression. To
determine the nature of the cleavage block in Xenopus, GST-Pin1 was
added to Xenopus egg extracts that had been arrested in second
meiotic metaphase due to the activity of cytostatic factor. These
extracts are arrested in mitosis (meiosis II) and reenter the cell
cycle in response to the addition of Ca.sup.2+. Extracts containing
demembranated sperm to monitor nuclear morphology and
rhodamine-tubulin to monitor microtubule spindle assembly, were
activated with Ca.sup.2+. Pin1 was added after the extracts had
entered interphase (15 min after the addition of Ca.sup.2+), and
the subsequent entry of the extracts into mitosis was followed by
nuclear morphology and Cdc2 kinase activity using histone H1 as a
substrate. Addition of either 10 or 40 .mu.M Pin1, approximately 20
or 80 fold higher than endogenous levels, completely blocked entry
into mitosis as detected by the persistence of interphase nuclei
and low Cdc2 kinase activity. In contrast, the same extracts
containing 40 .mu.m of either BSA or the mutant Pin1 proteins
entered mitosis by 70 to 80 min as detected by nuclear envelope
breakdown, spindle formation and high histone H1 kinase activity.
Thus, as was shown previously in HeLa cells, increasing the Pin1
concentration causes a cell cycle block in G2. More importantly,
Pin1 must bind mitotic phosphoproteins to elicit this
phenotype.
EXAMPLE 16
Pin1 Binds and Inhibits Mitotically Phosphorylated Cdc25
[0131] The above results indicate that overexpression of Pin1
inhibits mitotic entry in Xenopus, as is the case in HeLa cells and
yeast. Entry into mitosis is regulated by dephosphorylation of Cdc2
by the phosphatase Cdc25, and Cdc25 is activated by
mitosis-specific phosphorylation at the MPM-2 epitope at the G2/M
transition. Earlier results indicated that it is the mitotically
phosphorylated form of Cdc25 that interacts with Pin1 in vitro.
Therefore, it is conceivable that the inhibitory effects of Pin1 on
entry into mitosis could at least partially explained through
inhibition of Cdc25 activity.
[0132] To test this possibility, it was determined whether Pin1
interacts with Cdc25 in vivo and if so, whether this interaction is
cell cycle regulated. Xenopus eggs were collected at various times
after fertilization and subjected to immunoprecipitation using
anti-Xenopus Cdc25 antibodies as well as histone H1 kinase assay to
monitor cell cycle progression. When the resulting Cdc25
immunoprecipitates were immunoblotted with anti-Pin1 antibodies, we
found that endogenous Pin1 was precipitated by anti-Cdc25
antibodies. Furthermore, this interaction between Pin1 and Cdc25
was cell cycle-regulated, significantly increased just prior to
mitosis. Similar results were also obtained using synchronized HeLa
cells using anti-human Cdc25C. Unfortunately, we were not able to
detect Cdc25 in anti-Pin1 immunoprecipitates, probably because the
amount of Cdc25 precipitated is below the detection of the Cdc25
antibodies. It is worth of pointing out that the percentage of
coimmunoprecipitatable Pin1 and phosphorylated Cdc25 is not high.
This might be expected because the complex might not be stable to
the stringent immunoprecipitation conditions, the amount of Cdc25
phosphorylated on Pin1-binding sites might be low at this point,
and/or the complex might have a high off rate, since the
phosphorylated Cdc25 is a substrate of Pin1. Nevertheless, these
results suggest that Pin1 is associated with Cdc25 at a time when
Cdc25 is partially phosphorylated and yet its activity is low.
[0133] Since the interaction between Pin1 and Cdc25 is mediated by
phosphorylation of Cdc25, it was determined whether Pin1 interacts
with Cdc25 on important phosphorylation sites. At entry into
mitosis, Cdc25 is phosphorylated at multiple Thr/Ser-Pro (Peng, C.
Y. Graves, P. R., Thoma, R. S. Wu, Z. Shaw, A. S. and
Piwnica-Worms, H. Science, 277:1501-1505 (1997)).
[0134] Izumi and Maller (Izumi, T. and J. L. Maller, Mol Biol Cell
4:1337-1350 (1993)) have shown that the triple mutation of
conserved Thr48, Thr67 and Thr138 (T3 Cdc25), and the quintuple
mutation of the three Thr residues plus Ser205 and Ser285 (T3S2
Cdc25) prevent most of the shift in electrophoretic mobility of
Cdc25 after incubation with mitotic extracts. When they measured
the ability of the Cdc25 mutants to activate Cdc2 in the
Cdc25-depleted oocyte extracts and to initiate mitotic entry in
oocyte extracts, the activities of T3 and T3S2 mutants were reduced
about 70% and 90%, respectively (Izumi and Maller, 1993). These
results indicate that these Thr/Ser residues are essential for the
Cdc25 function. We examined the ability of Pin1 to bind the T3 and
T3S2 Cdc25 mutants. As shown previously (Izumi and Maller, 1993),
the T3 and T3S2 Cdc25 mutants failed to undergo the mobility shift
after incubation with mitotic extracts. Although Pin1 strongly
bound mitotically phosphorylated form of Cdc25, Pin1 almost (T3) or
completely (T3S2) failed to bind the Cdc25 mutants which were
incubated with either interphase or mitotic extracts. Although
further experiments are required to pinpoint which phosphorylation
site(s) play(s) the major role in mediating the Pin1 and Cdc25
interaction, these results show that Pin1 interacts with the
phosphorylation sites on Cdc25 that are essential for its mitotic
activation.
[0135] The above results indicate that Pin1 interacts with Cdc25
both in vitro and in vivo. Therefore, we tested whether Pin1 could
affect the physiological activity of Cdc25, which is to
dephosphorylate and activate the cyclin B/Cdc2 complex. To generate
the mitotically phosphorylated form of Cdc25, GST-Cdc25 was
incubated in Xenopus mitotic extracts, affinity purified on
glutathione agarose beads and eluted. This mitotic Cdc25 was at
least 9 fold more active than GST-Cdc25 purified in parallel from
interphase extracts (data not shown). This mitotic GST-Cdc25
activated cyclin B/Cdc2 complex that was kept inactive due to
inhibitory phosphorylations on Tyr15 and Thr14. If Pin1 (0.67
.mu.M) was included in the assay at amounts approximately
stoichiometric to mitotic Cdc25 (1 .mu.M), mitotic Cdc25 failed to
activate the Cdc2 complex. In contrast, neither the mutant
Pin1.sup.R68, 69A at the same concentration (0.67 .mu.M), or BSA at
a 25 fold higher concentration (15 .mu.M) had a significant
inhibitory effect on Cdc25 activity (FIG. 3A). Five fold higher
concentrations of Pin1.sup.R68, 69A could partially inhibit mitotic
Cdc25 activity (FIG. 3B), a result which is consistent with the
requirement for higher concentrations of this mutant protein to
arrest the Xenopus cell cycle. To rule out the possibility that
Pin1 could directly inhibit the cyclin B/Cdc2 complex itself, we
examined the effect of Pin1 and its mutants on the activity of
dephosphorylated-active cyclin B/Cdc2 under same conditions.
Neither Pin1 nor the Pin1 mutant had any effect on Cdc2 activity
(FIG. 3C). Taken together, these results indicate that Pin1 could
inhibit premature mitotic activation of Cdc25 by interacting with
the phosphorylation sites on Cdc25 that are essential for its
activation. This offers one explanation for the ability of Pin1 to
inhibit mitotic entry.
EXAMPLE 17
Screening of Peptide Libraries
[0136] The oriented peptide library approach (Z. Songyang et al.
Cell, 72:767 (1993) was used to screen for optimal peptides. All
amino acids except C were incorporated at equimolar amounts in each
degenerate position, yielding a total theoretical degeneracy for
both libraries of 19.sup.6=4.7.times.10.sup.7 distinct peptide
sequences. Pin1-GST beads and MPM2 antibody bound to protein-G
beads were incubated with the peptide library mixtures and washed
extensively. Bound peptides were eluted with 30% acetic acid and
sequenced.
[0137] The chromogenic oligopeptides were synthesized (A.
Bernhardt, M. Drewello, & M. Schutkowski, Int. J. Peptide
Protein Res. 50:143 (1997)) and confirmed by NMR. Standard peptides
were purchased from Bachem. PPIase activity were assayed and the
bimolecular rate constants k.sub.cat/K.sub.m were calculated
according to the equation
k.sub.cat/K.sub.m=(k.sub.obs-k.sub.u)/[PPIase], where k.sub.u is
the first-order rate constant for spontaneous cis/trans
isomerization and k.sub.obs is the pseudo-first-order rate constant
for cis/trans isomerization in the presence of PPIase, as described
in G. Fischer, H. Bang, C. Mech, Biomed. Biochim. Acta 43:1101
(1984); J. L. Kofron et al. Biochemistry 30:6127 (1991). Affinity
of Pin1 for peptides was measured as described in Schutkowski, M.,
Wollner, S., & Fischer, G. Biochemistry 34:13016, (1995).
[0138] Equivalents
[0139] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
54 1 8 PRT synthetic nucleotide PHOSPHORYLATION (4)...(5) VARIANT
(1)...(8) Xaa = Any Amino Acid 1 Xaa Xaa Xaa Ser Pro Xaa Xaa Xaa 1
5 2 12 PRT synthetic nucleotide PHOSPHORYLATION (6)...(6) VARIANT
(1)...(12) Xaa = Any Amino Acid 2 Met Ala Xaa Xaa Xaa Ser Xaa Xaa
Xaa Ala Lys Lys 1 5 10 3 6 PRT synthetic nucleotide PHOSPHORYLATION
(5)...(5) 3 Ala Ala Pro Leu Asn Ala 1 5 4 7 PRT synthetic
nucleotide PHOSPHORYLATION (6)...(6) 4 Ala Ala Ala Pro Arg Asn Ala
1 5 5 6 PRT synthetic nucleotide PHOSPHORYLATION (5)...(5) 5 Ala
Ala Pro Met Asn Ala 1 5 6 6 PRT synthetic nucleotide
PHOSPHORYLATION (5)...(5) 6 Ala Asp Pro Tyr Asn Ala 1 5 7 6 PRT
synthetic nucleotide PHOSPHORYLATION (5)...(5) 7 Ala Glu Pro Phe
Asn Ala 1 5 8 6 PRT synthetic nucleotide PHOSPHORYLATION (5)...(5)
8 Ala Tyr Pro Tyr Asn Ala 1 5 9 6 PRT synthetic nucleotide
PHOSPHORYLATION (2)...(2) PHOSPHORYLATION (5)...(5) 9 Ala Tyr Pro
Tyr Asn Ala 1 5 10 6 PRT synthetic nucleotide PHOSPHORYLATION
(5)...(5) 10 Ala Ser Pro Tyr Asn Ala 1 5 11 6 PRT synthetic
nucleotide PHOSPHORYLATION (2)...(2) PHOSPHORYLATION (5)...(5) 11
Ala Ser Pro Tyr Asn Ala 1 5 12 6 PRT synthetic nucleotide
PHOSPHORYLATION (5)...(5) 12 Ala Thr Pro Tyr Asn Ala 1 5 13 6 PRT
synthetic nucleotide PHOSPHORYLATION (2)...(2) PHOSPHORYLATION
(5)...(5) 13 Ala Thr Pro Tyr Asn Ala 1 5 14 7 PRT synthetic
nucleotide PHOSPHORYLATION (6)...(6) 14 Ala Ala Glu Pro Phe Asn Ala
1 5 15 7 PRT synthetic nucleotide PHOSPHORYLATION (6)...(6) 15 Ala
Ala Ser Pro Phe Asn Ala 1 5 16 7 PRT synthetic nucleotide
PHOSPHORYLATION (3)...(3) PHOSPHORYLATION (6)...(6) 16 Ala Ala Ser
Pro Phe Asn Ala 1 5 17 7 PRT synthetic nucleotide PHOSPHORYLATION
(6)...(6) 17 Ala Ala Thr Pro Phe Asn Ala 1 5 18 7 PRT synthetic
nucleotide PHOSPHORYLATION (3)...(3) PHOSPHORYLATION (6)...(6) 18
Ala Ala Thr Pro Phe Asn Ala 1 5 19 7 PRT synthetic nucleotide
PHOSPHORYLATION (6)...(6) 19 Ala Ala Ser Pro Arg Asn Ala 1 5 20 7
PRT synthetic nucleotide PHOSPHORYLATION (3)...(3) PHOSPHORYLATION
(6)...(6) 20 Ala Ala Ser Pro Arg Asn Ala 1 5 21 8 PRT synthetic
nucleotide PHOSPHORYLATION (3)...(3) PHOSPHORYLATION (7)...(7) 21
Trp Tyr Ser Pro Arg Thr Asn Ala 1 5 22 7 PRT synthetic nucleotide
PHOSPHORYLATION (3)...(3) PHOSPHORYLATION (6)...(6) 22 Ala Ala Thr
Pro Arg Asn Ala 1 5 23 8 PRT synthetic nucleotide PHOSPHORYLATION
(7)...(7) 23 Trp Phe Tyr Ser Pro Arg Asn Ala 1 5 24 8 PRT synthetic
nucleotide PHOSPHORYLATION (4)...(4) PHOSPHORYLATION (7)...(7) 24
Trp Phe Tyr Ser Pro Arg Asn Ala 1 5 25 8 PRT synthetic nucleotide
PHOSPHORYLATION (4)...(4) PHOSPHORYLATION (7)...(7) 25 Trp Phe Tyr
Ser Pro Arg Asn Ala 1 5 26 6 PRT synthetic nucleotide
PHOSPHORYLATION (4)...(4) 26 Trp Phe Tyr Ser Pro Arg 1 5 27 8 PRT
synthetic nucleotide 27 Tyr Val Gly Thr Pro Phe Tyr Met 1 5 28 8
PRT synthetic nucleotide 28 Phe Tyr Met Ser Pro Glu Ile Cys 1 5 29
8 PRT synthetic nucleotide 29 Ile Leu Asn Thr Pro Val Ile Arg 1 5
30 8 PRT synthetic nucleotide 30 Glu Ser Arg Thr Pro Phe Thr Arg 1
5 31 8 PRT synthetic nucleotide 31 Lys Ser Arg Ser Pro His Arg Arg
1 5 32 8 PRT synthetic nucleotide 32 Glu Met Pro Ser Pro Phe Leu
Ala 1 5 33 8 PRT synthetic nucleotide 33 Tyr Leu Gly Ser Pro Ile
Thr Thr 1 5 34 8 PRT synthetic nucleotide 34 Ala Asn Ile Thr Pro
Arg Glu Gly 1 5 35 8 PRT synthetic nucleotide 35 Gly Arg Arg Ser
Pro Arg Pro Asp 1 5 36 8 PRT synthetic nucleotide 36 Phe Leu Trp
Ser Pro Phe Glu Ser 1 5 37 8 PRT synthetic nucleotide 37 Ala Ser
Cys Ser Pro Ile Ile Met 1 5 38 8 PRT synthetic nucleotide 38 Leu
Arg Lys Ser Pro Phe Cys Arg 1 5 39 8 PRT synthetic nucleotide 39
Tyr Phe Ile Ser Pro Phe Gly His 1 5 40 8 PRT synthetic nucleotide
40 Arg Ser Arg Ser Pro Arg Arg Arg 1 5 41 8 PRT synthetic
nucleotide 41 Asp Ser Ala Ser Pro Arg Tyr Ile 1 5 42 8 PRT
synthetic nucleotide 42 Phe Phe Arg Ser Pro Arg Arg Met 1 5 43 8
PRT synthetic nucleotide 43 Asp Pro Tyr Ser Pro Arg Ile Gln 1 5 44
8 PRT synthetic nucleotide 44 Phe Gly Phe Ser Pro Ser Gly Thr 1 5
45 8 PRT synthetic nucleotide 45 Gln Leu Arg Ser Pro Arg Arg Thr 1
5 46 8 PRT synthetic nucleotide 46 Tyr Gly Lys Ser Pro Tyr Leu Tyr
1 5 47 8 PRT synthetic nucleotide 47 Lys Ile Arg Ser Pro Arg Arg
Phe 1 5 48 8 PRT synthetic nucleotide 48 Pro Cys Tyr Thr Pro Tyr
Tyr Val 1 5 49 8 PRT synthetic nucleotide 49 Gly Phe Phe Thr Pro
Arg Leu Ile 1 5 50 8 PRT synthetic nucleotide 50 Trp Tyr Arg Ser
Pro Arg Leu Leu 1 5 51 8 PRT synthetic nucleotide 51 Phe Met Met
Thr Pro Tyr Val Val 1 5 52 8 PRT synthetic nucleotide 52 Trp Gly
Ile Ser Pro Arg Gly Ala 1 5 53 8 PRT synthetic nucleotide 53 Asn
Trp Arg Ser Pro Arg Leu Arg 1 5 54 7 PRT synthetic nucleotide
VARIANT (1)...(7) Xaa = Any Amino Acid 54 Xaa Xaa Ser Thr Xaa Xaa
Xaa 1 5
* * * * *